Comparative interactome analysis of Chikungunya virus non-structural protein 2 (CHIKV-nsP2) in human (Huh7) and Aedes albopictus (U4.4) cells reveal conserved host dependencies

Abstract

Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is a multifunctional enzyme essential for viral replication and host immune evasion. While nsP2’s role in mammalian infection is well-established, its interactions within mosquito vectors remain poorly characterized. Here, we performed a comparative interactome analysis using co-immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry to identify nsP2 host protein interactions in human hepatoma-derived Huh7 cells and Aedes albopictus U4.4 cells. We identified 56 high-confidence nsP2 interactors in Huh7 cells and 46 in U4.4 cells, with pathway enrichment analysis revealing convergent targeting of translation machinery, proteasomal degradation, and metabolic pathways in both systems. Protein-protein interaction networks demonstrated distinct functional clustering, with mammalian networks emphasizing metabolic reprogramming and mitochondrial function, while mosquito networks prioritized translation and oxidative stress responses. Comparative analysis identified six conserved interactors across both cell types, highlighting fundamental host dependencies exploited by CHIKV across vertebrate and invertebrate hosts, providing insights for developing broad-spectrum antiviral strategies targeting virus-host interfaces critical for replication in both mammalian hosts and mosquito vectors.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13337-025-00941-x.

Introduction

Chikungunya virus (CHIKV) belongs to the family Togaviridae of the genus Alphavirus, causing chikungunya fever, characterized by severe polyarthralgia and myalgia in the infected individuals, which is transmitted through bites of female Aedes mosquitoes [1]. CHIKV exhibits a dual-host life cycle, replicating in both mosquito vectors and mammalian hosts, and has evolved strategies to exploit host cellular machinery for its replication and survival.

CHIKV possesses a ~ 11.8 kb positive-sense single-stranded RNA genome organized into two open reading frames (ORFs) [2]. The 5′ ORF encodes the non-structural polyprotein precursors P123 and P1234, which are processed into four non-structural proteins (nsP1–nsP4) that form the viral replication complex. The 3′ ORF encodes the structural polyprotein (capsid-E3-E2-6 K-E1), translated from a subgenomic RNA (sgRNA). During processing, the capsid protein remains in the cytoplasm, whereas the envelope proteins are trafficked through the endoplasmic reticulum and Golgi apparatus before assembly into virions [2, 3]. Thus, while structural proteins mediate host entry and virion assembly, the nonstructural proteins drive viral RNA synthesis and replication.

Among the nsPs, the nsP2 protein is a multifunctional enzyme essential for viral replication and host modulation. It contains an N-terminal domain with NTPase, RNA triphosphatase, and RNA helicase activities, and a C-terminal domain with protease activity required for processing the viral replicase polyprotein [4]. In mammalian cells, the expression of CHIKV non-structural polyproteins is limited to the early stages of infection, tapering off as infection progresses, potentially due to virus-induced host translational shutoff mechanisms [5]. Uniquely, nsP2 is the only CHIKV protein reported to localize in both the cytoplasm and the nucleus of infected cells [6], and its nuclear translocation has been implicated in host transcriptional shutoff and antagonism of type I interferon responses, contributing to immune evasion and pathogenesis [7, 8].

Although substantial efforts have focused on understanding CHIKV–host interactions in mammalian systems [8, 9], studies investigating these interactions in the mosquito vector remain limited. Given that Aedes mosquitoes serve as essential biological reservoirs and transmission agents, exploring virus–vector protein interactions are critical for a holistic understanding of CHIKV pathogenesis and transmission dynamics. Importantly, mosquito-derived cell lines offer physiologically relevant systems that can tolerate viral protein expression without the cytotoxicity often observed in mammalian cells.

The present study was undertaken to address the critical gap in our understanding of how CHIKV non-structural proteins interact with host cellular machinery across both its vertebrate host and invertebrate vector by analyzing CHIKV-nsP2 host interactomes in both mammalian (Huh7) and mosquito (Ae. albopictus) cell lines. We performed co-immunoprecipitation of nsP2 followed by high-throughput quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify its interacting partners in both human and mosquito cells. This comparative interactome analysis reveals both conserved and cell-type-specific host targets of nsP2, shedding light on its multifunctional role in viral replication and host manipulation across divergent biological systems.

Materials and methods

Cell lines and virus infection

CHIKV was isolated and purified from patient sera collected during an outbreak and propagated in C6/36 and Vero cell lines (Accession no JF950631.1) [10].

Human hepatoma-derived cells (Huh7) and Ae. albopictus (U4.4) cells were maintained and propagated in Dulbecco’s minimal essential medium (DMEM) and Leibovitz’s L-15 medium (Cat no. AL011S, HiMedia, India) respectively, supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 µg/ml streptomycin antibiotics, and in a tissue culture flask with 75 square centimeter growth surface. All cells were incubated and propagated under standard conditions (37 °C, 5% CO_2, and 95% humidity).

For infection, Huh7 cells and U4.4 were seeded and grown into a 25 mm flask in DMEM medium and Leibovitz’s L-15 medium (Cat no AL011S, HiMedia, India) supplemented with 10% FBS. Cells were infected at an MOI of 1 with the CHIKV strain. The virus was diluted in serum-free DMEM medium and incubated for 2 h at 37 °C. The cells were washed with phosphate-buffered saline (PBS) 2 h post-infection (hpi) and grown in 2% FBS-containing medium. Cells were collected at various time points, washed with PBS, and resuspended in RIPA lysis buffer for immunoblot analysis.

Plasmids and constructs

The CHIKV-nsP2 gene was amplified from CHIKV RNA using specific forward and reverse primers and cloned into the pET29 prokaryotic expression vector with an N-terminal His-tag and transformed into Escherichia coli (E. coli) codon plus cells for expression and purification of the protein.

Protein purification

CHIKV-nsP2 protein was purified using previously published methods with slight modifications in the protocol [11]. E. coli cultures having plasmid CHIKV- nsP2 were induced with 1 mM IPTG for 16–20 h at 18 °C (degree Celsius). The cultures were pelleted and lysed in the lysis buffer: 50 mM phosphate buffer, pH 7.5, 150 mM NaCl, 10 mM Imidazole, glycerol 5%, and 1 mM PMSF with lysozyme. This was followed by centrifugation. The clarified supernatant was mixed with freshly recharged Ni-NTA (Nickel-Nitrilotriacetic acid) agarose beads. The beads were eluted with elution buffer containing 50 mM phosphate buffer, pH 7.5, 150 mM NaCl, 100 mM imidazole, and 5 mM βMe.

Western blot analysis

U4.4 cells were harvested 24 hpi, while Huh7 cells were harvested after 12 hpi and lysed using RIPA lysis buffer supplemented with protease inhibitor cocktails (PIC). The lysate was collected after centrifugation, and protein concentration was estimated using the BCA reagent. Protein samples at equal concentration were prepared in 6X SDS loading dye and denatured at 95 °C for 15 min. The proteins were resolved on a 10% SDS-PAGE gel, followed by western blotting, and then transferred to a nitrocellulose membrane. Subsequently, the membrane was incubated with in-house-generated CHIKV-nsP2 primary antibodies (1:4000 dilution) prepared in 2.5% BSA and incubated at 4 °C overnight. The next day, the membrane was washed three times with 1X PBST for 10 min each and then incubated with HRP-conjugated secondary antibody (1:5000 dilution) in 2.5% BSA + PBST for 1 h at RT. The membrane was then washed three times with 1X PBST for 10 min each and visualized using a Chemidoc MP (Bio-Rad).

Co-immunoprecipitation assay

For identifying the host cellular interacting partners of the CHIKV-nsP2 protein, a co-immunoprecipitation assay was performed. Infected Huh7 cell lysate and U4 cell lysates were used to pull down the interacting partners of CHIKV-nsP2 using purified CHIKV-nsp2 antibody (generated in-house) using Pierce co-immunoprecipitation kit (Thermo Scientific, USA) as per the manufacturer’s protocol [12, 13]. Briefly, CHIKV-nsP2-specific antibody was incubated with 100 µl Protein A beads at 4 °C overnight. Next, the unbound antibody was washed off, and the antibody-labeled beads were further incubated with infected Huh7 cell lysate (containing 1 mg of total protein) and infected U4.4 lysate overnight at 4 °C. Subsequently, the beads were washed with IP wash buffer, followed by three sets of elution using elution buffer. The Co-IP elutions were subjected to mass spectrometry analysis.

Sample preparation for mass spectrometry

The Co-IP eluates were subjected to in-solution trypsin digestion. Firstly, eluates were adjusted with 50 mM ammonium bicarbonate buffer pH 8.0, followed by reduction with 10 mM Dithiothreitol (DTT) for an hour at RT. Next, it was alkylated with 40 mM iodoacetamide (IAA) for 1 h. Using trypsin-Gold (Promega) at a 1:50 ratio, protein eluates were digested by incubation in a water bath at 37 °C for 16 h. The trypsin digestion reaction was stopped using formic acid (0.1%). The digested peptides were concentrated in a speedvac and analyzed by LC-MS/MS.

Mass spectrometry

LC-MS/MS analysis was performed using an Orbitrap Velos Pro MS coupled to an Easy n-LC 1000 (Thermo Fisher Scientific). Tryptic digested peptide mixtures were first loaded onto a reverse-phase C18 pre-column (Acclaim PepMap, 75 mm x 2 cm, 3 mm, 100 A°, Thermo Fisher Scientific) and then separated using an analytical column (Acclaim PepMap, 50 mm x 15 cm, 2 mm, 100 A°) using a gradient of 5% solvent B (0.1% formic acid in 95/5 acetonitrile/water) to 35% solvent B in 35 min. The eluted peptides were injected into the mass spectrometer, and MS1 data were acquired using a mass range of 350–2000 Da in full scan mode at a resolution of 60,000. The top 15 precursors were allowed to fragment using CID (collision-induced dissociation) in the ion trap with a collision energy of 35. The raw data were analyzed using Proteome Discoverer 1.4 with the SEQUEST algorithm against the Uniprot database [13]. The identification of peptides was validated using Percolator at 5% FDR.

Bioinformatics analyses

Proteins with high FDR confidence were selected for further statistical analysis in XLSTAT. Common proteins between CHIKV uninfected and infected libraries were excluded, and unique proteins with high FDR were further assessed for functional annotation analysis using GProfiler (v 0.2.2), Shinygo, and String 12 [14, 15].

Protein-protein interaction network of the CHIKV-nsP2 human interacting partners

The STRING 12 database fetched the host-virus protein–protein interaction (PPI) and vector virus PPI network (STRING: functional protein association networks) [16]. The significant proteins and the CHIKV-nsP2 viral protein from the pull-down study were further taken for this analysis.

Results

Protein purification and expression profiling of CHIKV-nsP2 in CHIKV-infected cells

Firstly, to detect CHIKV-nsP2 in the infected cells, we expressed and purified recombinant CHIKV-nsP2 using Ni-NTA affinity chromatography. Polyclonal antibodies were subsequently raised in rabbits following an established protocol [11]. SDS-PAGE analysis followed by Coomassie Brilliant Blue (CBB) staining and western blotting confirmed the successful purification of nsP2, with a distinct band at 90 kDa corresponding to the expected molecular weight of the protein (Fig. 1a).

Fig. 1.

Expression profiling of CHIKV-nsP2 in Huh7 and U4.4 cells (a) Protein purification of CHIKV-nsP2 using Ni-NTA chromatography. CBB staining of the purified protein elutions collected at different imidazole concentrations (i.e., 100mM, 150mM, and 200mM.). The blot shows the expression of the nsP2 protein at 90 kDa. (b) Western blotting depicting the change in the expression pattern of CHIKV-nsP2 protein at 0-, 4-, 8-, and 12-hours post-infection (hpi) in infected Huh7 cells. (c) Western blotting depicting the change in the expression pattern of CHIKV-nsP2 protein at 0-, 6-, 12-, and 24-hours post-infection (hpi) in U4.4 cells. (d) Western blotting depicting the expression of nsP2 in Co-IP elution during infection in Huh7 cells (1st blot) and U4.4 cells (2nd blot) with IgG serving as a negative control (3rd blot)

To investigate the temporal expression pattern of nsP2 during infection, Huh7 cells were infected with CHIKV and harvested at early time points of infection, namely at 0, 4, 8, and 12 h post-infection (hpi). Here, 0 hpi refers to 2 h after viral adsorption and internalization. This initial 2-hour period allows the virus to attach to the host cell surface and undergo internalization, marking the completion of the entry phase. Immunoblot analysis revealed detectable expression of nsP2 at 4 hpi, with a progressive increase over time and a peak at 12 hpi (Fig. 1b).

Parallel experiments were conducted in Ae. albopictus U4.4 cells to gain insight into CHIKV replication dynamics in the mosquito vector. Time-course analysis revealed that nsP2 expression gradually increased over time, peaking at 24 hpi, while minimal expression was detected at earlier timepoints such as 6 and 12 hpi (Fig. 1c). Based on the observed peak expression, we selected 12hpi and 24 hpi to perform downstream Co-IP experiments in mammalian and mosquito cell lines, respectively. Immunoprecipitation from CHIKV-infected Huh7 and U4.4 cell lysates, followed by SDS-PAGE, confirmed successful pulldown of nsP2, with a prominent ~ 90 kDa band in the eluted samples (Fig. 1d).

These results confirm the temporal regulation of nsP2 expression in both mammalian and mosquito host cells, supporting its critical role in viral replication and pathogenesis. The peak expression timepoints were subsequently used to profile the nsP2 interactomes of both the systems via co-immunoprecipitation (CoIP) of nsP2, followed by quantitative mass spectrometry (LC-MS/MS).

CHIKV-nsP2 interacting partners in mammalian Huh7 cells

LC-MS/MS analysis of CHIKV-infected and control lysates, performed in triplicate, identified a total of 406 proteins in triplicate samples from infected cells, compared to 225 proteins detected in uninfected controls processed with the CHIKV-nsP2 antibody. Venn diagram-based comparison revealed 215 proteins shared between both conditions, while 191 proteins were uniquely present in the infected samples (Fig. 2a). Following a stringent filtering based on FDR of < 0.05 and presence of unique peptides, 56 proteins were identified as putative CHIKV-nsP2-specific interacting partners and subjected to downstream functional and pathway analyses (supplementary information 1).

Fig. 2.

Pathway and gene enrichment analysis of the CHIKV-nsP2 interacting partner in Huh7 cells: (a) A Venn diagram representing the identified common and unique number of interacting partners of nsP2 in Huh7 cells, plotted based on high confidence. (b) Bar graph representation of the top 20 significant pathways from the enrichment analysis identified using KOBAS 3.0. (c) GO enrichment analysis of crucial biological processes associated with CHIKV-nsP2 interacting proteins. (d) The top 10 molecular functions associated with the enriched genes based on p-value significance. (e) The top 10 cellular components plotted using String 12.0 (f). The protein domain associated with the proteins, based on p-value significance (p-value < 0.05), was identified using String 12.0

Functional enrichment of these 56 interacting proteins was performed using the ShinyGO platform (Fig. 2b). The top 20 significant pathways (corrected P-value ≤ 0.05) were identified after removal of gene redundancy. Among the most prominent pathways were those related to metabolism, proteasomal degradation, and neurodegenerative disorders. Notably, five of the enriched pathways were involved in metabolic processes, including the TCA cycle, ribosome, pyruvate metabolism, oxidative phosphorylation, and amino acid biosynthesis. Enrichment of neurodegeneration-associated pathways, such as those associated with Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis, underscores the potential involvement of nsP2 in modulating neuronal and mitochondrial functions.

To gain further insights into the nature of the host proteins targeted by nsP2, Gene Ontology (GO) analysis was conducted to assess biological processes, cellular localization, molecular functions, and protein domains. GO annotation and visualization using STRING v12.0 revealed that the predominant biological processes included cytosolic translation, apoptosis, amide biosynthesis, and peptide metabolic processes. (Fig. 2c). Further, the top 10 molecular functions enriched among nsP2 interactors were primarily associated with RNA binding, oxidoreductase activity, and acetyl transferase activity (Fig. 2d). In agreement with the related biological and molecular functions, the majority of proteins were localized to the ribosome, oxidoreductase complex, ribonucleoprotein complex, extracellular exosome, and secretory granules. (Fig. 2e). Furthermore, domain analysis using the InterPro database revealed that the most frequently represented domains among the interacting proteins were associated with the proteasome α and β subunits, nucleic acid binding, and NAD(P)-binding motifs (Fig. 2f).

Collectively, these findings suggest that CHIKV-nsP2 interacts with host pathways involved in translation, RNA metabolism, mitochondrial energy production, and proteasomal degradation, emphasizing its complex role in host manipulation during infection.

CHIKV-nsP2 interacting partners in Aedes albopictus (U4.4) cells

Mass spectrometry analysis of the interacting partners of CHIKV-nsP2 protein in Ae. albopictus (U4.4) cells at 24hpi identified 115 proteins uniquely present in infected samples, in contrast to 272 proteins shared between uninfected and infected cells, represented with a Venn diagram (Fig. 3a). Based on FDR of 0.05 and unique peptides, a list of 46 high-confidence CHIKV-nsP2 interacting proteins was curated for downstream analysis (Supplementary information 2).

Fig. 3.

Interacting partners of CHIKV-nsP2 in Ae. Albopictus cells: (a) A Venn diagram representing the identified common and unique number of interacting partners of nsP2 in U4.4 cells, plotted based on high confidence. (b) Bar graph representation of the significant pathways from the enrichment analysis identified using ShinyGO. (c) GO enrichment analysis of crucial biological processes associated with CHIKV-nsP2 interacting proteins in U4.4cells using STRING 12. (d) The significant molecular functions associated with the enriched genes are displayed with a colored border based on enrichment p-values using gprofiler. Terms with a blue border are connecting the enriched terms to the root of the domain. The additional crucial molecular functions associated with CHIKV-nsP2, such as ATP-dependent activities, are shown in a yellow background. (e) Cellular component of nsP2 interacting partners using STRING 12

Pathway enrichment analysis using the ShinyGO database revealed four significantly enriched pathways (p value < 0.05) that included metabolic pathways, biosynthesis of amino acid, proteasome, and ribosome-associated processes (Fig. 3b). Further Gene Ontology (GO) analysis of the 46 interacting proteins demonstrated that the majority of the interacting partners were involved in biological processes such as cellular processes like translation and metabolic processes such as aerobic respiration, electron transport chain and ATP synthesis (Fig. 3c). Enriched molecular functions comprised catalytic activity, protein chaperone function, and ATP-dependent activity suggesting nsP2 as a multifaceted protein that utilizes its catalytic activities and interaction strategy for its efficient replication and subvert host antiviral defense (Fig. 3d). CHIKV-nsP2 helicase domain possess enzymatic activities and utilizes ATP to unwinds the RNA for efficient virus replication. CHIKV-nsP2 may induce the degradation of catalytic activities of certain host enzymes, leading to transcriptional shutdown and allowing the virus to evade the innate immune response. In terms of subcellular localization, the majority of nsP2-associated proteins were found to be localized to the cytosol, ribosomes, and intracellular compartments (Fig. 3e).

Together, these data suggest that CHIKV-nsP2 interacts with key Aedes proteins involved in metabolic regulation, proteasomal degradation, and ribosomal function. Importantly, several of these pathways were also enriched among nsP2 interactors in mammalian (Huh7) cells, indicating a conserved viral strategy to exploit fundamental host processes across both vertebrate and invertebrate systems.

Protein-protein interaction network reveals conserved functional clusters of CHIKV-nsP2 interactors in mammalian and mosquito cells

To gain insights into the functional association of CHIKV-nsP2 interacting proteins in both mammalian and mosquito systems, protein–protein interaction (PPI) networks were constructed using STRING v12.0 with a high-confidence interaction score threshold of greater than 0.4. In CHIKV-infected Huh7 cells, the resulting network comprised 53 nodes and 171 edges, representing high-confidence host interactors of the nsP2 protein. Functional clustering revealed distinct groups of proteins based on pathway enrichment and network topology (Fig. 4a). The largest cluster, consisting of 18 proteins, was enriched for components involved in pyruvate metabolism, ribose biosynthesis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation, pointing to a central role for metabolic reprogramming during infection. A second major cluster of 12 proteins included molecules such as ARF3, CCT6A, AIFM1, CANX, CYC1, PHB, NDUFA13, DNAJC19, TUBB2A, UQCC2, HSP90B1, and PARK7, which are involved in mitochondrial functions such as ATP synthesis and protein folding, as well as in diseases (involving mitochondrial proteins). Another cluster, comprising 10 proteins, was enriched for components of the cytosolic translation pathway. Additional clusters were identified, including one containing 8 proteins with zinc finger domains, CHHC-type, and linked to multisystem proteinopathies, and another with 5 proteins, such as CAND1, PSMA2, S100A8, SUMO4, and UBA1, associated with the proteasomal degradation pathway. This suggests that CHIKV-nsP2 may manipulate the host protein turnover machinery.

Fig. 4.

(a) String Network visualization of CHIKV-associated interacting partners in the host and the vector to illustrate physical and functional connections during infection. Protein clusters are color-coded based on the obsolete k algorithm. Networks were generated using default settings for a comprehensive STRING network, displaying high-confidence edges with a minimum interaction score of 0.4, an FDR ≤ 0.05, a minimum interaction strength of 2, and at least two interactions per node, with no disconnected nodes. Blue circles represent host cell proteins, while orange circles indicate proteins in insect cells. Interacting proteins in both human and insect cells form five distinct clusters. In human liver cells, the first cluster (red) corresponds to metabolic pathways, the second (yellow) to mitochondrial proteins, the third (green) to translation and ribosomal proteins, the fourth (light blue) to RNA processing, and the fifth (Blue) to proteasomal pathway proteins. In Ae. albopictus cells, the largest cluster (red) represents proteins involved in translation, the second (yellow) includes metabolic proteins, the third (green) comprises proteins associated with oxidative stress, the fourth (light blue) includes helicases and chaperones, and the fifth (blue) includes proteasomal proteins. (b) The upper panel displays a Venn diagram of shared protein interactors in Huh7 and U4.4 cells. Huh7 cells contain 50 proteins, while Ae. albopictus cells have 40 proteins, with 6 proteins common to both cell types (listed in the lower panel’s first box). The second box displays a hierarchical clustering tree that illustrates the correlation among significant pathways, grouping pathways with shared genes together. Larger dots signify more significant p-values, analyzed using shinyGO 0.77. The third box presents a PPI network generated with String 12.0, with an expected interaction count of 3. Colored nodes represent the protein interactors of CHIKV-nsP2, while edges indicate protein-protein interactions

In Ae. albopictus U4.4 cells, the PPI network of nsP2 interactors was comparatively smaller, comprising 34 nodes and 55 edges, and organized into five functional clusters. The largest cluster (red in color) contained 12 proteins involved in cytoplasmic translation, including ribosomal subunits and translation-associated factors. A second cluster of ten proteins represented components of the mitochondrial respiratory chain complex, highlighting potential virus-induced alterations in energy metabolism. A third cluster (highlighted in green) comprised four proteins associated with the oxidative stress complex, namely RP20_CCG001423, RP20_CCG005913, RP20_CCG009927, and RP20_CCG012209. The fourth cluster (light blue color) consisted of additional helicases and chaperones, further supporting the replication machinery among mosquito cell interactors involved in the proteasomal pathway, consistent with similar findings in mammalian cells.

To compare nsP2 associated host proteins across systems, we generated a Venn diagram from the high-confidence interactors identified by Co-IP–LC-MS/MS in human Huh7 and Aedes albopictus U4.4 cells (Fig. 4b, upper panel). A total of 96 proteins were identified; six were shared between the two cell types (6.25%), 50 were unique to Huh7 (52.08%), and 40 were unique to U4.4 (41.67%), revealing a conserved core alongside species-restricted interactors. Notably, the CHIKV capsid was also detected as an nsP2 interactor in both datasets, suggesting potential intraviral coordination relevant in both vector and host.

The six common interactors the from both human (Huh7) and mosquito (Ae. albopictus U4.4) cells were identified to be DEAD-box helicase (DDX), succinate dehydrogenase (SDH), eukaryotic translation initiation factor 3 (eIF3E), glutathione S-transferase (GST), isocitrate dehydrogenase (IDH3B), and peptidyl-prolyl isomerase (PIN1) (Fig. 4b). It is noteworthy that these proteins represent core cellular pathways co-opted by CHIKV in both vertebrate and invertebrate hosts. For instance, DEAD-box helicases and eIF3 are central to RNA metabolism and translation initiation, suggesting that nsP2 directly interfaces with the host protein synthesis machinery to ensure efficient viral RNA translation. Similarly, identifying succinate dehydrogenase and isocitrate dehydrogenase, key enzymes of the tricarboxylic acid (TCA) cycle, as common interactors highlights the importance of metabolic reprogramming and energy production in sustaining viral replication. The identification of GST points to viral modulation of redox balance and stress responses, particularly relevant to persistent infection in mosquito cells. Peptidyl-prolyl isomerase, a molecular chaperone involved in protein folding and trafficking, further suggests that nsP2 may exploit host quality-control systems to stabilize viral or host protein complexes. The conservation of these six interactors across both host environments underscores their potential role as critical nodes in CHIKV replication and pathogenesis (Fig. 4b, lower first and second boxes). Additionally, we constructed the protein-protein interaction network of these conserved proteins using STRING 12.0. We found that the expected number of interactions is 3. The strongest association was observed between succinate dehydrogenase and isocitrate dehydrogenase, with a total of 4 edges. Additionally, peptidyl-prolyl isomerase, DEAD box helicase, and EIF3 each showed up to 3 potential interactions (Fig. 4b, lower third box).

Together, these PPI network analyses revealed that CHIKV-nsP2 interacts with a conserved set of cellular hubs involved in translation, proteasomal degradation, RNA regulation, and metabolic processes. The shared enrichment of these pathways in both mammalian and mosquito cells suggests that CHIKV employs a conserved host manipulation strategy to facilitate its replication and survival across diverse biological systems.

Discussion

CHIKV, like other alphaviruses, relies heavily on the host and vector cellular machinery to support its replication, assembly, and evasion of immune defenses. Among its nonstructural proteins, nsP2 is a multifunctional protein critical for polyprotein processing, replication complex formation, and modulation of host antiviral responses [17]. Despite its central role in the viral life cycle, the interactome of nsP2 has remained poorly defined, particularly in the context of both mammalian and mosquito cells. The present study aimed to bridge this gap, and we systematically mapped the CHIKV-nsP2 interactome using co-immunoprecipitation coupled with LC-MS/MS, followed by functional enrichment analyses in both human liver (Huh7) and Ae. albopictus U4.4 cells. Our findings provide novel insights into the host–pathogen interaction landscape and highlight key pathways commonly exploited by the virus in diverse biological environments.

Our data indicate that nsP2 engages a broad spectrum of host and viral proteins, many of which are involved in critical cellular processes such as translation, RNA metabolism, proteasomal degradation, and energy production. Notably, we identified the interaction of nsP2 with other CHIKV proteins, such as nsP1 and E1, consistent with previous findings suggesting a regulatory role of nsP2 in modulating nsP1 ATPase activity [18]. This intraviral crosstalk may coordinate the assembly and function of the replication complex. In agreement with previous work on CHIKV nsP3 [12, 19], nsP2 was also found to target RNA-binding proteins such as SNRPB and HNRNPA1, suggesting that CHIKV disrupts host splicing and RNA processing pathways to facilitate viral RNA translation and stability [20]. Furthermore, the interaction of nsP2 with cytoskeletal elements (e.g., actin, tubulin, filamin) and ER-associated proteins (e.g., calnexin) indicates a potential role in modulating intracellular trafficking and virion morphogenesis [21].

A significant enrichment of proteasomal subunits PSMC4, PSMA2, PSMA3, PSMA5, PSMA7, and PSMB4 was observed in mammalian cells, highlighting the virus’s capacity to manipulate the ubiquitin-proteasome system. This may enable CHIKV to degrade antiviral host proteins or to regulate viral protein turnover, as suggested for other viruses [22]. Interaction with UBA1 (ubiquitin-like modifier activating enzyme 1) further supports the role of nsP2 in subverting ubiquitin signaling.

Multiple ribosomal proteins, including MRPL12, RPL37A, RPL28, RPL38, RPL9, RPL9, RPL24, RPS27A, and RPL21, were identified as nsP2 interacting partners. Their association may reflect viral hijacking of the host translation machinery to selectively enhance the translation of viral RNAs, as observed in other alphaviruses [23] The interaction with eukaryotic translation initiation factor 3 (eIF3) components further underscores the importance of translation initiation in CHIKV replication.

Our analysis revealed a notable enrichment of metabolic enzymes, particularly those associated with the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Identified proteins include IDH3B, DLAT, SDHA, SDHB, ACLY, ATP5C1, NDUFA5, COX5A, and CYC1, highlighting the virus’s reliance on host energy metabolism. Previous studies have shown that viral infections, including CHIKV, reprogram host metabolism to generate biosynthetic precursors and ATP required for replication [24].Further, the ETC triggers OXPHOS to produce ATP. Given that viruses target fundamental cellular circuitry, it may not be surprising that the ETC is in the crosshairs during infection. Interestingly, many of these metabolic factors were also detected in mosquito (U4.4) cells, suggesting that CHIKV maintains a conserved mechanism of metabolic reprogramming across species. The virus may exploit TCA intermediates and lipid biosynthesis via ACLY to generate membranes required for the formation of viral replication complexes [25].

In mosquito cells, nsP2 interacted with key metabolic and redox-regulating proteins, including acyl-CoA dehydrogenase, triosephosphate isomerase, isocitrate dehydrogenase, cytochrome P450, glutathione peroxidase, and glutathione S-transferase. These proteins play crucial roles in ATP generation, redox homeostasis, and antiviral defense [25, 26]. The interaction of nsP2 with cysteine desulfurylase, DEAD-box RNA helicases, and ribosomal proteins such as S23, S28, and L37 suggests a role in modulating host translation, which may support persistent, non-cytolytic infection in the mosquito vector [27]. The overlap of key interactors, such as succinate dehydrogenase, glutathione S-transferase, DEAD-box ATP-dependent RNA helicase, peptidyl-prolyl isomerase, and eIF3, between mammalian and insect cells suggests conserved targets that may be critical for CHIKV replication in both environments.

Several of the pathways identified, such as those involving mitochondrial bioenergetics and proteasomal degradation, are also implicated in neurodegenerative diseases and metabolic syndromes, which have been associated with CHIKV infection [28, 29]. Given the role of mitochondria in apoptotic signaling, interaction with factors such as apoptotic protein AIFM1 may suggest a role of nsP2 in modulating cell death to enhance viral persistence [30].

We next interrogated the subset of six nsP2 interactors conserved between human Huh7 and Aedes albopictus U4.4 cells to define pan-host dependencies. The six conserved nsP2-associated proteins detected in both Huh7 and Aedes albopictus cells, DEAD-box RNA helicase, eukaryotic translation initiation factor 3 (eIF3), succinate dehydrogenase (SDH), isocitrate dehydrogenase (IDH), glutathione S-transferase (GST), and a peptidyl-prolyl isomerase, point to cross-species dependencies that CHIKV likely exploits during infection. Enrichment of a DEAD-box helicase and an eIF3 subunit is consistent with nsP2 engaging host RNA metabolism and initiation of translation, processes widely co-opted by RNA viruses to prioritize viral RNA expression [31, 32]. The repeated recovery of SDH and IDH indicates a conserved interface with mitochondrial TCA/OXPHOS metabolism, aligning with the broader theme of virus-driven metabolic rewiring to meet the energetic and biosynthetic demands of replication [25, 33–35]. Identification of GST supports a role for redox/stress-response pathways in sustaining infection, particularly relevant in the mosquito vector, where oxidative stress modulators influence arbovirus persistence [36]. Finally, the peptidyl-prolyl isomerase interactor suggests that foldase/chaperone activities may stabilize viral or host complexes required for efficient replication, echoing established contributions of PPIases across multiple RNA virus life cycles [37]. Furthermore, the involvement of the RIG-I/MDA5 signaling pathway suggests that nsP2 may suppress the host’s immune response, potentially facilitating the development of virus-specific treatments for CHIKV infection [38]. Together, the cross-species conservation of these interactors argues for fundamental nsP2–host interfaces that could be exploited as broad-spectrum antiviral entry points.

Although this provides a comprehensive interaction map of CHIKV-nsP2 and highlights several conserved host pathways, it is not without limitations. Replication patterns differ between the host and the vector, and mapping the interactome at defined time points may underrepresent transient or low-abundance interactions. Cross-system comparisons could also be influenced by differences in proteome coverage and isoform annotation. Future orthogonal validation and functional perturbation will refine individual assignments; however, the repeated enrichment of the same proteins and pathways across both hosts supports the robustness of our main conclusions.

Conclusion

To our knowledge, this work delivers the first cross-species map of CHIKV-nsP2 interactions profiled in parallel human (Huh7) and mosquito (Aedes albopictus U4.4) cells, revealing conserved engagement of translation initiation, proteostasis, and mitochondrial metabolism. The convergence on shared interactors highlights pan-host dependencies that can be prioritized for mechanistic follow-up. By anchoring comparative virology in proteomics, these findings refine current models of CHIKV replication across hosts and nominate tractable, host-directed points for antiviral intervention.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

NM: Formal analysis, Investigation, Bioinformatics analysis, Methodology, Validation, Writing- original draft, Writing-review & editing. SS: Conceptualization, Funding acquisition, Supervision, Writing- original draft, Writing-review & editing.

Funding

This work was supported by ICGEB core funds to SS.

Data availability

All relevant data are within the paper and its Supporting Information files.

Declarations

Conflict of interest

The authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics statement

Commercially available, established cell lines were used for the study.