Profiling HIV1-host protein–protein interaction networks in patient-derived exosome proteins: impact on pathophysiology and innate immune pathways

Abstract

Objectives

The objective of this research is to investigate the pathophysiological progression of HIV from acute infection to chronic immunodeficiency (AIDS) and to understand the host’s immunological responses, which are pivotal for elucidating disease aetiology and optimizing antiretroviral therapy (ART). Additionally, the study aims to explore the role of exosomes (40–130 nm bilipid-layered vesicles released by nearly all cell types) as key mediators of intercellular communication in the context of HIV infection.

Materials and methods

Recent research has uncovered that cells infected with HIV-1 release exosomes carrying a mix of viral and host components such as proteins, nucleic acids, lipids, and other metabolites. To decipher the plausible role of exosome-derived proteins in HIV disease progression, the exosomes isolated from HIV patient’s serum were subjected to LC–MS/MS analysis to identify exosome-derived human and viral protein sequences. The identified proteins were then investigated, annotated, and explored for protein–protein interaction (PPI) network between HIV and the human host’s proteins. Earlier experimental efforts focused on identifying PPI networks in host cells or only within the virus.

Results

The analysis showed that out of twelve exosome-derived host proteins identified from HIV-1 patient’s samples, only five of the proteins were associated with Toll-like receptors (TLR), inflammasome-activation, inhibition of apoptosis, innate immune response modulation, and autophagy pathways. In the TLR pathway, CDH5, ENO1, OGT, TJP1, and TRAF6 exosome-derived host proteins participated in regulation. Notably, CDH5, ENO1, OGT, and TRAF6 were shared among these pathways, BioGRID version 4.4 showed that HIV-1 Gag, Gag-pol, Env, and Nef proteins interact with 196, 162, 158, and 80 human proteins, respectively, associated with different innate immune response pathways.

Conclusion

These findings are a step ahead in comprehending the pathophysiology of HIV1 and the innate immune response pathways, providing excellent opportunities to explore further exosome-based biomarkers for theranostic approaches.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12985-025-02717-7.

Background

Human immunodeficiency virus (HIV), a member of the Retroviridae family, responsible for Acquired Immuno Deficiency Syndrome (AIDS), a global pandemic since 1981, inflicting 39 million individuals [1–3]. Out of two types, HIV-1 is the most common, highly virulent and pathogenic species worldwide; the second one, HIV-2, is slowly progressing and almost restricted to the West African population [4, 5]. There were 20.6 million HIV-positive people in Eastern and Southern Africa, compared to 6 million in Asia and the Pacific, 5 million in Western and Central Africa, and 2.3 million in Western and Central Europe and North America, attributing the disease to pandemic status [5]. HIV is structurally icosahedral, having two copies of 9–10 kb, non-segmented, + ss RNA genome possessing nine genes to express fifteen proteins, either directly translated or reverse transcribed to integrate into the host genome to establish latency [6–12].

The viral proteins play a pivotal role in immunopathology and disease progression, explicitly evading the innate immune system through various complex mechanisms. The env-gene codes for gp160, which gets cleaved into envelope glycoproteins-gp120, a ligand to interact with host CD4⁺ T-cells and gp41, which assists in viral fusion and entry into the host cell [13–15]. The gag-gene encodes P55, which is cleaved by viral protease (P10) to form p24 or capsid (CA), p7 or nucleocapsids (NC), p17 or matrix (MA), and C-terminal p6 proteins that are involved in viral structure, assembly, and trafficking [16–19]. The pol-gene codes P100, which is cleaved to form reverse transcriptase (RT), p10 protease (PR), and integrase (IN), cumulatively control and interact with cellular proteins and viral egress [20–22]. On the other hand, the Nef gene (Negative expression factors) encodes P27, an accessory protein that facilitates high viral loads and AIDS progression [23]. Notably, the Nef protein aids in evading the innate immune response through (i) upregulating proinflammatory cytokines expression, such as IL-1β, IL-12, IL-15, and TNF-α, as well as chemokines like macrophage inflammatory protein (MIP)-1α, -1β, and IL-8 [24, 25] (ii) precisely, HIV-1 Nef act as a tetherin antagonist and reduces NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) onslaught on HIV-infected cells. It also downregulates MHC-I molecules [26] (iii) assists HIV-1 budding, and (iv) generates Nef miRNA (miR367) in persistently HIV-1 infected cells, which modulates both viral and host gene expression to facilitate virus replication and the establishment of latency [27]. The trans-activator of transcription (tat)-gene and rev-gene encode for tat and rev proteins, responsible for viral transcription and translocation of unspliced or incompletely spliced mRNAs, respectively. The Vpu, an antagonism of tetherin, inhibits ADCC-mediated killing of HIV-infected cells by NK cells [28–30]. HIV evades human immune regulation via genetic mutation and recombination of parts of its genome, leading to several constantly varying epitopes that prevent the antibody-mediated recognition and neutralization of HIV virions at any given time [31]. The KEGG pathway analysis representing interactions of viral proteins (white boxes) with host proteins (green boxes). This figure also exhibits various signalling pathways involved in HIV pathogenesis (Supplementary Fig. 1).

Like all other cells, HIV-infected cells also release extracellular vesicles (EVs) called exosomes, primarily composed of proteins, lipids, various types of RNA and DNA. The presence of these tiny (40–140 nm) EVs in bodily fluids like blood, urine and saliva enables them as biomarkers and efficient cellular messengers by transferring their content to nearby and distant cells [32]. That redefines the disease progression and cancer spread mechanism by modulating virulence, pathogenesis and immune evasion [33–35]. For focused delivery of viral proteases and inhibitors to infected cellular reservoirs and latently infected cell populations in HIV-1 infection and similar viral diseases, exosome-mediated delivery systems—biogenic nanoscale vesicles—represent a viable treatment option [36–38]. This approach improves accuracy in tackling tissue-specific biodistribution problems and viral persistence inherent in traditional antiretroviral treatments.

The studies have revealed that viruses like EBV that establish latency consistently produce LMP-1-containing exosomes [39]. Because exosomes and viral particles are produced simultaneously in infected cells, these components can alter complex cellular signalling networks, extending their effects beyond infected cells to surrounding uninfected cells. In addition, the exosomes have been shown to promote and inhibit the activation of inflammasomes in various conditions [40–42]. The pivotal role of exosomes in various disease pathogenesis has been established. However, the role of HIV patient-derived exosome pathophysiology is poorly understood and largely remains to be explored in detail.

Crucially, prior studies have established the role of exosomes in HIV-1 pathogenesis, predominantly on in vitro models or engineered exosome systems, which fail to recapitulate the native molecular architecture under clinical settings. Modulation of exosomes by various factors, such as the extracellular matrix, tissue-specific microenvironmental cues, immune signalling, and systemic circulation, is challenging to imitate in vitro. Similarly, HIV-1 interacts with different immune cells, such as macrophages, CD4+ T-cells, and dendritic cells, in a highly complex and tightly controlled manner. In vitro studies show a lack of complexity, complicating the replication of the complete viral lifecycle and immune response [43]. Emphasising the unique molecular load seen in patient-derived exosomes, studies revealed that exosomes produced from the plasma of HIV-positive individuals on ART greatly boosted cancer cell proliferation and migration compared to exosomes from uninfected controls [44]. Whereas in vivo research maintains exosomes' standard composition and function, in vitro models can change their payload. A comprehensive comparison of latent HIV-1 reactivation across various cell models and resting CD4+ T cells from aviremic but infected patients demonstrated that no single in vitro model could adequately represent the ex vivo response characteristics of latently infected T-cells from patients [45]. Further studies indicate that HIV-positive patient’s exosomes contain proteins related to immunological activation and oxidative stress, affecting immune responses in ways standard in vitro cell culture cannot [46]. Therefore, patient-derived samples maintain virus-host dynamics, improving our understanding of HIV pathogenesis and therapeutic development.

In this study, we have tried to resolve this gap by directly interrogating exosomes isolated from HIV-positive patient’s serum, preserving their physiological complexity. Their size and biochemical characterizations were done and subjected to LC–MS/MS analysis. The LC–MS/MS study revealed that serum-derived exosomes predominantly contained twelve host and four major HIV proteins (Gag, Gag-pol, Env, and Nef). First, we identified and annotated human exosomal proteins or peptide sequences obtained from MS/MS assays using databases such as UniProt, the Human Protein Atlas, the Human Proteome Organization (HUPO), the Ensembl browser, and HIV-specific databases. Unlike prior studies, our proteomic and interactome analyses show multifaceted interaction between HIV-derived proteins (Env, Gag, Gag-Pol, and Nef) and host exosomal proteins (CDH5, ENO1, OGT, TJP1, and TRAF6) reveals a dynamic viral-host interaction landscape that propels immune evasion and disease progression. Particularly in systemic comorbidities such as cancer metastases, cardiovascular illness, or immunological dysregulation, the interaction between HIV proteins and patient-derived exosomal proteins—especially TJP1, TRAF6, CDH5, and DENND4A—remains poorly understood. The proteomics and interactome study show that exosomes are key mediators in HIV pathogenesis. These findings help us comprehend HIV's immune evasion methods and suggest new diagnostics and treatments targeting exosome-mediated processes.

Materials and methods

Ethical approval, inclusion and exclusion criteria

Ethical approval was obtained from Jamia Hamdard, New Delhi, India Human Ethics Committee (Aproval number: JHIEC/09/21,). For confirmation of HIV, three well-standardized techniques were used in this hospital for the detection of HIV using kits: (i)- Enzyme-Linked Immunosorbent Assay (ELISA), (ii)-Rapid tests, and (iii)-Immunoconcentration/Dot Blot assay. HIV-positive patients of the age group 30–52 years of both genders (7 males and 3 females), patient under ART 2–8 months, BMI range 22.7–31.2 and alcohol consumer 2 and non-alcoholic 8, have been included in this study. Patients undergoing antiretroviral therapy (ART) for longer than one month were excluded. Patients with other STDs, UTIs, all inflammatory, neurodegenerative disorders, diabetes, smoking, and cancer were also excluded. The flow diagram systematically illustrates the sequential methodological stages employed in this study (Fig. 1).

Fig. 1.

Working model of the manuscript: The flow diagram illustrates the sequential methodological stages of this study. The exosomes isolated from HIV patients’ serum were characterized biochemically, and size characterization was also performed. The exosomal proteins were identified using LC–MS/MS analysis. The protein–protein interaction analysis was performed using various software

Cell culture

The THP1 cells were cultured in DMEM supplemented with 10% FBS and incubated in cell culture incubator (Thermo Fisher Scientific, USA), at 37°C and 5% CO2 for 2–3 days. The cells were harvested then lysed in RIPA buffer to prepare whole cell lysate (WCL). The WCL later used as positive control for calnexin protein.

Sample collection and exosome isolation

Blood samples were collected from ten different HIV-positive patients at an early stage and ten control individuals using standard venipuncture techniques. Using vacutainers, 2 ml of blood was collected in red top tubes (554,101 Samplix). The blood was left to clot at room temperature for 15–30 min, and serum was isolated. Serum was centrifuged at 3,000×g for 15 min to remove cellular debris, then 250 µl serum was mixed with 63 ul of ExoQuick reagent (System Biosciences, CA, USA), incubated on ice for 30 min, centrifuged at 1,500xg for 30 min. Supernatant was removed and the exosome pellet was suspended in normal saline stored at − 80 °C in a freezer until experiments were performed.

Exosome-derived protein estimation

Isolated exosomes were lysed in RIPA buffer and BCA (Bicinchoninic Acid) assay, as per the manufacturer's (603,100,011,730 GeNei) instructions. Briefly, the sample and standards were prepared, and after specified incubation with the reagents, the optical density at 562 nm was obtained using a spectrophotometer (Microplate reader, Biotek, USA).

Size characterization by DLS and electron microscopy (SEM and TEM)

The exosomes were characterized for their size by dynamic light scattering (DLS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The isolated exosomes were diluted with normal saline,and 10–100 µL of the prepared exosome suspension in cuvette,and weremeasured using the instrument's software interface..

The exosomes were stained. Approximately 35 μl of the sample was placed on a carbon grid and allowed to rest for 60 s before blotting with filter paper. Using forceps, the carbon grid was dipped face down in a water droplet for 2–3 s and again blotted. One percent uranyl acetate (3.5 μl) was pipetted onto the grid and allowed to rest for 15 s before blotting and drying, and TEM images were taken.

SDS-PAGE and western blotting

SDS-PAGE was performed in a vertical mini-gel electrophoresis apparatus (BioRad, USA) using a 12% resolving gel with a 5% acrylamide concentration stacking gel. Equal amounts of protein samples (20 µg) prepared in lamellae buffer were loaded. The protein marker (loading control), precision plus protein standards (dual color), catalog number 161–0374, was from Bio-Rad, USA). The proteins were transferred to the 0.45 um pore size PVDF membrane using an electrophoresis transfer unit (BioRad, USA). The membranes were washed in TBST (Tris-buffer saline with 0.05% Tween-20) blocked by incubating in 5% skimmed milk for an hour. Further, the membrane was incubated with primary antibodies for one hour and then probed with HRP-conjugated secondary antibodies for one hour. Finally, employing the ECL method, images were developed using the MP Chemidoc imaging system (BioRad, USA).

Mass spectrometric analysis of peptide mixtures

To identify the proteins present in the HIV-positive patients’ serum and healthy control individual-derived exosome samples, LC–MS/MS analyses were performed on an Agilent 6550 UHD Accurate Mass Q-TOF–MS coupled with a Thermo QE Plus. Proteins were digested using trypsin to generate peptide fragments, and 1 ug of protein of each sample was loaded on the 50 cm long C18 of 3.0 μm Easy-spray column (Thermo Fisher Scientific). Peptides were eluted with a 0–40% gradient of buffer B (80% acetonitrile, 0.1% formic acid) at a 300 nl/min flow rate. Consequently, the sample was injected for MS analysis. LC gradients were run for 60 min. MS1 spectra were acquired in the Orbitrap at 70k resolution. Dynamic exclusion was employed for 10 s, excluding all charge states for a given precursor. MS2 spectra were acquired at 17,500 resolutions.

LC–MS/MS data processing

All samples were processed, and the RAW files generated were analyzed with Proteome Discoverer (v2.2) against the UniProt reference proteome database. For the sequester search, the precursor and fragment mass tolerances were set at 10 ppm and 0.5 Da, respectively. The protease used to generate peptides, i.e., enzyme specificity, was set for trypsin/P (cleavage at the C terminus of “K/R: unless followed by "P"), along with a maximum missed cleavage value of 2. Carbamidomethyl on cysteine as a fixed modification, oxidation of methionine, and N-terminal acetylation were considered variable modifications for database search. Both peptide spectrum match and protein false discovery rate (FDR) were set to 1% FDR, with Precursor Mass Tolerance of ± 20 ppm (monoisotopic), Accepted Precursor Mass Errors of 0Da, Product Mass Tolerance of ± 20 ppm (monoisotopic) and falsely considering modified forms as unique peptides.

PPI network analysis

BioGRID Version 4.4 is a critical and dynamic resource in bioinformatics, delivering high-quality, curated, and up-to-date molecular interaction data. BioGRID lists physical and genetic PPIs of raw and non-redundant interactions [47]. It contains details on 1,735 non-redundant interactions, 1,441 distinct genes or proteins, 2,529 raw interactions, 400 distinct publications, and 1,735 non-redundant interactions of HIV-1. The entire STRING network of human proteins interacting with Gag-pol, Gag, Nef, and Env proteins was separately reconstructed in the STRING database version 12.0 (https://string-db.org/), with the highest confidence score of 0.900 as the threshold [48]. The entire STRING network was clustered into three groups by k-means clustering [49]. These analyses help identify highly connected proteins, densely interconnected subgroups, and functional nodes or hub proteins within the PPI network. Cytoscape software was used to re-examine the PPI network [50]. The selected modules above the threshold value were subjected to GO functional enrichment analysis.

Viral-host interactome analysis

The Virus Mentha database was also included since it collects and provides data about viral PPI from published articles from various sources thoroughly and comprehensively for studying the interactome [51, 52]. The platform guarantees the dependability and correctness of the interactions offered and draws its data from manually curated PPI databases that have complied with the norms of the IMEx consortium.

Exploring exosome-derived human proteins involved in immune pathways

SIGNOR (Signaling Network Open Resource) version 3.0 (http://signor.uniroma2.it) is a comprehensive repository that contains extensively curated causal relationships between human proteins and other biologically relevant entities, including stimuli, phenotypes, enzyme inhibitors, complexes, and protein families. These relationships are supported by experimental evidence [53]. The platform offers a powerful search tool that enables customized searches across all available resources. The curated data is visually represented as signed directed graphs using a graph drawing tool. Exosome-derived UniProt IDs of human proteins, exclusively CDH5, DENND4A, ENO1, G3V1L9, GOLPH3, OGT, PABPC4, Q6DI00, Q9BXU9, RHOU, TJP1, and TRAF6 were entered in the search space provided by SIGNOR database to unravel the involvement of query proteins in different innate immunity pathways and cellular signalling networks.

Analysis for exosome-derived HIV1 protein-influenced Innate immune responsiveness and signal transduction pathways

Human immunodeficiency virus type 1 (entry nt06161) causing acute HIV infection (entry H01563) and chronic AIDS (entry H00406) was minutely explored in the KEGG (Kyoto Encyclopaedia of Genes and Genomes) database available at https://www.kegg.jp/kegg-bin/search?q=HIV. Moreover, the experimentally validated pathways affected by exosome-derived HIV1 proteins (Env, gag, gag-pol, and nef), primarily involved in signal transduction, cellular processes, and innate immunity, have been decoded through a circuit diagram at https://www.kegg.jp/pathway/hsa05170. Several innate immune responsive and signal transduction pathways such as MAPK (hsa04010), NF-kappa B (hsa04064), Cell cycle (hsa04110), Protein processing in endoplasmic reticulum (hsa04141), mTOR (hsa04150), PI3K-Akt (hsa04151), Antigen processing and presentation (hsa04612), Toll-like receptor (hsa04620), Cytosolic DNA-sensing pathway (hsa04623), and TNF signalling pathways (hsa04668) were revisited. Meanwhile, the Cell Signalling Technology platform (https://www.cellsignal.com/pathways/) was also used to cross-verify KEGG pathways and proteins involved in different pathways.

KEGG pathway analysis

Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, basically infects CD4+ (receptors) T lymphocytes (T helper cells), macrophages, monocytes, and dendritic cells. The primary cell surface receptor for HIV-1, the CD4 protein, and the co-receptor for HIV-1, either CCR5 or CXCR4, are found on macrophages and T lymphocytes. At the earliest step, sequential binding of virus envelope (Env) glycoprotein gp120 to CD4 and the co-receptor CCR5 or CXCR4 facilitates HIV-1 entry and can potentially trigger critical signalling that may favour viral replication. At advanced stages of the disease, HIV-1 infection dramatically induces T-cell (CD4+ T and CD8+ T cell) apoptosis in infected and uninfected bystander T cells, a hallmark of HIV-1 pathogenesis (Supplementary Fig. 1). In contrast, macrophages exhibit resistance to the cytopathic effects of HIV-1 and can continue to generate the virus over prolonged periods.

Functional enrichment analysis

Enrichment analysis identifies overrepresented functional terms within a group of proteins of interest, such as gene ontology (GO) terms, KEGG pathways, subcellular localization (compartments), Reactome pathways, and WikiPathways [54–58]. This study deciphers the biological processes and pathways linked to the PPI network and sheds light on the functional implications of protein interactions. The intensity of enrichment shows the relevance of a specific GO word enrichment within the set of proteins being studied. The most apparent GO concepts from three categories were offered by GO functional annotation: biological process (BP), cellular component (CC), and molecular function (MF) [59]. GO keywords (BP, CC, and MF) are often chosen based on the primary indicator of enrichment strength rather than False Discovery Rate (FDR) values [60]. Higher strength values indicate more significant enrichment, implying a possible functional relationship between the proteins and the annotated GO word. GO keywords with a prediction strength greater than 1.0 were shortlisted in this context. While FDR values are often employed to control for false positives when testing numerous hypotheses in multiple testing corrections, they are not directly used to shortlist GO keywords, KEGG pathways, Reactome pathways, or WikiPathways. Signalling pathways connected with immune-related proteins were discovered by KEGG enrichment analysis.

Statistical analysis

The statistical analysis of the data was performed using Version 19 of SPSS Software (SPSS Statistics: IBM Corporative, Chicago, Illinois, USA). To ensure reproducible comparisons across cohorts, we employed a rigorous adjustment process for our patient samples. We adjusted the data by accounting for potential confounding variables, including; age, gender, body mass index (BMI), height, alcohol consumption and economic stature. After satisfying the ANCOVA (analysis of covariance) assumptions in Levene's Test of Equality of Error Variances (p > 0.05), the ANCOVA analysis was performed (Confidence Interval: 95%). Data was considered statistically significant if p < 0.05 (Supplementary Table 2).

Results

Characterization of an HIV-1-infected patient-derived exosome by electron microscopy and dynamic light scattering (DLS)

The exosomes were subjected to size characterization by SEM and TEM to assess their integrity and morphology. The results for the SEM depict the size range for the HIV-infected patient's serum-derived exosome, ranging from 49.25 to 86.14 nm, showing the typical range of exosomes and excluding any other extracellular vesicle (Fig. 2A). The DLS analysis also suggests the exosomal size range (Fig. 2B). Similarly, the TEM image also predicts the size in the range of 48.26–143 nm (Fig. 2C).

Fig. 2.

Exosome characterization: The exosomes were isolated for HIV patient serum using the Exoquick kit. The isolated exosomes were subjected to A FE-SEM, B DLS and C TEM for their size characterization. D Exosome origin and purity confirmed by western blot analysis (Lanes 1–12): The exosomal lysate protein used from either healthy individuals or HIV-1 patients was characterized by western blot using α-CD63, α-Calnexin, and α-HIV (Nef) protein. THP-1 cell lysate (lane 1).,healthy individual serum-derived exosome (lane 2), HIV-infected patient-derived exosome (lanes 3–12). In each well, an equal amount of (20 µg protein) samples were loaded and subjected to western blot analysis using anti-CD-63 and anti-Calnexin antibodies and anti-Nef

Biochemical characterization by western blot analysis demonstrates HIV-1 infected cell origin of exosomes

After size characterization, the exosomes isolated from healthy individuals and HIV-1 patients were also characterized for their origin and purity by western blot analysis. Using appropriate detection antibodies against exosome-associated host proteins (CD63, mouse monoclonal antibody, SC-5275, MW 26 kDa; and Calnexin, mouse monoclonal antibody, SC-23954, MW 90kDa; Santa Cruz Biotechnology, USA)) or pathogen-origin protein Nef (mouse monoclonal, SC-65904, MW 27 kDa). To ensure the presence of pathogenic protein (Nef) in exosomes eventually validates their HIV-1 patient origin Fig. 2D (lanes 3–12). On the other hand, the absence of Nef protein in healthy individuals suggests antibody specificity (Fig. 2D lane 2). Also, the presence of calnexin in THP-1 whole cell lysate, a positive control (Fig. 2D lane 1), and its absence in the exosome preparation suggest that the antibody against calnexin is functional and clearly suggests its absence in exosomal preparations (Fig. 2D lanes 2–12 and Supplementary Figs. 7, 8 and 9). The presence of the tetraspanin family protein CD63 shows that the isolated vesicles are exosomes, and the absence of calnexin demonstrates the purity of these vesicles. These results suggest that the isolated exosomes are from HIV-1 infected cells of patients and are circulating in the blood.

The LC–MS-MS data reveals the presence of host and HIV-1 peptides in the exosome protein sample

The analysis of LC–MS-MS yielded a comprehensive overview of the protein landscape, encompassing 130 proteins. Among these, 65 were identified as target proteins, while an additional 65 were categorized as on-the-fly decoy proteins. A total of 44,352 non-unique peptides were identified, with 21,999 originating from target proteins and 22,353 from decoy peptides. This analysis was facilitated by capturing 2,666 MS/MS spectra, contributing to the depth of the investigation. The output was enriched with crucial information, including Protein Description, Protein Sequence, Protein Length, Number of Proteins in groups, Number of Peptide-Spectrum Matches, Number of Unique Peptides, Summed Peptide-Spectrum Match Precursor Intensity, Summed Unique Peptide Precursor Intensity, Protein Sequence Coverage (%), Summed Morpheus Score, and additional factors contributing to the comprehensive characterization.

Exosome-derived protein enrichment analysis using bioinformatics tool

Using the LC–MS/MS data, protein identification and enrichment analysis were performed. Interestingly, four HIV-1 proteins—Gag-Pol polyprotein (P04585), Gag protein (Q78639), Nef Protein (P04601), and Envelope glycoprotein gp160 (P03377)—were found in the exosome derived from HIV-1 positive serum samples (Table 1).

Table 1.

Identification of HIV-1-origin exosomal proteins and their primary functions

UniProt ID	Protein symbols or alternative names	Function	References
P04585	Gag-Pol polyprotein	Gag pol polyprotein, involved in virion assembly, translation, and regulation, is cleaved through proteolytic cleavage, releasing structural proteins and viral enzymes	[61]
P04601	Nef Protein (Immune evasion)	Nef is essential for HIV-1 replication in activated T cells, suggesting potential for therapies targeting its conserved activity in viral replication, immune evasion, cell signalling, trafficking, and viral entry	[62]
Q78639	Gag protein (Immune evasion)	Gag, a structural protein, is crucial in HIV-1 replication, controlling the late phase of the viral life cycle and serving as a long-term scaffoldingprotein	[63]
P03377	Envelope glycoprotein gp160 (Immune evasion)	HIV-1 envelope glycoproteins are crucial for the virus's replication cycle, as they facilitate the fusion of viral and cellular membranes during entry	[64]

This annotation table enlists exosomal proteins identified from HIV-1 infected samples, with their corresponding UniProt ID, protein symbols or alternative names, and primary functions. These proteins were found to be involved in various cellular processes such as virion assembly, membrane fusion, translation, regulation, immune evasion, and virus-host interactions

After extensive enrichment analysis of the exosome-derived full-length protein sequences, we got ten and twelve UniProt IDs or Ensembl IDs in the healthy control and HIV1 infected samples, respectively (Table 1). The Gag-pol, Nef, Gag and Env proteins play an important role in viral replication and assembly. Nef is a multiphase protein and helps in viral replication, immune evasion, cell signalling, cellular trafficking, cell surface expression, and viral entry (Table 1). Interestingly, only two unique exosome-derived proteins were found in the HIV1-infected exosome sample: Q07157 (ENSG00000104067), which signifies tight junction protein ZO-1, and Q7Z401 (ENSG00000174485) corresponding to C-myc promoter-binding protein. Table 2 presents a compilation of protein information, including UniProt IDs, Ensembl IDs, protein symbols or alternative names, and associated terms. These diverse proteins contribute to various cellular functions and molecular pathways (Table 2). Among the listed proteins are various key players in cellular processes. For instance, UniProt ID Q6DI00 corresponds to the homeobox protein Hox-A13, while Q13310 corresponds to the polyadenylate-binding protein. Notably, G3V1L9 represents the tight junction protein ZO-1, and P06733 corresponds to alpha-enolase. The cadherin-5 protein is attributed to UniProt ID P33151. Additionally, Q7L0Q8 signifies the Rho-related GTP-binding protein RhoU, and Q9H4A6 corresponds to Golgi phosphoprotein-3. UniProt ID Q9BXU9 corresponds to calcium-binding protein-8, while Q9Y4K3 represents TNF receptor-associated factor-6. UniProt ID O15294 corresponds to UDP-N-acetylglucosamine (Table 2).

Table 2.

Identification of human-origin exosomal proteins and their functions

S. No	UniProt ID	Ensembl ID	Protein symbols or alternative names	Term protein or gene name	Function	References
1	Q6DI00	ENSP00000222753	HOXA13, HOX1J, NP_000513.2, HXA13_HUMAN	Homeobox protein Hox-A13;	Hox proteins have few direct targets, and regulation mechanisms are unclear. The HoxA13 gene, expressed during embryogenesis, is crucial for skeletogenesis, interdigital programmed cell death, and autopod formation	[65]
2	Q13310	ENSG00000090621	PABPC4, LOC100996696, PABP4, APP1	Polyadenylate-binding protein 4	Poly (A) tails at the 3' ends of eukaryotic mRNAs are bound to PABPC and PABPN1, facilitating translation and the regulation of mRNA decay	[66]
3	G3V1L9	ENSG00000104067	TJP1, ZO1, NM_003257.4, uc001zcs.3	Tight junction protein ZO-1	This protein, part of the MAGUK family, regulates adherens junctions and acts as an adaptor at tight junctions, regulating ion and macromolecule movement between endothelial and epithelial cells	[67, 68]
4	P06733	ENSG00000074800	ENO1,ENO1L1, MPB1, MBPB1	Alpha-enolase	Alpha-enolase (ENO1) is a multifunctional enzyme involved in cellular stress, infections, cancer, parasitic infections, and growth, development, and reproduction, catalyzing the conversion of 2-phosphoglyceric acid to phosphoenolpyruvic acid	[69]
5	P33151	ENSG00000179776	CDH5, NX_P33151, Cadherin-5, uc002eon.1	Cadherin-5	VE-cadherin is a crucial endothelial-specific cadherin that regulates endothelial cell junctions, controlling vascular permeability and leukocyte recruitment. It also influences endothelial cell behaviour, proliferation, survival, migration, and blood vessel formation	[70]
6	Q7L0Q8	ENSG00000116574	RHOU, WRCH1, CDC42L1, ARHU	Rho-related GTP-binding protein RhoU	Dendritic spines are tiny protrusions on neurons that receive the most excitatory synaptic inputs in the brain. Before formation, dendrites are covered with actin-rich, filopodia-like protrusions, actively searching for presynaptic partners. Once formed, they mature into a mushroom-shaped spine	[71]
7	Q9H4A6	ENSG00000113384	GOLPH3, GPP34, OTTHUMT0,0000207363, MIDAS	Golgi phosphoprotein 3	Golgi phosphoprotein 3 (GOLPH3), a human protein-encoding the GOLPH3 gene, plays a crucial role in Golgi trafficking and is linked to poor prognosis in gastrointestinal tract cancers like gastric and esophageal squamous cell carcinoma	[72]
8	Q9BXU9	ENSG00000183166	CALN1, CABP8, XM_017012678.1, NP_113656.2	Calcium-binding protein 8	Calcium-binding proteins are crucial in maintaining calcium homeostasis, buffering, and potentially protecting against intracellular calcium fluctuations, and they are essential for neurotransmission and various cellular functions	[73]
9	Q9Y4K3	ENSG00000175104	TRAF6, RNF85, TRAF6_HUMAN, RING finger protein 85	TNF receptor-associated factor 6	TRAF proteins play a crucial role in regulating NF-κB and MAPK signalling pathways, highlighting their complex signalling functions in the immune system and their significant role in inflammatory responses	[74]
10	O15294	ENSG00000147162	OGT, OTTHUMT0,0000144139, 5HGV, NP_858058	UDP-N-acetylglucosamine-	MurA is a cytoplasmic enzyme that catalyzes the transfer of enol pyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, forming UDP-N-acetylglucosamine enol pyruvate and releasing inorganic phosphate	[75]
11	Q07157	ENSG00000104067	TJP1, ZO1, NM_003257.4, uc001zcs.3	Tight junction protein ZO-1	TJP1, TJP2, and TJP3 are scaffolding proteins that link tight junction transmembrane proteins to the actin cytoskeleton. They limit substance movement and serve as a boundary between apical and basolateral plasma membrane domains in epithelial and endothelial cells, essential for lumenogenesis and efficient epithelial polarization	[76]
12	Q7Z401	ENSG00000174485	DENND4A, IRLB, MYCPBP, XP_005254177	C-myc promoter-binding protein	MBP-1 is a DNA-binding protein that regulates the expression of the human c-myc gene by binding to a sequence that overlaps with the TBP binding site in the c-myc P2 promoter region	[77]

Complete annotation of proteins showing UniProt and Ensembl Identifiers, Protein Symbols or Alternative Names, Term Protein or Gene Name, and their functional roles in the Pathophysiology of HIV-1 infection processes

PPI network analysis (BioGRID version 4.4) reveals gag-pol versus host Protein interaction network

PPI shows that HIV-1 (UniProt ID: P04585) Gag-pol polyprotein physically interacts with 162 human proteins (Fig. 3A). Gag interacts with 196 proteins (Fig. 3B); Nef forms to be associated with 80 host proteins and two viral proteins (Fig. 3C), while Env interacts with 158 proteins (Fig. 3D). Concentric circles show that the nodes closest to the centre node are intimately related; however, the thickness of edges (lines connecting two nodes) indicates the degree of interaction. The yellow edges reflect physical interaction, whereas the purple edges depict genetic interaction. Light blue ovals represent a cluster of related and connected nodes. The concentric circles' layout and Arbor dynamic layout of Cytoscape V 3.10.0 show the most interactive host proteins, which are also listed in this study and encompass a range of functions crucial to cellular and viral processes. These include EIF3I, EIF3H, EIF3K, EIF3E, EIF3L, EIF3M, EIF3B, and EIF3A, subunits of the EIF3 complex of human proteins, which are responsible for viral protein translation initiation. EP300, a human transcriptional coactivator, interacts with viral proteins to regulate transcription. Host STAU1, an RNA-binding protein, contributes to viral RNA stability and translation (Table 3).

Fig. 3.

HIV-1 protein–protein interaction networks for Gag-Pol, Gag, Nef, and Env proteins (represented by the centre brown node) with human and other proteins of HIV1. Yellow nodes represent human proteins, and blue nodes represent other HIV-1 proteins in the interaction network. A The Gag-pol protein interactome. B The Gag protein interactome. C HIV-1 NEF protein interactome. D HIV’s Env protein interactome

Table 3.

HIV-1 Gag-Pol interacts with proteins of broad-spectrum pathways

S. No	Proteins	Uniprot ID	Ensembl ID	Functions	Referencess
1	EIF3I, EIF3H, EIF3K, EIF3E, EIF3L, EIF3M, EIF3B, EIF3A, EIF3D	Q13347, O15372, Q9UBQ5, Q9C5Z3, Q9Y262, Q7L2H7, Q8JZQ9, Q9VN25, O15371	ENST00000678162, ENSG00000147677, ENSG00000178982, ENSG00000104408, ENSG00000100129, ENSG00000149100, ENSG00000106263, ENSG00000107581, ENSG00000100353	EIF-3 subunits are part of the eukaryotic translation initiation factor 3 (elf3) complex, which initiates viral protein translation. They target mRNAs involved in cell proliferation and use RNA stem-loop binding to activate or repress translation. EIF3D is required for nonsense-mediated mRNA decay (NMD) and may work with UPF2 to divert mRNAs from translation to the NMD pathway	[78–83]
2	EP300	Q09472	ENSG00000100393	The transcriptional coactivator interacts with viral proteins and regulates their transcription, functions as a transcriptional coactivator for SMAD4 in the TGF-beta signalling pathway, and regulates receptor signalling pathways, androgen receptor signalling pathways, and autophagy	[79, 84–89]
3	STAU1	O95793	ENSG00000124214	The human RNA-binding protein supports viruses like HIV-1, HERV-K, Ebola, and influenza by stabilizing their RNA and increasing viral protein production. It also plays a crucial role in cellular activities like autophagy, inflammation control, cell structure organization, microtubule movement, chromosome separation, cell adhesion, cell shape maintenance, and programmed cell death	[79, 90–93]
4	VIM (vimentin)	P08670	ENSG00000026025	VIM, an intermediate filament protein, is crucial for cellular structure and viral assembly during viral replication. Its impact on HIV-1 replication is complex, with some studies suggesting it promotes viral transcription and protein production, while others suggest it hinders virion release	[79, 94–96]
5	KIAA1429	Q69YN4	ENSG00000164944	KIAA1429, a m^6A methyltransferase complex, modifies RNA and influences the function of long non-coding RNA (lncRNA) and circular RNA (circRNA), potentially causing disease development	[79, 97–101]
6	YWHAB, YWHAE, YWHAG, YWHAZ	P31946, P62258, P61981, P63104	ENSG00000166913 ENSG00000108953, ENSG00000170027, ENSG00000164924	Human 14–3-3 proteins interact with HIV-1 proteins, affecting the virus's life cycle. HIV-1 gp120 upregulates YWHAB, while Vpr and retropepsin inhibit YWHAE. Integrase interacts with YWHAG, and Tat upregulates YWHAZ. These interactions demonstrate viral protein manipulation during infection	[79, 102–104]
7	HERC2	O95714	ENSG00000128731	HIV-1 PR interacts with HERC2, an E3 ubiquitin ligase, regulating protein degradation, and retropepsin interacts with HERC2 in human HEK293 and Jurkat cell lines	[79, 105, 106]
8	GRB2	P62993	ENSG00000177885	GRB2, an adaptor protein involved in signal transduction, interacts with HIV-1 proteins Pol and RT. Nef and Tat upregulate GRB2 expression, while Vif downregulates it, suggesting GRB2 manipulates host cell signalling during infection	[79, 107]
9	MCM7	P33993	ENSG00000166508	Minichromosomal Maintenance Complex 7 (MCM7) is crucial to DNA and viral replication licensing factors. It interacts with Envelope Surface Glycoproteins gp120, Gag (pr55), Gag-pol, and Nef and is upregulated by Pol and Tat. MCM7's expression is regulated by PI3K/AKT/GSK3B/CCND1/RB1 axis and E2F transcription factors. Aberrant MCM7 expression can contribute to tumour development and progression	[79, 108–110]
10	OSBPL3	Q9H4L5	ENSG00000070882	Pol and retropepsin interact with OSBPL3, enhancing HIV-1 virus replication by interacting with oxysterol-binding protein-related protein three isoform a	[79]

This is an annotated table of HIV-1 Gag-Pol protein interacting with different sets of human proteins, which influences several molecular processes (activation or repression of viral mRNA translation, autophagy, inflammation control, cell structure organization, microtubule movement, chromosome separation, cell adhesion, cell shape maintenance, and programmed cell death, modifies RNA and influences the function of long non-coding RNA and circular RNA) and pathways (TGF-beta signalling pathway, androgen receptor signalling pathways, and autophagy) in the pathophysiology of HIV-1 infection

Gag (Pr55), Gag-pol, EIF3D, and envelope surface glycoprotein gp120 complexes with EIF3A, EIF3D, and EIF3E. HIV Pol interacts with all subunits of eukaryotic translation initiation factor 3 (elf3)-EIF3A, EIF3B, EIF3C, EIF3D, EIF3E, EIF3F, EIF3G, EIF3H, EIF3I, EIF3K, and EIF3L [93]. HIV-1 virus replication is enhanced by the human gene histone acetyltransferase p300 (EP300) expression, which enhances the turnout of envelope surface glycoprotein gp120, acetylation of integrase, and activation of Tat and Vpr. HIV-1 gp120 and EP300 synergistically increase TGF-β, ATF-2 and activating protein-1 (AP-1) expression, leading to tubular cell apoptosis; in addition, HIV-1 gp120 treatment causes phosphorylation of Smad2 and downregulation of c-Jun [103, 104, 111]. Tandem affinity purification and mass spectrometry analysis revealed that Gag (Pr55), Gag-pol, Nef, nucleocapsid, and envelope surface glycoprotein gp120 interact with double-stranded RNA-binding protein Staufen homolog one isoform a (STAU1), as Staufen1 ribonucleoprotein complexes are isolated from HIV-1 infected cells [93]. VIM (vimentin), an intermediate filament protein, maintains the cellular structure and influences viral assembly and cell signalling during replication. Indeed, HIV-1, like many viruses, engages with the human cytoskeleton through various mechanisms to facilitate its replication and induce significant alterations in the host cell. One notable strategy employed by HIV-1 involves the protease (PR)-mediated cleavage of cytoskeletal proteins, especially vimentin (VIM). This viral-induced cytoskeleton modification plays a crucial role in the viral life cycle, contributing to viral entry, assembly, and release processes. Nef, gp120, Gag (Pr55), and Gag-pol are complexed with VIM, where gp120 decreases the phosphorylation of VIM. Matrix protein upregulates while Vpr downregulates VIM expression in infected cells. Meanwhile, retropepsin cleaves and degrades host VIM protein [112–114]. KIAA1429, also known as VIRMA (vir-like m6A methyltransferase associate), represents an isoform of methyltransferases and stands out as the largest protein within the methyltransferase complex. Its influence on related RNAs extends beyond m6A-dependent mechanisms, encompassing m6A-independent processes such as alternative splicing, RNA maturation, translation, degradation, and stability [97, 99, 101, 115]. Human 14–3-3 proteins YWHAB, YWHAE, YWHAG, and YWHAZ regulate diverse cellular processes. Envelope surface glycoprotein gp120 upregulates 14–3-3 beta/alpha (YWHAB) protein [102]. HIV-1 gp120 downregulates expression of c-Myc, Max, and 14–3-3 epsilon protein (YWHAE) and decreases phosphorylation of ATP-dependent tyrosine kinases (Akt) at Ser-473 in humans. Affinity tagging and purification mass spectrometry analyses show that HIV-1 integrase (IN) physically interacts with 14–3-3 beta/alpha (YWHAB), 14–3-3 epsilon (YWHAE), 14–3-3 gamma (YWHAG), and 14–3-3 zeta/delta (YWHAZ) proteins in human HEK293 and Jurkat cell lines [104] (Fig. 3). Meanwhile, HERC2, an E3 ubiquitin ligase, modulates protein degradation. GRB2 functions as an adaptor protein in signal transduction pathways. MCM7, a helicase complex component, is essential for DNA replication and potentially vital for viral replication. Host OSBPL3, involved in lipid metabolism, affects viral membrane dynamics, shaping their behaviour (Table 3). Interaction of the Gag-pol complex and Gag protein and virion host proteins present in exosomes signify the pathophysiological involvement of exosomes derived from HIV 1 infection cells in the host.

Gag versus host protein interaction network

The interaction between the HIV-1 Gag protein and 196 human proteins yields valuable insights into their collaborative pathophysiological roles (Fig. 4B). The Gag protein's engagement with various human proteins is highlighted below, showcasing their contributions. For instance, DDX5 and DDX17 proteins operate in RNA metabolism and participate in binding viral RNA during the assembly of Gag (Table 4).

Fig. 4.

The Venn diagram depicts the intersectional study of HIV1 Gag-Pol, Gag, Nef, and Env PPI with human proteins. Coloured boxes represent the symbolic names of common human genes

Table 4.

HIV-1 Gag protein interacting with Human proteins and their respective roles

S. No	Proteins	UniProt ID	Ensembl ID	Function	References
1	DDX5 DDX17	P17844, Q92841	ENSG00000108654 ENSG00000100201	These proteins participate in RNA metabolism and viral RNA binding during GAG assembly	[116]
2	FLNA	P21333	ENSG00000196924	It interacts with the HIV-1 protein GAG and is involved in GAG localization and viral assembly	[117, 118]
3	LARP1	Q6PKG0	ENSG00000155506	It plays a role in coordinating viral RNA packaging into gag particles during assembly	[117, 118]
4	RSBN1L	Q6PCB5	ENSG00000187257	It interacts with the viral protein Vif, which participates in viral replication and evasion of the host immune system	[116]
5	ACTB	P60709	ENSG00000075624	It plays a role in gag localization and viral assembly. It may be involved in stabilising the gag lattice during particle formation	[119]
6	TCEB2	Q15370	ENSG00000232285	TCEB2 is a component of the elongation factor complex, which participates in transcription and RNA processing. Its exact role in assembly is not well-defined	[79, 120]
7	EPRS	P07814	ENST00000433816	Also known as glutamyl-prolyl-t RNA synthetase, it is responsible for aminoacyl t RNA synthesis	[79]

Annotated table of Human Proteins Engaging with HIV-1 Gag protein involved in different types of Molecular Processes such as RNA metabolism and viral RNA binding, viral RNA replication and evasion of the host immune system

Their involvement is pivotal in orchestrating the formation of Gag structures. FLNA, in association with HIV-1 protein Gag, FLNA plays a crucial role in Gag localization and the subsequent viral assembly process. Its interaction plays a role in ensuring effective Gag placement. LARP1 assumes a role in coordinating the packaging of viral RNA into Gag particles during assembly. This participation facilitates the strategic incorporation of RNA molecules into Gag structures. RSBN1L interacts with the viral protein Vif, and RSBN1L contributes to viral replication and aids in evading the host immune system. This interaction plays a pivotal role in the viral life cycle. ACTB's influence on Gag localization and viral assembly is significant. It potentially stabilizes the Gag lattice during particle formation, contributing to the overall structural integrity of the viral particles. TCEB2 is a component of the elongation factor complex, which engages in transcription and RNA processing. Although its exact role in assembly is not fully delineated, its presence in this context is noteworthy. EPRS, also known as glutamyl-prolyl-tRNA synthetase, EPRS is responsible for aminoacyl-tRNA synthesis (Table 4). Its involvement adds a layer of complexity to the interplay between gag and human proteins. These interactions underline the intricate collaborations between the HIV1 Gag protein and various human proteins, each contributing uniquely to the viral assembly and replication processes.

Nef versus host protein interaction network

The study examined the interaction of Nef protein with a diverse array of about eighty human proteins identified in HIV-1 patient serum-derived exosomes proteomics analysis and their associated functions, shedding light on their roles in various signalling pathways. Notably, TRAF6, acting as an adaptor protein, was found to mediate both Toll-like receptor (TLR) and NF-KB signalling, crucial components of the immune response (Table 5). SRC, belonging to the non-receptor tyrosine kinase family, has emerged as a critical player in signal transduction pathways, contributing to intricate cellular communication. PARK2, an E3 Ubiquitin ligase, showcased its significance in protein degradation and homeostasis maintenance. TP53's role as a pivotal regulator of cell cycle arrest and apoptosis underlines its importance in cellular health. LYN, a member of the SRC kinase family, was identified as participating in immune cell signalling. TNIP1, on the other hand, emerged as a negative regulator of the NF-KB signalling pathway, contributing to immune response modulation. CXCR4, a chemokine receptor, gained prominence as a co-receptor facilitating HIV entry into target cells, while CDKN2A, encoding P16INK4a and P14ARF proteins, displayed its role as a cyclin-dependent kinase inhibitor, critical for cell cycle regulation. GABARAPL2's function was linked to autophagy pathways, which modulate autophagy-related processes. Lastly, ARHGEF6's involvement in regulating RHO GTPases, vital for cytoskeletal dynamics and cell migration, highlighted its contribution to cellular movement and organization. This comprehensive exploration of human proteins and their diverse functions provides valuable insights into their intricate roles within cellular processes (Fig. 3C, Table 5). Being one of the most prominent proteins in pathophysiological regulation, the presence of Nef in exosomes demonstrates that exosomes are not only their vesicles derived from infected cells but also involved in disease progression.

Table 5.

HIV-1 Nef protein interacting with human proteins and their respective roles

S. No	Protein	UniProt ID	Ensembl ID	Functions	References
1	TRAF6	Q9Y4K3	ENSG00000175104	An adaptor protein that mediates TLR and NF-KB signalling	[121]
2	SRC	P12931	ENSG00000197122	Non-receptor Tyrosine kinase is involved in signal transduction pathways	[122]
3	PARK2	O60260	ENSG00000185345	It is an E3 Ubiquitin ligase involved in protein degradation and homeostasis	[123]
4	TP53	Q12888	ENSG00000067369	It regulates cell cycle arrest and apoptosis	[124]
5	LYN	P07948	ENSG00000254087	It belongs to the SRC kinase family and is involved in immune cell signalling	[125]
6	TNIP1	Q15025	ENSG00000145901	It is a negative regulator of the NF-KB signalling pathway	[126, 127]
7	CXCR4	P61073	ENSG00000121966	Chemokine receptor and co-receptor for HIV entry into the target cell	[128]
8	CDKN2A	Q8N726	ENSG00000147889	Cyclin-dependent kinase inhibitor 2A that encodes P16INK4a and P14ARF proteins	[79]
9	GABARAPL2	P60520	ENSG00000034713	It is an autophagy-related protein modulating the autophagy pathway	[129, 130]
10	ARHGEF6	Q15052	ENSG00000129675	Involved in regulating RHO GTPases, controlling cytoskeletal dynamics and cell migration	[131]

Annotated table of Human Proteins Engaging with HIV-1 Gag protein involved in controlling cytoskeletal dynamics and cell migration, modulating the autophagy pathway, TLR and NF-KB signal transduction pathways

Functional enrichment analysis of Nef-mediated pathways

Enrichment analysis identifies overrepresented functional terms within a group of proteins of interest, such as gene ontology (GO) terms, KEGG pathways, Reactome pathways, and WikiPathways. Our analysis yielded significant enrichments across various types of Nef protein-mediated pathways. In our analysis, we found significant enrichments in various categories. There were 361 enriched GO-terms in Biological Processes (BP), 22 in Molecular Function (MF), and 77 in Cellular Component (CC). All the 361 enriched GO-terms in BP were further filtered based on prediction strength (> 1.0) calculated by [Log10(observed/expected)] and FDR values (< 0.00014) for the top 100 GO-terms (Supplementary Fig. 2). Stimulation of the macrophage migration inhibitory factor signalling pathway exhibited a value of 2.41, while the chemokine receptor CXCR4 signalling pathway scored 2.11. The interleukin-17-mediated signalling pathway also displayed a score of 1.93 (Supplementary Fig. 2). Furthermore, we identified enriched pathways, with 42 KEGG pathways, 122 Reactome pathways, and 45 WikiPathways. Additionally, our analysis highlighted significant enrichment in tissue expression, specifically in 68 tissues. Notably, a substantial enrichment was observed in publications, encompassing more than 10,000 significantly enriched publications supporting this analysis.

All 22 GO terms involved in Molecular Function (MF) were selected based on prediction strength (> 0.5) and FDR values (0.012) (Supplementary Fig. 3). The result shows that various GO terms related to Molecular Function (MF) were examined exclusively: transmembrane transporter binding (GO:004432), phosphoprotein binding (GO:0051219), protein tyrosine kinase binding (GO:1,990,782), ubiquitin-like protein ligase binding (GO:0044389), ubiquitin protein ligase binding (GO:0031625), protein kinase binding (GO:0019901), protein domain-specific binding (GO:0019904), and signalling receptor binding (GO:0005102) (Supplementary Fig. 3). The p-strength ranged from 0.69 to 1.18, and the number of observed proteins varied for each term, ranging from 5 to 29. These findings provide insights into the molecular functions associated with the observed proteins.

The top 57 GO terms involved in Cellular Component (CC) were selected based on prediction strength (> 0.5) and FDR values (0.012) (Supplementary Fig. 4). The results encompass various Gene Ontology (GO) terms related to Cellular Component (CC). These terms include the T cell receptor complex (GO:0042101), clathrin adaptor complex (GO:0030131), autophagosome membrane (GO:0000421), clathrin-coated endocytic vesicle membrane (GO:0030669), proteasome complex (GO:0000502), clathrin-coated vesicle membrane (GO:0030665), autophagosome (GO:0005776), plasma membrane (PM) signalling receptor complex (GO:0098802), clathrin-coated vesicle (GO:0030136), endosome (GO:0005768), early endosome (GO:0005769), and late endosome (GO:0005770). The p-value strengths for these terms range from 0.64 to 1.56, and the corresponding FDR values provide further insight into their significance in Nef-mediated HIV1 pathogenesis, revealing their potential roles in cellular components and processes.

The analysis of results encompasses the top 58 Nef-mediated Reactome terms and their associated prediction strengths above the threshold value of p-strength of > 0.5 and the FDR value of < 0.012. These terms include Nef and signal transduction, with a prediction strength of 2.2; FLT3 signalling through SRC family kinases; and binding and entry of HIV virion, both with a p-strength of 2.11. Nef-mediated CD4 down-regulation has a prediction strength of 2.06, PECAM1 interactions score 2.03, and FCGR activation is noted with a p-strength of 2.01. Furthermore, the role of Nef in HIV-1 replication and disease pathogenesis exhibits a prediction strength of 1.98, regulation of KIT signalling is rated at 1.9, and Nef-mediated down modulation of cell surface receptors by recruiting them to clathrin adapters has a prediction strength of 1.89. Lastly, activated NTRK2 signals through FYN are associated with a prediction strength of 1.86. These findings show these processes' relevance, potential roles, and interactions in biological pathways (Supplementary Fig. 5).

The analysis of results encompasses all 42 Nef-mediated KEGG pathways and their associated prediction strengths, as indicated by the default value of p-strength of > 0.6 and the FDR value of < 0.0112 (Supplementary Fig. 6). The KEGG enrichment analysis identified the Nef-mediated PPI network's prediction strengths for the different KEGG keywords. Autophagy (hsa04136) scored 1.39, while mitophagy (hsa04137) displayed a prediction strength of 1.5. Furthermore, the prediction strength for the T cell receptor signalling route (hsa04660) was 1.19, the HIV1 infection (hsa05170) was 1.1, and the NF-kappa B signalling pathway (hsa04064) was 1.1. Additionally, the prediction strengths for animal autophagy (hsa04140) and ubiquitin-mediated proteolysis (hsa04120) were 0.99 and 0.98, respectively. The NOD-like receptor signalling route (hsa04621) and the ErbB signalling pathway (hsa04012) had scores of 0.98. Furthermore, prediction strengths of 0.95 and 0.93 were demonstrated by the IL-17 signalling route (hsa04657) and the sphingolipid signalling pathway (hsa04621). Finally, prediction strengths for the Apelin signalling route (hsa04371), endocytosis (hsa04144), actin cytoskeleton regulation (hsa04810), and chemokine signalling pathway (hsa04062) ranged from 0.84 to 0.87. These results offer insightful information on the possible importance of these KEGG pathways in HIV1 pathogenesis.

HIV-1 Env versus host protein interaction network

The HIV-1 origin Env protein was predominantly detected in patient-derived exosomal proteomics analysis. Further analysis of the provided proteomics data reveals the intricate functions associated with various human proteins. CANX, a chaperone protein, is pivotal in aiding the proper folding and quality control of glycoproteins, ensuring their structural integrity. VCP, or Valosin-containing protein, is an ATPase associated with protein degradation and membrane fusion, contributing to cellular maintenance. BCA P31 is a significant regulator in HIV RNA cyclization stability and translation, highlighting its role in viral RNA processing (Fig. 3D).

PHB's involvement in transcriptional regulation and mitochondrial function underscores its importance in cellular processes. TFRC, functioning as a cell surface receptor, is crucial in iron uptake, contributing to essential metabolic processes. RPN1, on the other hand, contributes to the proper N-glycosylation of nascent proteins, a critical step for protein maturation. APP, a transmembrane protein, has implications for normal neuronal function, emphasizing its role in maintaining neural health. Lastly, PGRNC1's association with steroid receptor signalling and its presence in mitochondrial membranes reinforce its significance in cellular signalling and energy production (Fig. 4D, Table 6). This comprehensive exploration of human proteins and their diverse functions enhances our understanding of their intricate roles within various cellular pathways.

Table 6 .

The role of HIV-1 Env protein in interacting with critical human host proteins

S. No	Protein	Uniprot ID	Ensembl ID	Functions	References
1	CANX	P27824	ENSG00000127022	Chaperon protein assists in proper folding and quality control of glycoprotein	[79]
2	VCP	P55072	ENSG00000165280	Valosin-containing protein is an ATPase associated with protein degradation and membrane fusion	[79]
3	BAP31	P51572	ENSG00000185825	Involved in the regulation of HIV RNA cyclization stability and translation	[79]
4	PHB	P35232	ENSG00000167085	It is involved in transcriptional regulation and mitochondrial function	[79]
5	TFRC	P02786	ENSG00000072274	It is a cell surface receptor involved in iron uptake	[1, 2]
6	RPN1	P04843	ENSG00000163902	It assists in the proper N- glycosylation of nascent protein	[79]
7	APP	Q92624	ENSG00000062725	It is a transmembrane protein involved in normal neuronal function	[79]
8	PGRMC1	O00264	ENSG00000101856	Mitochondrial membrane protein associated with steroid receptor signalling	[79]

A catalogue of the complete set of host proteins assists in proper folding and quality control of viral glycoprotein, protein degradation and membrane fusion, HIV RNA cyclization stability and translation, iron uptake, N-glycosylation of nascent protein, and protein associated with steroid receptor signalling

Intersectional analysis of PPIs

The intersectional analysis of protein–protein interactions (PPIs) unveils intriguing insights, -\

highlighting common proteins that interact with essential components of HIV1. The intersectional investigation of PPI networks of HIV-1 envelope (Env) and Nef proteins showed eight human proteins with overwhelmingly common contact partners: CXCR4, CD4, SQSTM1, BCAP31, LCK, AP1M1, TFRC, and CALM1 (Figs. 4 and 5). HIV-1 enters host cells through cell surface receptors known as CXCR4 and CD4 [132]. Their interaction with Env (directly engaged in entrance) and Nef (possibly affects receptor expression) indicates a concerted attempt by the virus to facilitate entry. The SQSTM1 (p62) protein is involved in selective autophagy and cellular signalling. It interacts with various signalling molecules and can influence HIV-1 replication and assembly by modulating autophagic pathways and protein degradation processes. An endoplasmic reticulum (ER) protein, BCAP31, transports proteins to the Golgi apparatus from the ER. It has been implicated in regulating apoptosis and may affect HIV-1 protein trafficking and assembly [133]. The LCK (A Src family tyrosine kinase) and AP1M1 (adaptor protein complex 1) proteins are important in T-cell activation and signal transduction pathways. LCK is crucial for efficiently replicating HIV-1, as it phosphorylates key proteins in the viral life cycle. AP1M1 is involved in sorting cargo proteins in the trans-Golgi network (TNG) and endosomes [134]. It can influence the trafficking and release of HIV-1 particles. Nef's interaction with them may aid the virus in evading immune responses or creating an environment favourable to viral replication. The TFRC (transferrin receptor) protein is involved in iron uptake in cells [135]. HIV-1 may use iron for viral activities, and its interaction with TFRC via Env or Nef could be a way to facilitate HIV-1 entry into cells. It may also affect viral replication and pathogenesis by modulating cellular iron levels. CALM1 (Calmodulin 1) is a multifunctional calcium-binding messenger protein that participates in various physiological functions, including signal transduction and apoptosis. Its association with Env and Nef shows that HIV-1 may manipulate calcium signalling to its advantage [136].

Fig. 5.

The interaction between human proteins (depicted as green circles) and HIV-1 proteins (represented by orange squares) highlights the cellular compartments where these interactions occur. The interaction scores involving the Gag-pol, Gag, Nef, and Env proteins of HIV-1 with human proteins ranged from 0.2 to 0.9. This analysis identifies potential interaction sites within various cellular compartments, including the nucleus, cytoplasm, cell membrane, and extracellular space. This investigation is facilitated using the Mentha Java Desktop Version 1.0 tool, which aids in visualizing these putative interactions

When comparing the HIV-1 PPI networks of Env with those of Gag, eight human proteins—CD59, TLR1, DDX6, CD63, THY1, FLOT1, TLR2, and MR1—were identified as common interaction partners (Figs. 3 and 4). These Toll-like receptors (TLR1 and TLR2) are critical for recognizing pathogen-associated molecular patterns (PAMPs). Their activation can trigger immune responses that both restrict and, paradoxically, facilitate HIV-1 infection through inflammatory processes. The CD59 protein protects cells from complement-mediated lysis. HIV-1 integrates CD59 into its viral envelope, which helps it evade the host immune system by blocking the complement cascade. CD63, a tetraspanin that controls cell adhesion and motility, is in HIV-1 particles and impacts viral budding and entrance into host cells. FLOT1, a protein linked with lipid rafts, plays a role in various cellular activities, such as endocytosis and signal transduction. It has been linked to the assembly and budding of HIV-1. DDX6, an RNA helicase involved in mRNA degradation and control, has been linked to regulating HIV-1 RNA stability and translation, influencing viral replication and latency [137]. THY1, or CD90, is involved in cell-to-cell contacts and signalling. It could play a role in HIV-1 infection by influencing cell adhesion and migration, potentially altering virus spread. The significance of MR1 in HIV-1 pathogenesis is unclear; however, it may modulate immunological responses.

The observed association of six human proteins—THRAP3, EED, UBE2I, DAXX, CUL2, and EEF1A1—between the PPI networks of Gag and Gag-Pol in HIV-1 is intriguing since it suggests potential functions for these proteins in viral assembly and host interaction (Figs. 3 and 4). The THRAP3 (Thyroid Hormone Receptor Associated Protein 3) protein is involved in mRNA splicing and nuclear export. Gag interacts with mRNA from the host cell during virion assembly. The interaction between Gag and THRAP3 may be critical for integrating viral RNA into the assembling virion particle. UBE2I (ubiquitin-conjugating enzyme E2I) promotes ubiquitination, a biological mechanism involved in protein breakdown. Gag-Pol may use UBE2I to ubiquitinate its own or host-specific proteins during viral assembly or maturation. The EEF1A1 (Eukaryotic Translation Elongation Factor 1alpha1) protein is essential for the elongation phase of protein translation. The interaction between Gag-Pol and human EEF1A1 may be required to efficiently translate viral RNA, aiding its stability and translation. The EED (Embryonic Ectoderm Development) protein is a Polycomb Repressed Complex 2 (PRC2) component that maintains genes' transcriptionally repressed status via histone methylation. Gag may interact with EED to modify the host's chromatin structure, enhancing viral latency and evading the host’s immune response by silencing proviral DNA. The DAXX (Death-Associated Protein 6) protein is involved in various cellular functions, including apoptosis. Gag-Pol's association with DAXX may be a strategy for inhibiting apoptosis in host cells, thereby extending cell survival and increasing viral production by allowing the virus to retain viral latency by suppressing viral gene expression. The CUL2 (Cullin 2) protein is a component of E3 ubiquitin ligase complexes that tag proteins for destruction by the proteasome. The association of Gag-Pol with CUL2 suggests that it may play a function in modulating the ubiquitination pathway to benefit the virus. This could include the breakdown of host proteins, which could interfere with viral replication or immunological responses.

STAU1 (Staufen1) presence in all three Gag, Gag-Pol, and Nef PPI networks shows that it may serve as a central hub, promoting interactions between viral proteins and perhaps regulating their functions (Figs. 4 and 5). STAU1 is an RNA-binding protein involved in post-transcriptional control of gene expression. It is involved in several essential pathways that promote viral replication and persistence during HIV infection. STAU1 binds to the HIV-1 RNA, stabilizing it and facilitating its transfer from the nucleus to the cytoplasm, ensuring that the RNA is translated efficiently into viral proteins. This interaction is critical for forming Gag and Gag-Pol polyproteins, which are required components of the viral particle.

Furthermore, STAU1 can affect the location and stability of cellular mRNAs, particularly those implicated in the immunological response. STAU1 helps HIV-1 evade the immune system by regulating host immune gene expression. This prevents the host immune system from detecting and eliminating the virus, resulting in viral persistence and chronic infection. STAU1 interacts with other biological components involved in mRNA degradation and translation, resulting in a complex protein–protein interaction network that promotes viral replication. STAU1 aids HIV-1 in manipulating the host cell machinery to generate an environment favourable to viral replication while evading immune surveillance. These findings reveal that HIV-1 proteins, including Gag-Pol, Gag, Nef, and Env, in conjunction with various host proteins, may play roles in HIV-1 pathogenesis and are critical in the viral life cycle and immune evasion.

This analysis further explores the cellular compartments where interactions between HIV-1 and human proteins might occur. The sites of interactions were simulated by Mentha Java Version 1.0, which provides a comprehensive view of the interplay between HIV-1 and human proteins. These predictions showed that interactions between human proteins (green circles) and HIV-1 proteins (orange squares) mostly happen inside the cytoplasm (Fig. 5). Meanwhile, a few interactions occur inside the nucleus, cell membrane, and extracellular space. Additionally, a scoring system estimates the strength of these potential interactions, ranging from 0.2 (weak) to 0.9 (strong). The analysis reveals that only a few host proteins-EP300 and NFKB1 interact with the Gag-Pol protein inside the nucleus, while most of the interaction between host and viral proteins occurs inside the cytoplasm (Fig. 5). This visualization aids in understanding the specific microenvironments and physiological conditions in which critical protein–protein interactions occur.

The interaction scores involving the Gag-pol, Gag, Nef, and Env proteins of HIV-1 with human proteins ranged from 0.2 to 0.9. This analysis identifies potential interaction sites within various cellular compartments, including the nucleus, cytoplasm, cell membrane, and extracellular space. This investigation is facilitated using the Mentha Java Desktop Version 1.0 tool, which aids in visualizing these putative interactions.

PPI network analysis

The K-means clustered network analysis at a moderate confidence threshold of 0.40 reveals several essential statistics. In the PPI network, 162 human proteins and twelve exosome-derived experimental proteins were found in the HIV-1-positive patient serum, demonstrating functional associations and physical interactions with the HIV1 Gag-Pol protein (Fig. 6). Three distinct clusters are identified within this set of 162 proteins based on their interaction levels. These clusters are denoted by red, green, and blue, representing the first, second, and third shells of interactors, respectively. Notably, the green cluster of PPI proteins exhibits a higher degree of interconnectivity. This implies that a subset of highly interconnected proteins (including EIF3I, EIF3H, EIF3K, EIF3E, EIF3L, EIF3M, EIF3B, EIF3A, EIF3D, STAU1, and EP300) are biologically associated with one another and are influenced by the presence of the HIV-1 Gag-Pol protein (Figs. 5, 6, and Table 5).

Fig. 6.

PPI network of 162 human proteins. Twelve exosome-derived experimental proteins identified in the HIV-1 positive samples showed functional associations and physical interactions with HIV-1 Gag-Pol protein. K-means clustering identifies three separate clusters characterized by red, green, and blue colours, each determined by their respective interaction levels (STRING version 12.0)

There are 195 nodes and 348 edges within the PPI network of host protein interactions with Gag protein at the highest confidence threshold of 0.90 (Fig. 7). On average, each node is connected to approximately 3.57 other nodes, indicating a moderate level of connectivity. The average local clustering coefficient, which measures how interconnected the neighbours of a node are, is 0.388, suggesting that they are clusters of nodes with high interconnectivity. The expected number of edges based on the network size is 127. Through K-means clustering, we observe the emergence of three distinct clusters, distinguished by the colours red, green, and blue. Notably, within these clusters, we identify two that display a high degree of interconnection. One is the red cluster, composed of 17 human proteins; the other is the green cluster, comprising 14 human proteins. These clusters offer insights into the respective interaction patterns when encountering the Gag protein of HIV1 (Fig. 8). Additionally, the p-value for protein–protein interaction (PPI) enrichment is extremely low, less than 1.0e-16, indicating a highly significant enrichment of protein interactions within this network. These statistics provide valuable insights into the structural characteristics and significance of the network at this confidence threshold, highlighting its complexity and biological relevance.

Fig. 7.

PPI network of 196 proteins. Including twelve exosome-derived experimentally identified proteins in HIV-1 positive samples showing functional associations and physical interactions with HIV1 Gag protein (STRING V 12.0). Three separate clusters are observed by K-means clustering; they are indicated by the hues red, green, and blue. Interestingly, we find two with high interconnectivity inside these clusters. There are two types of clusters: the red cluster contains 17 human proteins, while the green cluster has 14 human proteins

Fig. 8.

PPI network of 77 human proteins. Twelve exosome-derived experimentally identified proteins in HIV-1 positive samples showed functional associations and physical interactions with HIV1 Nef protein (STRING V 12.0). Three separate clusters are observed by K-means clustering; the red, green, and blue nodes indicate them. We find two with a high degree of interconnectivity inside these clusters- the red cluster contains 14 human proteins, while the green cluster has 13 human proteins

The network involving host protein interactions with the Nef protein is established at the highest confidence threshold of 0.90, comprising 77 nodes and 56 edges (Fig. 8). On average, each node is connected to approximately 1.45 other nodes, indicating a moderate level of connectivity. The average local clustering coefficient, which measures the interconnectedness of a node's neighbours, is 0.305, signifying the presence of clusters of nodes with moderate interconnectivity. Within this network, we can identify three main clusters: the first cluster, represented by red nodes, consists of 23 human proteins; the second cluster, denoted by green nodes, includes 21 human proteins; and the third cluster, characterized by blue nodes, encompasses 33 human proteins. Of particular note, within these clusters, we observe two that exhibit a notably high degree of interconnectivity. The red cluster, for instance, comprises 14 human proteins (SRC, YES1, FYN, LCK, LYN, CD247, CD4, GNAI2, HCK, ITGB1, NCK1, PAK2, ARHGEF6, and ARHGEF7), while the green cluster encompasses 13 human proteins (TP53, CDKN2A, ARF1, AP1M1, HSPA9, SQSTM1, GABARAP, GABARAPL1, GABARAPL2, PARK2, TRAF2, TRAF5, and TRAF6). In contrast, the third cluster, composed of blue nodes, includes 33 human proteins, but limited interactions among its members characterize it.

Pathway analysis of exosome-derived proteins from HIV-1 positive samples

Upon querying the SIGNOR 3.0 database with the twelve exosome-derived proteins identified from HIV1-positive samples (namely Q6DI00, Q13310, G3V1L9, P06733, P33151, Q7L0Q8, Q9H4A6, Q9BXU9, Q9Y4K3, O15294, Q07157, and Q7Z401), it was observed that three to five of these proteins were associated with Toll-like receptors (TLR), inflammasome activation, inhibition of apoptosis, innate immune response, and autophagy pathways. The Toll-like receptor (TLR) pathway identified the following proteins: CDH5, ENO1, OGT, TJP1, and TRAF6. Similarly, these proteins were found to activate inflammasomes and inhibit apoptosis pathways. CDH5, ENO1, OGT, and TRAF6 were shared between these pathways, suggesting their versatile roles in regulating immune responses (Table 7). These proteins were also implicated in the innate immune response and autophagy pathways. Notably, three exosome-derived proteins, CDH5, ENO1, and TRAF6, participate in innate immune responses like Toll-like receptors (TLR), inflammasome activation, inhibition of apoptosis, and autophagy, indicating their versatile role in mediating these pathways. This comprehensive exploration of human proteins highlights their interconnected roles in multiple immune pathways, underscoring their significance in orchestrating the immune response in the context of HIV infection.

Table 7.

Exosome-derived proteins from an HIV-1 positive sample and their involvement in innate immune response pathways

Innate immune response pathways	Human proteins (HIV-positive sample)
Toll-like receptors (TLR)	CDH5, ENO1, OGT, TJP1, and TRAF6
Inflammasome Activation	CDH5, ENO1, OGT, and TRAF6
Inhibition of Apoptosis	CDH5, ENO1, OGT, and TRAF6
Innate Immune Response	CDH5, ENO1, and TRAF6
Autophagy	CDH5, ENO1, and TRAF6

The most distinguished shortlisted proteins—CDH5, ENO1, OGT, TJP1, and TRAF6—are associated with primary innate immune mechanisms, including Toll-like receptor (TLR) signalling, inflammasome activation, inhibition of apoptosis, innate immune response, and autophagy

Innate Immune Response network analysis

Exploring the putative human protein network associated with the innate immune response (IIR) sheds light on the involvement of specific proteins, particularly CDH5 and ENO1, during HIV-1 infection. In this network, the sample protein CDH5, highlighted as a pink node, stands out with a score of 0.748, indicating its significant role in the immune response. Additionally, ENO1, another sample protein, exhibits a score of 0.377, further emphasizing its potential involvement in this immune context. The P-values of 6.52E-02 and 1.02E-01 for CDH5 and ENO1, respectively, suggest their significance in the context of HIV-1 infection within the IIR pathway. These findings provide valuable insights into the intricate interplay between these proteins and their potential contributions to the immune defence mechanisms during HIV-1 infection, as illustrated within the Signor Version 3.0 network (Fig. 9).

Fig. 9.

Signor Version 3.0. The analysis illustrates the inferred network of human proteins associated with the IIR, emphasizing the involvement of exosome-derived sample proteins CDH5 and ENO1 (pink node) in HIV-1 infection

Inflammasome activation network analysis suggests the role of viral proteins

Signor Version 3.0. exploration depicts conceivable connections among human proteins involved with Inflammatory pathway activation, emphasizing the involvement of exosome-derived sample proteins CDH5, ENO1, and OGT in the background of HIV-1 infection (Fig. 10). The pink nodes represent the sample proteins involved in inflammasome activation. Notably, CDH5 has a p-value of 8.17E-02 and a score of 0.743, ENO1 has a score of 0.377 and a p-value of 1.24E-01, and OGT has a score of 0.425 and a p-value of 1.26E-01. These results provide insight into the possible function of proteins produced from exosomes in the complex network of inflammatory events that occurs during HIV-1 infection (Fig. 11).

Fig. 10.

The potential human protein network activates the inflammasome pathway. This visualization highlights the participation of exosome-derived sample proteins CDH5, ENO1, and OGT in the context of HIV1 infection, as analysed using Signor Version 3.0

Fig. 11.

Signor image illustrates human proteins' putative network associated with the Toll-Like-Receptor (TLR) pathway. This visual representation focuses on the participation of exosome-derived sample proteins CDH5, ENO1, and TRAF6 within the context of HIV1 infection

The investigation unveiled a complex interplay of proteins governing inflammasome activation during HIV-1 infection. Notably, CDH5 was found to initiate the activation of the CTNNB1 protein, subsequently leading to upregulation of the POU5F1 and EP300 proteins. This concerted action was further associated with the downregulation of PPARG protein, collectively contributing to the intricate orchestration of inflammasome activation. This network of interactions highlights the multifaceted roles of these proteins in modulating the host's response to HIV-1 infection. Interestingly, the exosome-derived protein ENO1 was observed to indirectly modulate MYC protein, a pivotal player in inflammatory activation. The regulation of the immune response becomes messy due to this indirect inhibition. Moreover, OGT emerged as a critical player, activating TET1, which indirectly prompted the activation of the PTEN protein through a cascade of protein–protein interactions. The subsequent upregulation of the NFkb-p65/p50 protein complex underscores the intricate regulatory mechanisms governing inflammatory activation during HIV-1 infection. In the context of viral infection, the findings offer important insights into the complex network of proteins that regulate the host's immune response.

Toll-like-receptor (TLR) pathway analysis

The Signor-based pathway analysis visually represents the putative human protein network associated with the Toll-Like-Receptor (TLR) pathway. The analysis is centred on the involvement of exosome-derived sample proteins CDH5, ENO1, and TRAF6 within the context of HIV1 infection. The presence of these sample proteins is evident when examining the TLR signalling pathway. CDH5, with a score of 0.743 and a P-value of 7.11E-02, displays its potential to influence TLR pathway activation. Similarly, ENO1 exhibits its role with a score of 0.377 and a P-value of 8.97E-02. The participation of OGT and TJP1, marked with scores of 0.425 and 0.45, respectively, further adds to the intricate network of TLR signalling during HIV1 infection (Fig. 12). This analysis underscores the potential impact of these proteins on the TLR pathway's dynamics and highlights their relevance within the broader context of immune responses to HIV-1 infection.

Fig. 12.

A putative network of human proteins associated with the Inhibition of Apoptosis. This visualization highlights the participation of exosome-derived sample proteins CDH5, ENO1, and TRAF6 within the context of HIV-1 infection. The intricate connections and relationships within the inhibition of the apoptosis pathway are depicted, emphasizing the potential roles of these sample proteins in modulating apoptosis during HIV-1 infection

Inhibition of apoptosis

Signor V 3.0 exploration visual represents the human protein network associated with the process of inhibition of apoptosis, particularly in the context of HIV-1 infection (Fig. 12). This illustration highlights the intricate interactions among human proteins in inhibiting the apoptosis pathway during HIV-1 infection. Notably, including exosome-derived sample proteins CDH5, ENO1, and TRAF6 within this network suggests their potential roles in modulating apoptosis during HIV1 infection. The quantitative data provided further emphasizes the significance of these proteins in this context. Specifically, CDH5 demonstrates a score of 0.748, ENO1 has a score of 0.377, OGT is associated with a score of 0.425, and TRAF6 exhibits a score of 0.640 (Fig. 13). These scores and their respective p-values signify these proteins' potential interactions and influences on the inhibition of the apoptosis pathway during HIV-1 infection. Whatever is considered, this quantitative and visual approach advances our knowledge of the intricate relationship between viral infection and host proteins, offering insights into potential mechanisms underlying the modulation of apoptosis in the context of HIV-1 infection. Here, results suggest that exosomes derived from the HIV1 patient serum can inhibit apoptosis. This signifies the pathological role of exosome reduction in apoptosis, which may help establish latency, one of the significant events in the HIV life cycle. Further studies are required to depict the role of HIV patient serum-derived exosomes in the apoptosis of various kinds of cells.

Fig. 13.

A network of exosome-derived sample proteins CDH5, ENO1, and TRAF6 associated with autophagy during HIV1 infection. The network consists of various molecular entities, including proteins, protein families, complexes, and chemical molecules, with their interactions mapped. Exosome-derived proteins involved in autophagy are CDH5, ENO1, and TRAF6 (Pink Nodes) andtheir respective scores and p-values are provided in the table, indicating their statistical significance in the network

Involvement of exosome-derived proteins in autophagy during HIV-1 infection

In the context of autophagy during HIV-1 infection, the finding provided here includes investigating the interaction network, including the sample proteins CDH5, ENO1, and TRAF6 produced from exosomes. The illustration is part of Signor Version 3.0, which successfully conveys the complex network of relationships between these proteins and their putative functions in controlling the autophagy pathway during HIV-1 infection. Focusing on the individual contributions of these proteins, CDH5 emerges with a score of 0.743, suggesting its potential significance in modulating autophagy. Similarly, ENO1 displays a score of 0.377, indicating its involvement in the autophagy process. TRAF6, with a score of 0.640, also appears to play a notable role in autophagy regulation (Fig. 13).

The p-values provided (7.30E-02, 6.01E-02, and 1.83E-02) offer insights into the statistical significance of these protein interactions within the context of autophagy during HIV-1 infection. The lower p-values indicate a higher level of confidence in the observed associations. The network illustrated by Signor Version 3.0 provides an invaluable visual representation of the potential effects of the exosome-derived proteins CDH5, ENO1, and TRAF6 on the autophagy pathway during HIV-1 infection. The HIV patient serum-originated exosomal proteins show their possible role in controlling autophagy and eventually in HIV 1 pathogenesis regulations, adding to our understanding of the control of autophagy and its consequences in HIV-1 pathogenesis, adding to our understanding of the intricate interactions between host proteins and viral infection. Heat map analysis and Volcano plot.

The heatmap represents a correlation matrix of proteins identified in exosome samples from control and HIV-infected individuals using mass spectrometry. It visualizes pairwise correlations, where yellow indicates strong positive correlations, blue represents negative correlations, and intermediate shades suggest moderate associations. Tight junction proteins (ZO-1), Rho-related GTP-binding protein RhoU, and Golgi phosphoprotein 3 exhibit strong correlations, suggesting their co-regulation in membrane organization. Conversely, metabolic and structural proteins like Alpha-enolase and Cadherin-5 show weak or negative correlations with viral proteins (Gag-Pol polyprotein, Nef protein, and Envelope glycoprotein gp160), indicating possible HIV-induced alterations in exosomal composition (Fig. 14A). The presence of viral proteins in exosomes suggests a role in HIV pathogenesis, immune modulation, and potential biomarker discovery.

Fig. 14.

Heat map analysis showing the intensity of host and viral protein expressions, the yellow colour boxes depicts high expression of proteins with positive correlation and blue colour boxes shows low expression of proteins with negative correlation (A). The volcano plot analysis depicts the intensity of host And viral protein expressions, the red colour dots represents significantly high fold change of proteins and cyan colour dots shows relatively low fold change of proteins (B)

The volcano plot illustrates the differential expression of proteins in exosome samples from control and HIV-infected individuals. The x-axis represents fold change (Log₂), indicating upregulation (positive values) or downregulation (negative values), while the y-axis shows statistical significance (-Log₁₀ p-value), with higher values representing stronger significance. HIV-associated viral proteins, including Envelope glycoprotein gp160, Partial gag protein, Nef protein, and Gag-Pol polyprotein, are significantly upregulated, suggesting their active incorporation into exosomes during infection. In contrast, proteins such as Golgi phosphoprotein 3, Calcium-binding protein 8, and Alpha-enolase exhibit moderate downregulation, indicating potential disruption in host cellular functions. The presence of structural and signaling proteins (Cadherin-5, Rho-related GTP-binding protein RhoU) among differentially expressed proteins suggests alterations in cytoskeletal organization and vesicle trafficking (Fig. 14B).

Discussion

The exosomes isolated from HIV-1 patient serum subjected to proteome analysis emphasize protein interactions to uncover the plausible role of exosomes in HIV-1 pathogenesis and immune response (Fig. 14). The exosome proteins from pathogen origin or host origin were subjected to the study of protein–protein interaction. Our goal with protein profiling was to go beyond surface-level observations and into the molecular orchestra that orchestrates the response to HIV-1 infection. Cellular compartment localization was evaluated for its plausible roles in innate immune responses, inflammation, autophagy, and apoptosis. The bioinformatics analysis suggests HIV-1 predominantly infects CD4 + T-lymphocytes, then replicates and spreads to other CD4 + T-cells near the initial site of infection. The specialized lymphoid tissue, for instance, lymph nodes or gut-associated lymphatic tissue (GALT), is the most favourite site of HIV infection where CD4 + lymphocytes are concentrated, and their explosive growth and dissemination occur after a few days to weeks [3, 24, 25, 138–140]. The chronic persistence of HIV infections is attributed to the involvement of cells of the immune system itself since activation of cellular immunity is a critical factor that paradoxically helps HIV dissemination and persistence on a long-term basis [141]. Acute HIV syndrome witnesses a high viral load, reaching 10⁶ –10⁷ copies/mL in plasma [142]. This phase usually lasts about 2–4 weeks and occurs in almost half of patients with primary infection. After the acute stage, viral load decreases (to approximately 10³ –10⁴ copies/mL plasma) and remains at roughly that level for around 8–10 years [143, 144], known as the clinical latency stage (CLS) [24, 145–147]. The virus binds to the network of follicular dendritic cells, trapping the infectious virus in a concentrated state within the B-cell follicles of lymph nodes and replicating inside the lymph nodes during the CLS [148]. This reservoir is also the origin of virus resurgence in individuals who have been effectively treated but discontinued or paused antiretroviral therapy [149, 150]. According to a non-linear relation, increased viral load leads to a decreased number of CD4⁺ lymphocytes and vice versa [151]. The secondary diseases due to the loss of CD4⁺ lymphocytes impair immune function, leading to opportunistic infections and the development of malignancies (AIDS) that cause death [151, 152]. The primary reason HIV has remained uncured for decades is its remarkable capacity to elude the immune system with several immune evasion mechanisms [153, 154]. Most infected cells secret pathogen-associated molecule patterns (PAMP) containing exosomes. The virus targets explicitly CD4⁺ T cells, macrophages, and dendritic cells, causing their depletion and dysfunction, and a CD4⁺ T cell count below 200 cells/mm3 is diagnostic of AIDS [155, 156]. The HIV-1 accessary protein, Nef, was predominantly delivered on exosomes. Nef is multifunctional and known for immune evasion by decreasing MHC-I and MHC-II expression and the degradation of MHC molecules by endocytosis [27]. MHC-I downregulation protects infected cells from cytotoxic T cells onslaught.

In our proteomics analysis of HIV patient’s serum-derived exosomes, we found Gag-pol, Gag, Nef, and Env proteins interact with almost 162, 196, 80, and 158, respectively, and these proteins were found to be associated with various cellular and immune functions. Particularly CDH5, ENO1, OGT, TJP1, and TRAF6, exosome-derived host proteins from HIV-positive patients dramatically alter the natural immunological terrain. These proteins help immune evasion and viral persistence by adjusting TLR signalling, inflammasome activation, death inhibition, immune response control, and autophagy. TRAF6, a vital adaptor molecule in the (TLR) signalling pathway, increases NF-κB and MAPK signalling, producing pro-inflammatory cytokines. At the same time, TRAF6 promotes inflammasome activation through NF-κB priming, facilitating IL-1β and IL-18 production. TRAF6 activates the PI3K-AKT pathway, preventing apoptosis in immune and endothelial cells. TRAF6 facilitates autophagy induction by modulating Beclin-1 interactions, a crucial step in clearing viral particles.

ENO1 acts as a TLR4 ligand, triggering pro-inflammatory responses and enhancing monocyte and macrophage activation. ENO1 enhances inflammation by interacting with plasminogen and TLRs, leading to the activation of inflammasomes. ENO1 interacts with PI3K signalling, promoting cell survival and resistance to apoptosis. ENO1 is linked to stress-induced autophagy, affecting immune cell metabolism and survival.

CDH5 (VE-cadherin), although primarily an adhesion molecule, is involved in regulating endothelial responses to inflammation by modulating leukocyte transmigration. It is also known for maintaining endothelial integrity, which modulates leukocyte transmigration and vascular permeability in response to immune activation. Affecting vascular permeability and leukocyte flow, CDH5 modulates endothelial reactions to inflammasome activation. Reducing immune cell-mediated death, CDH5 improves endothelium survival and stabilises cell junctions. CDH5 controls extravasation and recruitment of immune cells, therefore influencing general innate immunological dynamics. Contributing to endothelial homeostasis, CDH5 affects autophagic reactions in vascular tissues.

OGT (O-GlcNAc transferase) regulates inflammasome assembly by modifying key components via O-GlcNAcylation, modulating cytokine release. By modifying apoptotic regulators like Bcl-2 and p53 post-translationally, OGT improves cell survival. Important for post-translational protein regulation, OGT affects cytokine release, inflammasome assembly, and apoptotic signalling in immune proteins.

TRAF6, ENO1, OGT, and CDH5 mediate the prevention of death, a characteristic of chronic infections, via activating the PI3K-AKT pathway and post-translational changes of death-regulating agents like Bcl-2 and p53. Moreover, these proteins support autophagy, a crucial mechanism in viral persistence whereby TRAF6 promotes Beclin-1-mediated autophagic flux while ENO1 and CDH5 control stress-induced autophagy and endothelium homeostasis. Experimental pieces of evidence are required to validate these findings.

Due to the high degree of variability in gp120, the antibodies lose neutralization efficacy. The non-neutralization antibodies activate Fc receptor-mediated signalling, suppressing immune effector NK cells and macrophages. Eventually, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis of infected cells [64] remain hampered. Apart from compromised ADCC, the gp120 recruits factor H that reduces complement-mediated lysis of virus infection cells [157, 158] and provokes inflammatory cytokines and chemokines to attract lymphocytes, DC, and macrophages [159–161].

HIV possesses intrinsic capabilities for evading detection by pathogen recognition receptors (PRRs) to establish persistent infections. It employs viral proteins, such as Nef and Vpr, which can hinder PRR signalling by reducing PRR expression or impeding downstream signalling pathways [162]. Cells infected with HIV also can inadvertently induce the destruction of uninfected T helper cells by releasing viral proteins like Tat, Nef, and gp120.

These viral proteins activate apoptosis pathways in neighbouring cells through various mechanisms, including enhancing Fas, FasL, and TNFα expression [156], reducing Bcl-2 expression, and activating p53 [163]. During viral infections such as HIV, the JAK/STAT pathway is crucial in regulating inflammation [164]. Compounds that activate this pathway hold promise for controlling HIV, but HIV Vpu and Nef disrupt JAK/STAT activation in response to IFN-α stimulation, thereby diminishing its initiation [165].

Nef protein's role in the exosome has been shown to induce apoptosis in CD4⁺ T cells by interacting with the CXCR3 receptor. However, the roles of Nef containing exosome on Fas, FasL, P53 activation and Bcl-2 reduction in other uninfected cells have not been deciphered.

Inflammatory disease in persons with HIV is marked by clinical deterioration and localized tissue inflammation after starting antiretroviral therapy (ART). It is believed to be brought on by the immune system starting to work again, which causes inflammatory reactions at the infection sites. Within 4–8 weeks of starting ART, a remarkable and rapid fall of viral RNA occurs, or an increase in CD4 count is marked. However, the symptoms of an infectious or inflammatory condition cannot be explained by the expected clinical course of a known infection, a side-effect of ART, a new infection, or autoimmunity. Regardless of CD4 count, patients who start ART are at risk of developing complications related to the recovery of their immune system. This condition arises due to a remarkably high viral load, very low CD4 count (50 CD4 + cells/mm3), low CD4 percentage, and lower CD4:CD8 ratio at ART initiation. Immune Reconstitution Inflammatory Syndrome (IRIS) is an inflammatory syndrome that can occur after highly active antiretroviral therapy (HAART) initiation and consists of either the appearance of a new condition or the worsening of a pre-existing condition. This heightened immune response can develop or worsen various inflammatory conditions, including infections or autoimmune disorders. Common examples of IRIS manifestations include the reactivation of latent infections like tuberculosis or the worsening of pre-existing conditions such as skin rashes or inflammatory lesions [166–168]. It occurs in ~ 15–25% of patients who started ART. The bystander role of exosomes in the HIV-mediated pathogenesis during ART needs to be deciphered in detail. The comprehension of host–pathogen interaction remains crucial to controlling the disease's progression. Deciphering EV content isolated from a patient’s serum during ART and their further analysis using bioinformatics should reveal crucial information like pathological changes and host response to the treatment. Nevertheless, the Compilation of experimentally verified human-HIV interactions should pave the way for comprehension of HIV biology and, eventually, vaccine and drug development.

In normal conditions, CD4⁺ cell count varies from 500 to 1500 cells/mm3. Three categories for CD4⁺ cell count ≥ 500 cells/mm3 are A1 (clinical category A, asymptomatic, acute HIV, or PGL), B1 (Clinical category B and Symptomatic conditions, not A or C), and C1 (clinical category C, AIDS-defining conditions). Similarly, three classes of CD4⁺ cell count for 200–499 cells/mm3 are A2 (Clinical category A, asymptomatic, acute HIV or PGL), B2 (Clinical category B, and symptomatic conditions, not A or C), and C2 (Clinical category C, AIDS-defining conditions). Finally, CD4⁺ cell count ≤ 200 cells/mm3 also have three categories: A3 (Clinical category A, asymptomatic, acute HIV, or PGL), B3 (Clinical category B, and symptomatic conditions, not A or C), and C3 (Clinical category C, AIDS-defining conditions). Any patient belonging to categories A3, B3, or C1-C3 is considered to have AIDS. It means < 400 CD4⁺ cells/mm3 indicate moderate immunocompromised while < 200 CD4⁺ cells/mm3 reflect AIDS-defining illnesses (e.g., Pneumocystis pneumonia) have emerged [155, 156, 169]. Most patients who do not receive treatment eventually die of complications of HIV infection. The complications during AIDS and eventual death of the patient are not only from HIV infection but also host immune moderation either by virus given the fact that EVs have the presence of viral proteins capable of interfering with various cellular and immune functions may be proved as bystander players in complications as well the death of the patients.

The BioGRID version 4.4 PPI network analysis reveals a substantial physical contact between the HIV1 Gag-Pol protein (UniProt ID: P04585) and a varied pool of 162 human proteins. This delicate interplay was further illustrated using Cytoscape 3.10.0, which depicts the concentric circles, pattern, and Arbor dynamic layout, highlighting the host proteins with the most extensive interactions. These include a wide range of functions essential to cellular and viral processes. Notably, EIF3 complex components such as EIF3I, EIF3H, EIF3K, EIF3E, EIF3L, EIF3M, EIF3B, and EIF3A are implicated in viral protein translation initiation. Furthermore, the transcriptional coactivator EP300 works with viral proteins to exert regulatory control over transcription, and the RNA-binding protein STAU1 helps viral protein stability and translation of viral RNA. The complicated web of connections also includes the HIV1 Gag protein, which interacts with a wide range of 196 human proteins. The article highlights these relationships' specific contributions, highlighting their functional consequences. For example, the collaboration between the Gag protein and the RNA metabolism proteins DDX5/DDX17 is important in binding viral RNA during Gag assembly.

This study, which characterizes the exosome for HIV patient sample and their plausible role in physiology the predictability using bioinformatics tools, has plunged into all possible dimensions of networks, IIR and signalling pathways, reactomes, GO terms and KEGG between human proteins and HIV1 proteins- Gag-pol, Gag, Nef, and Env, emphasizing the complicated web of interdependencies that arise. This investigation is being expanded to understand the immune evasion strategies of HIV1 proteins, especially Nef, gp41, gp120 and Env. This thorough data analysis-driven study complements the preexisting information on HIV1 pathogenesis and gives a simulated knowledge of the dynamic interactions between human and viral components. However, it is imperative to validate these interactions and their physiological roles. This work also highlights the potential importance of these interactions in HIV 1 pathogenesis, immune evasion and counter-defensive cellular processes. The Comprehensive study would decipher the interaction and help understand the possible role of HIV protein-containing exosomes in pathogenesis and HIV biology. This work remains out of the scope of this manuscript.

These investigations help clarify the several functions of exosome-derived proteins in controlling innate immune responses during HIV-1 infection, guiding possible therapeutic approaches and supporting our knowledge of viral persistence. Their roles imply possible treatment targets for altering innate immunity in HIV infection and related comorbidities, including cancer and chronic inflammatory diseases. New treatments targeted at interrupting exosome-mediated immune regulation in HIV-1 pathogenesis could result from further studies into these processes. Moreover, these exosomes can precisely be used as diagnostic as well as prognostic markers. The detailed study of protein and metabolic markers on exosomes during the ART therapy could be helpful to follow the disease pathophysiology. Furthermore, the host markers on exosomes can also reveal which body part or organ is inflicted with the disease. The immunomodulation studies using the coincubation of exosome with immune cells studies would also be helpful in determination of impact of these exosomes in various immune responses. Having all abovementioned facts, this study has some limitations like small sample size of ten patients, more heterogeneity among cohort is required to achieve more concrete conclusion. The isolation of exosome in terms of yield varies batch to batch, hence the protein content. The proteomics analysis of exosomes isolated from large sample size studies would be highly useful in determination of differential protein profile between healthy and HIV patients.

Conclusions

Four HIV-1 proteins- Gag-Pol (P04585), Gag (Q78639), Nef (P04601), and Envelope glycoprotein (P03377) were found in the exosome-derived HIV-1 positive samples. The twelve exosome-derived human proteins identified from HIV1-positive samples are namely Q6DI00, Q13310, G3V1L9, P06733, P33151, Q7L0Q8, Q9H4A6, Q9BXU9, Q9Y4K3, O15294, Q07157, and Q7Z401. The detailed analysis suggests that three to five of these proteins were associated with cell differentiation, intracellular signalling cascade, IIR pathways, especially Toll-like receptors (TLR), inflammasome activation, Inhibition of apoptosis, and autophagy pathways. This analysis revealed an intricate regulatory pathway involving CDH5, CTNNB1, POU5F1, EP300, and PPARG proteins, contributing collectively to the orchestration of the innate immune response during HIV-1 infection. Notably, CDH5 has a p-value of 8.17E-02 and a score of 0.743, ENO1 has a score of 0.377 and a p-value of 1.24E-01, and OGT has a score of 0.425 and a p-value of 1.26E-01. These findings shed light on the potential role of exosome-derived proteins in the intricate network of inflammatory events that occurs during HIV-1 infection.

The ability of various HIV-1 pathogenic proteins, including gag, gag-pol, nef, and env proteins, to hijack immune response pathways has diverse pathogenic potential. Meanwhile, PPI interactome networking, GO term, KEGG, and Reactome enrichment investigations have redefined the crucial function of host proteins in HIV-1 infection. Through PPI networking, the pivotal role of such human proteins in HIV infection has been developed. By deciphering this network, we aimed to reevaluate the mechanisms and pathways that become interwoven with HIV1 pathogenesis. Understanding the molecular processes that underlie the acute and chronic phases of HIV pathogenesis brings up new research avenues. The importance of these host proteins in CD4⁺ T cell-mediated dysfunction and HIV pathogenesis prevention in vitro and in vivo remains untested. We strongly believe that this work will enrich our comprehension of HIV-1 infections and pave the way for transformative solutions that can shape the future of HIV-1 management.

Author contributions

Conceptualization: MAA and NF. Formal Analysis MSB, NF. Data Curation: NF, MSB, MS, AHR. Methodology: NF, MSB, AA, MS, KKM, AKD, MDAA, MAA. Software and validation: MSB, NF, AHR. Supervision, Funding and project administration: MAA. Resources: MAA, KKM, MDAA, AKD, VVB, EA. Investigation: NF, MSB; Original draft: MSB, NF, MAA. Review and editing: AHR, MS, MDS, AA, AKD, EA, MDAA, MAA.

Funding

MAA acknowledges Ramalingaswami Fellowship (BT/RLF/Re-entry/15/2015) from DBT, Govt. of India.

Availability of data and material

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

Ethical approval was obtained from Jamia Hamdard, Human Ethics Committee (New Delhi, India), and each individual's consent was obtained before sample collection.

Consent for publication

Consent for publication was taken from each individual.

Competing interest

The authors declare no competing interests.