Structure, Attachment and Transmembrane Internalisation of Peste Des Petits Ruminants Virus

ABSTRACT

Peste des petits ruminants virus (PPRV), a single‐stranded negative‐sense RNA virus with an envelope, belongs to the Morbillivirus in the Paramyxoviridae family and is prevalent worldwide. PPRV infection causes fever, stomatitis, diarrhoea, pneumonia, abortion and other symptoms in small ruminants, with a high mortality rate that poses a significant threat to the sustainability and productivity of the small ruminant livestock sector. The PPRV virus particles have a diameter of approximately 400–500 nm and are composed of six structural proteins: nucleocapsid protein (N), phosphoprotein (P), envelope matrix protein (M), fusion protein (F), haemagglutinin protein (H) and large protein (L). Each protein has a distinct role in the virus's life cycle. Although the life cycle activities of PPRV have been widely reported, they are still limited. Research has demonstrated that PPRV has distinct adhesion factors on various cell surfaces, such as the epithelial cell adhesion factor nectin‐4 or the lymphocyte adhesion factor SLAM. After attaching to the cell, the F and H proteins on the PPRV membrane interact with each other, resulting in a conformational change in the F protein. This change allows the F protein to enter the cell through direct fusion with the host cell membrane. The virus enters the host cell via the outer vesicle endocytosis strategy and replicates and proliferates through the role of caveolin, actin, dynein and cholesterol on the host cell membrane. This review summarises the viral structure, attachment mechanism and transmembrane internalisation mechanism of PPRV. The aim of this review is to provide theoretical support for the development of PPRV inhibitors and the prevention and control of PPR.

The structural and non‐structural proteins of PPRV and the mechanism of transmembrane internalization via clathrin‐cediated endocytosis.

1. Introduction

Peste des petits ruminants virus (PPRV) is an infectious disease agent that causes peste des petits ruminants (PPR) in small ruminants, such as sheep and goats. It belongs to the Morbillivirus in the Paramyxoviridae family, which also includes canine distemper virus (CDV), rinderpest virus (RPV) and measles virus (MV) (Liu et al. 2015b). Clinical signs in small ruminants after infection include fever, stomatitis, diarrhoea, pneumonia, abortion and other symptoms. PPRV has been reported to cause infection in cattle (Schulz et al. 2019), buffalo (Jones et al. 2021), camel (Woma et al. 2015), Asian lion (Balamurugan et al. 2012) and wild ungulates (Pruvot et al. 2020). The first report of PPR was in Côte d'Ivoire in 1942, and since then, high incidence has been reported in African, Asian and European countries. In July 2007, PPRV infection was first reported in the Ali region of China. In 2013, an outbreak of PPRV occurred on a farm in Huocheng County, Yili, Xinjiang. Subsequently, four infected farms were found in the same province (Z. Wang et al. 2009). The disease quickly spread to 21 provinces in China through the trade of goats and sheep, resulting in the deaths of over 16,000 animals and significant economic losses for local animal husbandry (Bao et al. 2017). Currently, the use of the PPRV vaccine has effectively controlled PPR in China. However, in some developing countries, the presence of PPR is a major hindrance to the growth of local animal husbandry (Kumar et al. 2017). Therefore, the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organisation (FAO) have prioritised the complete elimination of PPR, with an expected deadline of 2030 (Baron et al. 2016). This paper summarises the structure and function of structural and non‐structural proteins and the cell attachment and potential cell invasion mechanism of PPRV in the early stage of its cell life cycle. The aim is to enhance understanding of the biological structure characteristics of PPRV and provide assistance for early prevention and control of PPR.

1. Summary

The widespread epidemic of Peste des Petits Ruminants Virus (PPRV) and its persistent infection have caused significant economic losses to small ruminant farming.

Farms also contend with co‐infections involving PPRV and various other pathogens. The emergence of new PPRV strains has led to a decrease in vaccine efficacy.

Moreover, PPRV's reliance on exosomes enhances its ability to evade the host's natural immune system, complicating complete eradication efforts.

This article outlines the structure of PPRV, detailing the functions of its structural and non‐structural proteins, as well as its mechanisms of cell adhesion and potential cell invasion during the early stages of its life cycle.

The aim is to deepen our understanding of PPRV's biological structure and provide insights for its early prevention and control.

2. Structure and Protein Function of PPRV

2.1. The Structure of PPRV

PPRV is a single‐stranded, negative‐stranded RNA virus with an envelope. The virus particle has a diameter of approximately 400–500 nm, and the genomic RNA is approximately 15,948 nt in length. Different strains may have variations, but they all conform to the typical characteristics of the ‘six‐base principle’ of other paramyxoviridae (Albina et al. 2013). The genome of PPRV comprises a 3′ non‐coding region genome promoter (GP), six structural proteins, namely nucleocapsid protein (N), phosphoprotein (P), envelope matrix protein (M), fusion protein (F), haemagglutinin protein (H) and large protein (L), and the 5′ non‐coding region is composed of the anti‐genome promoter region (AGP) (Kwiatek et al. 2022). The viral RNA is wrapped by the N (Figure 1). The P gene also encodes for two non‐structural proteins, V and C, which are produced as a result of RNA editing and the use of an alternative open reading frame (ORF) in the P gene's transcript, respectively (L. Li et al. 2023).

FIGURE 1.

1 (A) Pictorial representation of the structure of the PPRV particle, an enveloped, negative sense, single stranded RNA virus. (B) Genome structure of PPRV in 5′ to 3′ direction. PPRV codes for six structural genes (N, P, M, F, H and L) and two non‐structural genes (C and V).

2.2. Structural and Non‐Structural Proteins of PPRV and Their Functional Characteristics

The first structural protein encoded by PPRV is the N, which has a molecular size of approximately 58 kDa. It is highly immunogenic and conserved but does not induce the host to produce neutralizing antibodies (Choi et al. 2005). The core domain of the protein is responsible for RNA encapsidation and the formation of the viral spiral nucleocapsid. Together with the M protein, it promotes virus assembly and the encapsidation of genomic RNA (Servan de Almeida et al. 2007). The genetic evolution of PPRV can be divided into four lineages based on the sequence difference of the N gene (1287–1638 nt): I, II, III and IV (Shahriari, Khodakaram‐Tafti, and Mohammadi 2019). These lineages exhibit geographical specificity, with lineage I and II mainly reported in West and Central Africa, lineage III restricted to East Africa and the Arabian Peninsula and lineage IV distributed from Southeast Asia to the Middle East. IV strains have been identified in some areas of Africa and are gradually replacing III strains (Kinimi et al. 2020). Furthermore, the N protein of PPRV plays a crucial role in both the virus's pro‐inflammatory response and its evasion of the host's innate immune system. The N protein can directly interact with myeloid differentiation factor 88 (MyD88), thereby activating the NF‐κB signalling pathway. In addition, its interaction with the NOD‐like receptor thermal protein domain‐associated protein 3 (NLRP3) promotes the assembly and activation of the NLRP3 inflammasome, leading to the cleavage of pro‐caspase‐1 and the secretion of interleukin‐1β (IL‐1β), thus initiating the host's inflammatory response (L. Li et al. 2022). The N protein also interacts with vacuolar protein sorting 34 (VPS34) and vacuolar protein sorting 15 (VPS15), active components of the phosphatidylinositol 3‐kinase complex I (PI3K complex I), inducing autophagy in cells to evade the host's innate immune system and facilitate viral replication and proliferation (Chaudhary et al. 2023). Furthermore, the N protein can interact with interferon regulatory factor 3 (IRF3), blocking the interaction between TBK1 and IRF3, which inhibits the expression of IFN‐β and interferon‐stimulated genes (ISGs). This interaction prevents the phosphorylation of IRF3, thereby inhibiting the activation of the RIG‐I‐like receptor (RLR) signalling pathway and the production of Type I interferon (IFN), helping PPRV evade the host's innate immune system and promoting its replication (Zhu et al. 2019). In addition, the N protein, particularly within the C‐terminal domain spanning residues 376–525, can inhibit signal transduction that induces IFN‐γ production, thereby suppressing Type II IFN production. However, the specific mechanism underlying this action requires further investigation (P. Li et al. 2019).

The molecular size of the P is approximately 60 kDa, and its size varies with phosphorylation. The ORF of the P protein can be translated into two non‐structural proteins, C or V (Mahapatra et al. 2003). The P protein is multifunctional, acting as a cofactor in the transcription complex by interacting with the C‐terminus of the N protein to form a P–N homologous envelope complex, thereby maintaining the solubility of the N protein (Alfred et al. 2021).The P protein serves two critical functions: it interacts with the L protein to form a heterologous P–L complex, and it acts as a bridge between N‐RNA and the L protein to form a P–N–L heterologous tripartite complex. This tripartite complex constitutes the basic component of RNA‐dependent RNA polymerase (RdRp), which drives the replication and transcription of the PPRV genome (Yunus and Shaila 2012). And the P protein is essential in the life cycle of PPRV, regulating transcription, translation and cell cycle control. However, the specific molecular mechanisms underlying its regulation remain unclear. Studies have shown that the in vitro expression of the PPRV P protein inhibits the activation of protein kinase RNA‐like endoplasmic reticulum kinase (PERK). Concurrently, the gene GADD34, which is responsible for protein growth arrest and DNA damage induction, is activated to prevent the phosphorylation of eIF2α. This promotes viral replication through the phosphorylation of both the P protein and eukaryotic initiation factor‐2α (eIF2α) (Ma et al. 2015). In addition, the V protein has been shown to interact with signal transducers and activators of transcription 1/2 (STAT1/2), causing nuclear translocation of the STAT1/2 protein. This blocks IFN signalling and contributes to the evasion of host innate immunity, although this approach is dependent on the concentration of the V protein. The V protein has also been found to bind to the RIG‐I receptor, inhibiting IFN production. The specific molecular mechanisms behind these interactions are still under investigation (Runge et al. 2014). Data from Gerlier and Valentin (2009) identified tyrosine 110 (Y110) of the P protein as a specific amino acid residue that interacts with STAT1. This interaction significantly affects the phosphorylation of STAT1, aiding in the evasion of the host immune response. Furthermore, the interaction between the P protein and IRF3 inhibits the phosphorylation of IRF3, preventing IRF3 dimerisation and nuclear translocation, thereby inhibiting the production of Type I IFN (P. Li et al. 2021).

The C protein, which is encoded by the P protein gene, also regulates the activation of IFN. According to Linjie et al.'s (2021) data, the C protein can downregulate the downstream cytokines of ISG56, ISG15, C‐X‐C motif chemokine (CXCL10) and RIG‐I‐mediated interferon stimulated response element (ISRE) and IFN promoter element of NF‐κB. It can also inhibit the phosphorylation of STAT1 to interfere with signal transduction in the JAK–STAT pathway, resulting in blocked IFN production. Reverse genetics experiments demonstrated that the knockout of the V or C protein showed differing effects on IFN induction. Viral infection could still inhibit IFN induction after the V protein was knocked out, whereas viral infection could not inhibit IFN production following the knockout of the C protein (Schuhmann, Pfaller, and Conzelmann 2011). The early stages of PPRV infection are primarily mediated by the C protein, while the later stages are mainly mediated by the V protein. Specifically, knocking out the V protein did not induce IFN production during the early stages of PPRV infection, whereas knocking out the C protein did result in IFN production (Shaffer, Bellini, and Rota 2003).

The M protein of the envelope has a molecular size of 38 kDa and is mainly responsible for the assembly and release of PPRV. According to W. Li et al.'s (2014) data, co‐expression of PPRV N protein and M protein by baculovirus showed that blocking the synthesis of M protein in PPRV‐infected cells resulted in broader cell–cell fusion and reduced virus release. This suggests that M protein plays a role in regulating virus assembly. Studies have shown that while other proteins such as H protein and F protein promote the packaging and budding process of PPRV, the main protein responsible for the assembly and release of the virus is the M protein (Q. Wang et al. 2017). The C‐terminus of the M protein binds to the viral envelope to form the inner layer of the envelope. It acts as an adapter that connects the viral N protein, L protein and the cell membrane. The M protein can interact with envelope glycoproteins (H and F) and RNP complexes in the cytoplasm to promote virus assembly and release (Moll et al. 2004). Three different siRNAs targeting the translation region of the 75/1 M mRNA of the Nigerian PPRV strain were designed and transfected into Vero‐SLAM cells. The study found that siRNA‐mediated knockdown of the M protein altered its interaction with viral glycoproteins, increased cell–cell fusion and inhibited viral replication (Liu et al. 2015a).

The F protein has a molecular size of 60 kDa. It is activated upon virus attachment to the host and participates in forming spikes on the viral envelope surface. The F protein then fuses the viral envelope with the host cell membrane, releasing the viral genetic material into the cytoplasm (Kumar et al. 2014). Studies have shown that the F protein plays a crucial role in the specific immune response of PPRV. In Liu et al.'s (2015a)s data, the replicative DNA vaccine expressing PPRVF protein induced humoral and cell‐mediated immune responses in mice. The immune effect was good, providing a new strategy for the development of a PPRV vaccine based on the F protein to prevent and control PPR (Y. Wang et al. 2015). In addition, Devireddy et al. (1999) found that the F protein also exhibits haemolytic characteristics. When the F protein of PPRV, purified by immunoaffinity chromatography, was incubated with chicken red blood cells, it caused them to lyse. This indicates that the PPRV F protein acts as a haemolysin and plays a crucial role in the pathogenesis of PPRV infection.

The H protein, which has a molecular size of 60 kDa, is the initial structural protein that interacts with the host during the viral replication life cycle. It possesses haemagglutinin and neuraminidase activity and is also referred to as the haemagglutinin–neuraminidase protein (HN) (Agrawal et al. 2023). The H protein appears as a spike on the surface of virus particles and is responsible for binding the virus to the host receptor. It cleaves the sialic acid residues in the carbohydrate moiety of the host glycoprotein, regulates virus adsorption and entry and contributes to pathogenicity (Rojas et al. 2021). Nectin‐4 is the primary receptor for the H protein in epithelial cells (Atmaca and Kul 2012), while the signalling lymphocyte activation molecule (SLAM) is the primary receptor for the H protein in lymphoid cells. These two receptors interact with the H protein during the early stages of PPRV infection (Meng et al. 2020). During the early stages of a viral infection, PPRV is initially found in the intraepithelial space and lamina propria of the respiratory tract mucosa. It is then taken up by the antigen‐presenting cell (APC) and transported to the lymphoid tissue where viral replication occurs. The virus then spreads throughout the body via lymphocytes (Zhao et al. 2020). Furthermore, the H protein not only participates in the release of newly generated viral particles (Kumar et al. 2014) but also plays a crucial role in promoting viral replication and proliferation. Xue et al.'s (2020) data identified the 35–58 region of the H protein as essential for inhibiting the expression of cyclophilin A (CypA), although the specific molecular mechanism of this interaction remains unknown. Research has demonstrated that CypA can impede PPRV replication through its peptidyl propionyl isomerase enzyme activity.

The L protein, which has a molecular size of 247.3 kDa, is a crucial component of the viral polymerase and replicase (Ansari et al. 2019). It is a multifunctional catalytic protein that forms a complex with the P protein, responsible for the transcription and replication of genomic RNA. This includes initiation, elongation and termination, as well as capping, methylation and polyadenylation of viral mRNA (Malur et al. 2002). The L protein is divided into three domains. The first domain of the protein consists of the conserved sequence KEXXRLXXKMXXKM from residue 1–606, which primarily serves as the binding region with RNA. The second domain spans residues 650–1694 and contains the conserved sequences GDDD and QGDNQ, which are flanked by hydrophobic regions and serve as functional sites of RdRp. The third domain, which has kinase activity, including RNA triphosphatase (RTPase), guanylate transferase (GTase) and methyltransferase activity, is a residue from 1717–2183. However, the specific mechanism of action is still under investigation (Blumberg et al. 1988).

3. Attachment of PPRV

3.1. Dual Tropism in PPRV

PPRV is primarily transmitted through contact but can also be transmitted through the respiratory tract to small ruminants, such as sheep and goats. Symptoms include high fever, pneumonia, diarrhoea and inflammation of the respiratory and gastrointestinal mucosa (SowjanyaKumari et al. 2021). During the early stages of infection, PPRV primarily colonizes the respiratory tract. After breaching the host's respiratory barrier, the virus invades host cells and spreads to the tonsils, pharynx, jaw and other local lymph nodes for replication and proliferation, causing viremia (Gautam et al. 2021). Subsequently, the virus spreads throughout the host's abdominal visceral lymph nodes, bone marrow, spleen and other organs, resulting in numerous replications and proliferations (Wen et al. 2022). Finally, a large number of viruses were produced and released from the epithelial cells of the respiratory and digestive tracts, which were then excreted and spread again (Jelsma et al. 2021).

3.2. Host Cell Attachment Receptors

The attachment of PPRV to the host cell surface is the first step in the virus's life cycle. This interaction is crucial for the virus's rapid replication and is primarily mediated by the glycoprotein F and the H on the envelope surface (Sinnathamby et al. 2004). The glycoprotein interacts with attachment receptors on the surface of the host cell, anchors itself to the surface of the host cell and recruits related proteins to form complexes and enter the cell. Studies have identified nectin‐4 and SLAM receptor proteins as attachment receptors or related proteins that interact with PPRV (Yang et al. 2018).

The nectin‐4 receptor protein, also referred to as poliovirus receptor‐related 4 (PVRL4), is an intercellular adhesion protein that belongs to the nectin family of the immunoglobulin superfamily. This family comprises four nectin molecules and five nectin‐like molecules, all of which are typical transmembrane proteins (Chatterjee et al. 2021). The molecular weight of the nectin‐4 receptor protein is 66 kDa, and it is composed of 510 amino acid transmembrane proteins (Rendon‐Marin et al. 2019). Nectin‐4 is expressed throughout the human body, particularly in epithelial cells and some tumour cells, and is crucial for cell adhesion and intercellular interactions, including cell adhesion, intercellular junctions and extracellular matrix exchange (Zhang et al. 2018). In addition, nectin‐4 participates in various biological processes, such as embryonic development, tissue repair and immune cell activation (Jelsma et al. 2021). Recently, nectin‐4 has gained significant attention in the field of oncology. Due to its high expression on certain tumour cells, it is considered a potential therapeutic target for anti‐tumour treatment (Wong and Rosenberg 2021). Mühlebach et al. (2011) identified nectin‐4 as an epithelial cell receptor of MV involved in nectin‐4‐mediated PPRV replication. However, the specific molecular mechanism requires further exploration (Birch et al. 2013). To investigate the mechanism of action of nectin‐4, we identified its action region based on the interaction model of MV and nectin‐4. The action region mainly includes three Ig‐like extracellular domains: an N‐terminal V‐type and two C2 domains that mediate ligand binding, a transmembrane region, and a cytoplasmic tail structure. The V‐type domain is crucial for homotypic or heterotypic interaction with nectin‐1 and primarily binds to the viral envelope glycoprotein H protein. The C2 domain enhances the affinity of these interactions (Prajapati et al. 2019). Furthermore, nectin‐4 has been proposed as an exit receptor, mainly acting in the late stage of infection when the virus is released from the epithelial cells after completing viral replication (Mühlebach et al. 2011). However, Delpeut, Noyce, and Richardson (2014) shows two protein variants of nectin‐4, with its V domain mediating CDV entry and spread between cells. Further study is required to fully understand the interaction mechanism between nectin‐4 and the virus.

The SLAM receptor protein is a member of the CD2 family and functions as a cell surface receptor in the immune system. Other members of this family include SLAM1 (CD150), SLAM2 (CD48), SLAM3 (Ly‐9/CD229), SLAM4 (CD244/2B4), SLAM5 (CD84), SLAM6 (Ly108/CD352), SLAM7 (CRACC/CS1/CD319), SLAM8 (CD353) and SLAM9 (CD84‐H1/CD2F10) (Farhangnia et al. 2023). SLAM receptors are expressed on the surface of various immune cells, including T cells, natural killer cells and macrophages. They play a crucial regulatory role in immune cell interactions (Gartshteyn, Askanase, and Mor 2021). SLAM receptors can bind to ligands on homogeneous or heterogeneous cells, triggering signalling pathways between cells and affecting physiological processes such as cell activation, proliferation, differentiation and adhesion (Tojjari et al. 2023). During their study of the mechanism of the SLAM receptor in cell adhesion, Tatsuo et al. discovered that the SLAM receptor is a major receptor of MV. It can strongly interact with the MV H protein and facilitate virus adhesion and invasion into host cells. Research has demonstrated that the SLAM receptor is a significant receptor for Paramyxoviridae viruses, including CDV, RPV and PPRV. The receptor interacts with the H protein on lymphocyte surfaces, facilitating virus adhesion and invasion into host cells (Tatsuo, Ono, and Yanagi 2001). Upon analysis of the protein structure of SLAM receptors, it was discovered that they are primarily composed of two Ig‐like extracellular domains: an N‐terminal V‐type and a C2 domain that mediates ligand binding.

In addition, they contain a transmembrane region and a cytoplasmic tail structure. However, the SLAMF3 receptor differs in that it has four Ig‐like extracellular domains, consisting of two V‐type and two C2‐type (Navaratnarajah et al. 2008). MENG X shows that the interaction between H protein and SLAM receptor protein and their mutants (SLAM1, SLAM2 and SLAM3) resulted in no interaction between H protein and SLAM3 (Atmaca and Kul 2012). However, H protein did interact with SLAM and SLAM2, with varying intensities, and had a low level of interaction with SLAM1. H‐protein mutants were unable to interact with SLAM receptors and their mutants. Agrawal's data showed that the invasion of PPRV was blocked by constructing the homologous peptide of the SLAM receptor protein. This suggests the potential for developing anti‐PPR inhibitors based on the H protein. In addition, the study confirmed that the SLAM receptor is one of the important receptors for PPRV to invade host cells (Agrawal et al. 2023, 2024).

Toplu, Oguzoglu, and Albayrak (2012) detected sheep border virus (BDV) and PPRV antigens in the brain, oral mucosa, intestine and lung of infected animals using immunohistochemistry. The authors suggested that intrauterine BDV infection caused brain injury, which promoted PPRV entry into the brain and resulted in infection of neurones and glial cells. PPRV adhesion and invasion cells identify SLAM receptor and nectin‐4 receptor as receptor proteins. There are currently no reports of SLAM receptor and nectin‐4 receptor expression in brain tissue. Some scientists speculate that there may be hypothetical receptors in brain tissue, but they have not yet been identified. In addition, there are few reports of PPRV infection in small ruminant brain tissue, indicating a need for further research (de Witte et al. 2008).

4. Transmembrane Internalisation Pathway of PPRV

PPRV is a strict intracellular parasite that requires cell entry to complete its replication cycle. The replication cycle can be divided into viral attachment, transmembrane internalisation, genome replication, early protein synthesis and viral particle assembly and release. Immune escape cascade reactions occur during the replication cycle (Manjunath et al. 2019). Enveloped viruses enter host cells through two molecular mechanisms: direct fusion and receptor‐mediated endocytosis. According to Yang's data, PPRV invades host cells using various strategies depending on the specificity of the host cell type. These strategies include, but are not limited to, reliance on dynein‐mediated endocytosis or direct cell fusion (Yang et al. 2018).

4.1. Invasion Mechanism of PPRV Direct Fusion With Cells

PPRV and MV are members of the Paramyxoviridae family. The envelope glycoproteins F and H share similarities in structure and function, particularly in transmembrane internalisation. Studies have shown that the structure of the MVF protein includes a hydrophobic fusion peptide (FP), two 7‐peptide repeat regions (Heptad repeat regions A and B), a transmembrane domain (TM) and a C‐terminal cytoplasmic tail (CT) (Smith et al. 2009). The F protein is initially inactive as the F0 precursor and only becomes active after the F1 and F2 subunits are linked by a disulphide bond. At this point, its fusion activity is exerted. Upon activation of the F protein fusion, the heptapeptide repeat region undergoes rearrangement, resulting in irreversible conformational changes that form a stable six‐helix bundle structure. This structure mediates the internalisation of MV across the cell membrane (Sun et al. 2015). The activation of F protein fusion is currently being studied by scholars. According to Mirza's research, the H protein plays a crucial role in activating the fusion of the F protein by interacting with it and promoting its activation. However, the specific molecular mechanism requires further investigation (Mirza et al. 2011). Two models have been proposed by scholars to explain the interaction mechanism between the H protein and F protein. The first model is the clamp model, where the F protein and H protein interact within the cell and are transported to the cell surface as a complex. The H protein clamps the F protein, causing it to be in a metastable state. Upon binding to the receptor, the H protein releases and activates the F protein, initiating the membrane fusion process (Brindley, Chaudhury, and Plemper 2015). At first, the F and H proteins are transported independently to the cell surface and do not interact. When the H protein binds to the receptor, it transmits an activation signal to the F protein, causing a conformational change and subsequent fusion of the cell membrane (Marcink et al. 2020). There are currently limited reports on the molecular mechanism of cell membrane fusion mediated by PPRVF protein. However, most of the biochemical characteristics of Paramyxoviridae viruses support the clamp model. Although there are similarities in the glycoprotein structures among members of the Paramyxoviridae family, notable differences also exist. Consequently, the transmembrane internalisation mechanisms of various Paramyxoviridae family members require further investigation (Plemper, Brindley, and Iorio 2011). For example, while the CD46 protein plays a crucial role in the invasion of MV, it is not one of the primary receptors of PPRV. Hence, additional research is required to investigate the invasion mechanism of PPRV F protein‐mediated direct cell membrane fusion (Melia et al. 2014).

4.2. Internalisation Mechanism of PPRV Endocytosis Pathway

These pathways include clathrin‐dependent endocytosis, clathrin‐independent endocytosis and macropinocytosis‐dependent endocytosis (Nabi and Le 2003). Enveloped viruses, such as CSFV (Guo et al. 2023), ASFV (Chen et al. 2023) and dengue virus (Cruz‐Oliveira et al. 2015), use various endocytosis pathways for transcellular internalisation. HIV uses dynein‐dependent endocytosis for intercellular transmission (Chauhan and Khandkar 2015), whereas the vaccinia virus (Schroeder et al. 2012), Ebola virus (Aleksandrowicz et al. 2011) and influenza virus (Ni et al. 2024) use macropinocytosis to enter cells. In the data from Yang et al. (2018), PPRV enters goat endometrial epithelial cells through a dynein‐mediated endocytosis pathway that requires membrane cholesterol, phosphatidylinositol 3‐kinase, pH changes and dynein participation and does not depend on clathrin. After PPRV infects epithelial cells, the H protein strongly binds to the V‐shaped structure of the nectin‐4 receptor on the host epithelial cell membrane and becomes anchored on the cell surface or in microbubbles (Birch et al. 2013). Microbubbles are raft‐like structures found on the cell surface, typically measuring 50–100 nm in size. They are primarily composed of lipids, including cholesterol, sphingolipids, glycosphingolipids and caveolins (Guo et al. 2023). The caveolae are then internalised to form vesicles. A spiral ring is assembled around the vesicles by dynein, causing their contraction and distortion. This leads to the detachment of the vesicles from the cell membrane, forming early endosomes in the cytoplasm. The internalisation of PPRV during this period was successful due to the acidic pH environment, as noted in reference (Figure 2) (Jimah and Hinshaw 2019). Furthermore, Yang's et al. (2018) data emphasises the crucial role of phosphatidylinositol 3‐kinase in PPRV's invasion strategy. It is worth noting, however, that PI3K is typically linked to the endocytosis pathway of macropinocytosis. In lymphoid tissues, PPRV primarily adheres to lymphocytes through the SLAM receptor. Nevertheless, the molecular mechanism behind PPRV's invasion of lymphocytes remains unreported (Chekwube Chukwudi et al. 2021). The existence of an endocytosis pathway and the involvement of macropinocytosis are unclear. In addition, the process by which the early endosome of PPRV matures into a late endosome in the cell and is transported to the replication site requires further research. It is also necessary to identify any other proteins involved during this period.

FIGURE 2.

Schematic model of PPRV cell adhesion and transmembrane internalisation. (1) PPRV is present on the surface of epithelial cells. (2) PPRV interacts with adhesion receptors on the surface of epithelial cells and mediates the adhesion of the virus. Currently, two adhesion factors of PPRV have been identified: Nectin‐4 and SLAM. (3) The internalisation of PPRV into epithelial cells is primarily dependent on the conformational change of the F protein, which enables membrane fusion. The PPRV envelope fuses with the epithelial cell membrane, allowing the virus to invade the cells. This process also involves the binding of the H protein and F protein to activate the membrane fusion ability of the latter. (4) PPRV RNA replicates and proliferates within cells. (3) PPRV RNA replicates and proliferates within cells. PPRV binds to the microbubble structure on the cell membrane surface. The virus enters cells through a dynein‐mediated endocytosis pathway, which involves membrane cholesterol, phosphatidylinositol 3‐kinase, pH changes and dynein. (4) The caveolae present on the cell membrane surface are internalised to form vesicles. Dynein mediates this process by assembling into a spiral ring and gathering around the ring structure of the internalised vesicles. (5) The sliding of dynein causes the spiral ring to contract and distort, while the vesicles move inside the cell membrane and form early endosomes in the cytoplasm. PPRV invades the cytoplasm.

5. Conclusions

The structure of PPRV is crucial to its life cycle, with both structural and non‐structural proteins playing a role in regulating its activities. Early events in the virus's life cycle include cell adhesion and internalisation across the cell membrane, which are necessary for viral replication and proliferation. This paper focuses on the structure and function of PPRV proteins. It includes an analysis of the N, P, M, F, H and L (Figure 1 and Table 1). The text describes the possible mechanism of PPRV F protein invading cells by directly fusing with the cell membrane. It also discusses the nectin‐4 receptor protein and SLAM receptor protein associated with PPRV adhesion, as well as the identified and potential cross‐membrane internalisation strategies. Following infection of epithelial cells, the H protein binds strongly to the V‐shaped structure of the nectin‐4 receptor on the host epithelial cell membrane, anchoring it on the cell surface or in microbubbles. Under the action of actin and caveolin, the cave is invaginated, and dynein cleavage is required. The internalised vesicles formed by PPRV become early endosomes in the cytoplasm, allowing for complete invasion of the host cell. It is important to note that PPRV has various methods of cell invasion, each with unique characteristics. Complete elimination of PPRV cannot be achieved by blocking any single method. The functions of PPRV structural and non‐structural proteins are closely related to PPRV replication and immune evasion. Therefore, understanding their functional roles is necessary for studying the molecular mechanisms of viral replication, virulence and immune system evasion. Constructing a network of interactions between viral and host cell proteins can provide a comprehensive understanding of the mechanism of PPRV protein in viral replication and infection. This can serve as a reference for developing new PPRV diagnostic methods and effective genetic engineering vaccines.

TABLE 1.

Protein function of PPRV.

Viral proteins	Protein function
Nuclear capsid protein (N)	1. High immunogenicity
	2. High conservation
	3. Genome RNA shelling
	4. Participate in the assembly of virus particles
	5. Promoting host inflammatory response
	6. Evading host innate immune response
	7. Promoting virus replication
	8. Basic components of RNA‐dependent RNA polymerase (RdRp)
Phosphoprotein (P (V/C))	1. Basic components of RNA‐dependent RNA polymerase (RdRp)
	2. Phosphorylation
	3. Promoting virus replication
	4. Evading host innate immune response
Envelope matrix protein (M)	1. Participate in the assembly of virus particles
	2. Promoting virus replication
	3. Immunogenicity
Fusion protein (F)	1. Participate in the assembly of virus particles
	2. Involved in the fusion of viral cell membranes
	3. Pathogenicity
	4. Immunogenicity
Haemagglutinin protein (H)	1. Participate in the assembly of virus particles
	2. Involved in the fusion of viral cell membranes
	3. Pathogenicity
	4. Immunogenicity
	5. Promoting virus replication
Large protein (L)	1. Basic components of RNA‐dependent RNA polymerase (RdRp)
	2. Participate in viral post‐transcriptional modification

Currently, PPR has been effectively controlled through the use of live attenuated PPRV vaccines and effective virus monitoring mechanisms. However, the epidemic situation in some developing countries remains complex and severe (Wong and Rosenberg 2021). The use of live attenuated PPRV vaccine has led to the emergence of new problems, such as mixed infections of multiple pathogens in farms and persistent PPRV infections. In addition, vaccine‐driven or antigenic drift has resulted in the emergence of new PPRV strains with new genetic compositions and stronger adaptability, leading to a decrease in the current vaccine titer. The presence of exosomes facilitates PPRV's evasion of the host's natural immune response and promotes viral transmission (Mühlebach et al. 2011). These factors have made complete eradication of PPRV more difficult (Kumar et al. 2014). Agrawal et al. (2023) found that the synthetic HN homologous peptide competes with the natural HN protein of the PPR virus and binds to the SLAM receptor, thereby blocking the cell adhesion of PPRV. This study demonstrates the potential of synthetic HN homologous peptides for PPR prevention and control. Therefore, it is significant to investigate the cell invasion mechanism of PPRV and develop new antiviral blockers to reduce PPR infection at the initial stage for prevention and control purposes.

Author Contributions

Hong Zou: writing–review and editing, writing–original draft. Zheng Niu: writing–review and editing, writing–original draft. Peng Cheng: methodology. Chunxia Wu: methodology. Wenjie Li: methodology. Gan Luo: writing–review and editing, writing–original draft. Shilei Huang: writing–original draft, writing–review and editing, funding acquisition.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

All relevant data are within the paper.