A short N-terminal glycosylated ectodomain—exposed either to the exterior of the virus or to the lumen of intracellular organelles—spans the first 1–19 amino acid residues at the N-terminus. In some α-CoVs, the M protein contains an additional hydrophobic segment that functions as a signal peptide. Research on Transmissible Gastroenteritis Virus (TGEV) has shown that this signal peptide fragment is located within the first 50 amino acids at the N-terminus of the M protein (Escors et al., 2001a). In PDCoV, hydrophobicity analysis reveals that the N-terminal region of the M protein is highly hydrophilic (Wu et al., 2023).

Glycosylation is the process by which sugar chains are covalently attached to specific amino acid residues of proteins via glycosyltransferases, and it is an important post-translational modification. The M protein is always glycosylated at its N-terminal ectodomain (Fung and Liu, 2018). According to the site of modification, it can be classified into N-glycosylation and O-glycosylation. Many experiments have shown that almost all CoV M proteins are exclusively N-linked glycoproteins, especially in α-CoVs and γ-CoVs. After exiting the ER, the M protein reaches the Golgi apparatus, where its N-glycans are further modified (Nal et al., 2005; Perrier et al., 2019). The N-glycosylation of the M protein is not mediated by the virus itself but relies on the host cell’s ER and Golgi apparatus glycosylation modification enzymatic systems operating through the secretory pathway. When M protein mRNA is translated on ribosomes attached to the ER, its N-terminal signal peptide directs the nascent polypeptide into the ER lumen. If the amino acid sequence contains the N-glycosylation consensus sequence (Asn-X-Ser/Thr, where X is any amino acid except proline), the oligosaccharide transferase complex catalyzes the transfer of the core oligosaccharide to the target asparagine residue, initiating glycosylation (Aebi, 2013; Shrimal et al., 2015). Subsequently, glucosidases I and II catalyze the initial processing and folding of the glycoprotein. The glycosylated M protein is then transported to the Golgi apparatus via vesicles, where a series of glycosyltransferases further modify the oligosaccharide chains, ultimately yielding mature, structurally diverse N-linked glycans.

Studies on the N-glycosylation of MERS-CoV and SARS-CoV-2 have revealed that their M proteins are modified with polylactosamine chains, and this modification relies on acidic residues located near the first transmembrane domain (Juckel et al., 2023). During the glycosylation of the M protein, certain acidic residues in its N-terminal region are essential for this modification, in addition to the intrinsic N-glycosylation site. For instance, researchers have confirmed that residues E11, E12, and E18 in the SARS-CoV-2 M protein, a residues E9 and D18 in the MERS-CoV M protein are crucial for the proper glycosylation of these proteins (Juckel et al., 2023). In contrast, the M proteins of certain lineage A β-CoVs such as MHV, BCoV, and HCoV-OC43, are modified by O-linked glycosylation (Liang et al., 2019; Yamada et al., 2000). Studies on MHV have shown that its M protein undergoes O-glycosylation in the Golgi apparatus, with the glycosylation sites located on Ser/Thr residues in the extracellular N-terminal domain (de Haan et al., 2000). Since its identification, O-linked glycosylation has been served as a valuable indicator for investigating the maturation, membrane insertion, and intracellular trafficking of the M protein in these three β-CoVs (Fung and Liu, 2018).

Glycosylation plays a crucial role in maintaining the function of the M protein during viral assembly, release, and interaction membrane interaction. Through deletion mutations of glycosylation sites, it has been found that the N-glycosylation of the SARS-CoV M protein is essential for viral assembly and infectivity. Deletion of this site impairs progeny viral particle assembly (Voss et al., 2006). Deletion mutations of the N3 and N6 glycosylation sites in IBV significantly affect viral virulence (Liang et al., 2019). In addition, the M protein influences viral particle assembly. Substituting O-glycosylation sites in MHV with N-glycosylation sites reduces the efficiency of viral particle recombination (de Haan et al., 2003). Currently, one to four potential N-glycosylation sites have been identified in the M proteins of various CoVs. Generally, most CoV M proteins contain a single N-glycosylation site, and some CoVs have been confirmed to possess multiple glycosylation sites on their M protein (Table 1).

The transmembrane domain consists of three transmembrane (TM) helices that anchor the protein in the viral envelope and are labeled TM1, TM2, and TM3 from the N-terminal (Ujike and Taguchi, 2015). The three transmembrane domains of the CoV M proteins are critically important for virus to target the plasma membrane. Moreover, the 50 amino acid residues at the amino terminus containing the TM1 domain, are crucial for achieving effective M–M protein interaction (Tseng et al., 2010). The plasma membrane trafficking signal of the TM3 domain of the SARS-CoV M protein contains the highly conserved residues phenylalanine 95 (F95) and serine 110 (S110), which are essential for viral assembly.

There is a larger C-terminal intracellular structural domain in the M protein, featuring a prominent cytoplasmic tail approximately 6–8 nm in length, which has been identified as the major functional region for interactions between host cell components and other viral proteins (Hsieh et al., 2008). The C-terminal domain, which follows the TM3 region, is organized into a tightly membrane-associated amphipathic structural domain and a distal short hydrophobic structural domain (Ujike and Taguchi, 2015), resulting in a topology comprising both the N-terminal ectodomain and C-terminal cytoplasmic domain (Voss et al., 2009). When investigating the intracellular trafficking of the MERS-CoV M protein, researchers have identified a region in the cytoplasmic tail of its C-terminal domain as the determinant for localization to the trans-Golgi network (TGN). This localization process is mediated by a specific set of four residues (Lys199, Gly201, Tyr203, and Arg204) within the MERS-CoV M protein (Perrier et al., 2019). Furthermore, the presence of the C-terminal domain of MERS-CoV M protein is important for inducing its specific localization.

The M protein in the virion constitutes the majority of the viral envelope, and the overall scaffold of the envelope is formed by M–M interactions (Arndt et al., 2010; Neuman et al., 2011; Rodríguez-Enríquez et al., 2022; Figures 1d,e). Research indicates that the M protein, which exists as a dimer, adopts two distinct conformations. This structural flexibility enables it to facilitate membrane bending and bind to the nucleocapsid (Rodríguez-Enríquez et al., 2022). The two conformations of the M protein are termed the long and compact (or short) forms (Figure 3), which influence the curvature of the viral envelope. The long form (M-long) confers rigidity, uniformity, and a narrow curvature to the viral envelope, whereas the short form (M-short) plays a contrasting role by providing greater flexibility and reducing spike density (Neuman et al., 2011). Both the long and short forms of the M protein are critical for viral assembly. The M protein forms dimers through interactions with corresponding regions of its structural domains, and in this binding process, specific sites in the three-transmembrane domain play a key role. These dimerizations and subsequent polymerizations are conserved processes. The dimerization of the M protein is a cooperative process involving its transmembrane helices and a C-terminal β-sandwich domain (BD) oriented toward the interior. The dimerization gives rise to a dome-like structure that covers the inner surface of the transmembrane region via the internal sheets of the BD (Zhang Z. et al., 2022). The M protein is structurally similar to the ion channel protein ORF3a, but it does not function as an ion channel by itself. In the two conformations of the M protein dimer (long form and short form), the internal conformation of each monomer remains essentially unchanged, whereas the relative arrangement between the two BDs undergoes significant changes. During the conversion from the long form to the short form, the two monomers approach each other on the extracellular side and separate on the cytoplasmic side. Concurrently, the dimerization interface between the two BDs also undergoes rearrangement, resulting in an increase in the opening angle of the dome-like BD structure from approximately 100–140 (Zhang Z. et al., 2022). The interconversion between the two conformations is regulated by the hinge region, which may be closely linked to modulation of membrane environment, lipid binding, and membrane curvature during viral assembly.

The lattice-like, densely packed matrix structure of the viral envelope results in the lateral interactions of homotypic M–M dimers, which constitutes the fundamental structural components of the viral structure formed by the M protein (de Haan et al., 2000). In SARS-CoV-2, the dimer interface composes 38 residues: 17 are from one M protein molecule (W55, P59, L62, V66, A69, V70, W75, I82, A85, W92, L93, F96, F100, F103, R107, M109, and F112) and 21 are from the other M protein molecule (P59, L62, V66, A69, V70, Y71, I82, A85, W92, L93, F96, I97, F100, F103, A104, R107, S108, M109, S111, and F112) (Marques-Pereira et al., 2022). Experiments on SARS-CoV show that residues W19, W57, P58, W91, Y94, F95, and C158 are essential for M protein homodimer interaction (Tseng et al., 2013). In this context, some host membrane proteins are excluded from the viral envelope through the interaction network formed by M–M dimers. Certain residues of the M protein (K14, Y39, R42, N43, R44, F45, Y71, R72, W75, S94, R101, R107, W110, S173, and R174) participate in the formation of polar, contacting with membrane lipids (Marques-Pereira et al., 2022). SARS-CoV may rely more on cysteine-mediated disulfide bonds to stabilize the dimer, whereas SARS-CoV-2 depends more on electrostatic interactions (Table 2). Hydrophobic interactions underpin M protein membrane intercalation and dimerization across all CoVs. A more stable M protein dimer may enhance the virus’s environment tolerance, improve assembly efficiency, and promote immune escape, thereby increasing transmissibility and pathogenicity. However, the specific mechanism requires further validation through in vivo experiments.

The reticular matrix serves as a scaffold for the assembly of either the E or the N protein. When interacting with the N protein, the M protein packages ribonucleoproteins (RNPs, composed of the viral RNA genome and the N protein) into virions, and this function primarily relies on the intracellular C-terminal domain of the M protein. Moreover, M proteins interact pairwise to form dimers, constituting the viral envelope scaffold; such specific interactions can also incorporate the S or HE protein into lattice vacancies, positioning these proteins regularly within the lattice (Ujike and Taguchi, 2015). Research indicates that it is the collective interactions among M protein dimers that primarily induce membrane curvature, whereas E protein pentamers affect membrane deformation, prevent curvature, and maintain membrane flatness (Collins et al., 2021; Monje-Galvan and Voth, 2021).

The CoV genomes replicate in the cytoplasm and assemble into viral particles by budding into the smooth membrane of the ERGIC, then traffic to the ER, and are released via exocytosis to infect surrounding cells (Liang et al., 2019; Steiner et al., 2024; Figure 4). The M protein of CoVs is a central player in virion assembly, particularly during replication and transcription, serving as the key organizer that transforms host cell membranes into sites for viral particle production (Bracquemond and Muriaux, 2021; Kuo et al., 2016a; Steiner et al., 2024). The M protein dynamically cycles between the Golgi complex and the ER, and returns to the Golgi from the plasma membrane via the endocytic recycling pathway. The synthesis of the M protein increases during CoV infection (Wang R. et al., 2020). Immunofluorescence and Western blot analyses reveal that M protein expression can be detected as early as post-infection and persists throughout the course of infection until cell death (Wu et al., 2023). CoV assembly is critically dependent on the proper and accurate intracellular transport of the M protein (Desmarets et al., 2023). Through M–M interactions, a scaffold for viral assembly is formed, and host cell membrane proteins are excluded, thereby conferring specificity to the viral envelope (Liang et al., 2019). Heterotypic interactions facilitate the recruitment of other structural proteins and the viral genomic RNA to the assembly site. The M protein not only self-associates but also assists in virion assembly and interacts with other structural proteins (N, E, and S), as well as with accessory proteins (including ORF3), host proteins, and non-structural proteins (Mahtarin et al., 2022; Rodríguez-Enríquez et al., 2022). By recruiting other structural proteins to the budding site, the M protein organizes a specific membrane structural domain for viral assembly (Neuman et al., 2011). Through these interactions, the M protein ensures the efficient assembly and release of new viral particles (Castaño et al., 2021).

The M protein triggers caspase-dependent apoptosis via the mitochondrion-mediated pathway. Specifically, in SARS-CoV, the M protein promotes apoptosis by modulating the release of mitochondrial cytochrome c and affecting the cellular Akt pro-survival signaling pathway (Chan et al., 2007). Akt, also known as protein kinase B (PKB), is a central player in the cellular kinase cascade, regulating diverse cellular functions and influencing the viral life cycle. The PKB/Akt pathway is involved in multiple viral infections, including but not limited to SARS-CoV (Mizutani et al., 2006a; Mizutani et al., 2006b). Caspase-9 is a target of PKB/Akt-mediated phosphorylation, and the activity of caspase-8 is also indirectly regulated by PKB/Akt. Overexpression of the M protein suppresses PKB/Akt phosphorylation, leading to downregulation of pro-survival signaling and ultimately triggering apoptotic cell death. The PKB/Akt signaling pathway plays a pivotal role, as it is critical for maximal viral production and apoptosis regulation. Through the PKB/Akt signaling cascade, the expression of M protein activates caspase-8 and caspase-9 (Tsoi et al., 2014).

Depending on caspase-9, transient overexpression of the SARS-CoV-2 M protein initiates mitochondrial apoptosis by engaging B-cell lymphoma 2 (BCL-2) ovarian killer (BOK). According to the studies on SARS-CoV-2, the M protein has been identified as an inducer of mitochondrial apoptosis in pulmonary epithelial cells (Yang et al., 2022). The M protein triggers caspase-associated cell death, leading to the increased pulmonary barrier permeability (Wang F. et al., 2024; Yang et al., 2022). Studies investigating the interaction between SARS-CoV-2 M protein and human proliferating cell nuclear antigen (PCNA) have shown that the M protein induces cytoplasmic translocation of PCNA from the nucleus. This finding suggests that the M protein may promote DNA damage by involving PCNA (Zambalde et al., 2022).

A study has demonstrated that the M protein of SARS-CoV-2 induces neurodegeneration by disrupting the interaction between the Golgi apparatus and mitochondria (Wang F. et al., 2024). This study also confirms that the M protein interacts with two regulatory factors of the Arf signaling pathway, ArfGEF1 and ArfGAP1, in the Golgi apparatus. Since the PI4KIIIβ/Arf1 pathway is involved in the interaction between the Golgi complex and mitochondria, expression of the M protein may disrupt Golgi structure, thereby leading to structural abnormalities and functional impairment of mitochondria. The M protein promotes retrograde transport from the Golgi apparatus to the ER, inducing mitochondrial fragmentation and functional impairment, which results in decreased ATP production, excess reactive oxygen species (ROS), and ultimately cell death. Additionally, the autophagy cargo receptor (ACR) SQSTM1 plays a dual role in host-pathogen interactions and is often context-dependent. By phosphorylating SQSTM1 at a special site, the M protein promotes antiviral activity (Li et al., 2024). Another study shows that the M protein can also mediate and promote mitophagy. M protein contains a β_3–5 domain, which plays a significant role in promoting mitochondrial autophagy through PDPK1-mediated phosphorylation of SQSTM1 site. The β_3–5 domain can counteract the mitophagy promoted by the M protein. The M interferes with the function and distribution of the Golgi apparatus, leading to mitochondrial abnormalities and dysfunction, thereby contributing to neurodegeneration.

Genome packaging is a critical process arising from the parasitic status of CoV (Li et al., 2024). During the replication process of the CoV, large amounts of RNA are produced, including positive-strand genomic RNA (gRNA), negative-strand genomic RNA and positive-strand subgenomic RNA (Narayanan et al., 2000). Both the M and N proteins are known viral molecular partners that mediate packaging signal (PS) recognition. To ensure selective packaging of gRNA into viral particles, the CoV distinguishes its full-length gRNA from other cellular RNAs through genome-specific PS and specifically recruits M and N proteins (Bai et al., 2021). There are three types of models describing how the PS, N, and M proteins interact with each other to help CoVs achieve packaging selectivity. The N protein of the CoV contains two highly conserved, independently folded domains (the N-terminal domain and the C-terminal domain), and a third, predominantly acidic C-terminal region termed the N3 domain (Zhao et al., 2024). The genetic information model integrates the known functions of the N protein domain. In this model, the CTD serves as an RNA-binding module, whereas the N3 domain is hypothesized to be the exclusive site for interaction with the M protein (Chang et al., 2014; Kuo and Masters, 2013; Kuo et al., 2014; Kuo et al., 2016b). The second model explains how the M protein primarily recognizes PS. Evidence indicates that the M protein is the primary and perhaps the exclusive PS recognition element (Masters, 2019). The internal domain of the M protein is oligomerized at the assembly site of the cell membrane, enabling it to mediate specific PS recognition. This implies that each assembled virion contains an individual and specific contact site between the M protein and the gRNA PS (Neuman et al., 2011). Therefore, the M protein drives their interaction and condensation by initiating the nucleation of M-N and N-N protein complexes. The presence of C-tail is vital for the effective encapsidation of gRNA by the N protein, and this process is regulated by M protein (Han et al., 2024).

Studies on MHV have demonstrated that the M protein modulates PS recognition. Evidence indicates that the specific interaction between the M protein and the intracellular defective interfering ribonucleoprotein (DI RNP) complex involves the PS (Narayanan and Makino, 2001). The M protein mediates efficient packaging of DI RNP complexes into MHV particles by specifically recognizing and binding to those complexes that carry packaging signals. Researchers have concluded that the PS is responsible for the specific interaction between the M protein and particular intracellular RNP complexes, thereby drives the efficient and selective packaging of MHV RNAs containing these signals into viral particles.

The M protein plays a significant role in the viral life cycle and also modulates host cells activity. Its functions are closely related to its structural features. The major functions of M protein are elaborated in detail in Table 4.

Due to its crucial role in the viral life cycle, the M protein of CoVs has significant potential for drug development. The M protein plays a key role in the assembly and budding of the virus, and its structure and function are essential for viral maturation and release. Therefore, the M protein can serve as a potential target for the research and development of antiviral drugs, facilitating the creation of specific therapeutic drugs against CoVs and providing robust strategies for epidemic prevention and control. For example, as mentioned above, the PDPK1-targeted peptide effectively eliminates CoV infection by redirecting SQSTM1 from the viral M protein to mitochondria, thereby restoring viral phagocytosis and simultaneously inhibiting mitochondrial autophagy (Li et al., 2024). HSC70 has been identified as a host factor required for the internalization of TGEV, dependent on its interaction with the viral M protein. Consequently, disrupting the M-HSC70 interaction presents a viable strategy for designing antiviral therapies against TGEV (Ji et al., 2023). Through the study of the M protein, researchers have identified a novel drug target within the CoVs replication cycle, along with a potent small-molecule inhibitor. It is essential to consider the membrane environment in drug design when targeting the M protein as a viral drug target. Research has shown that the ₁₉₉KxGxYR₂₀₄ motif in the C-terminal tail of the MERS-CoV M protein may represent such a CoV-specific target (Desmarets et al., 2023). Recently, a study has found that a structural expansion below the transmembrane domain and above the β-sheet intercalation domain within the M protein dimer is observed in all M proteins. This site is located in the β-sheet sandwich domain near the C-terminus and may serve as a potential drug-binding site (Yegnaswamy et al., 2025). Apart from the M protein, certain host cell components can also serve as targets for antiviral drug development. Research has found that PEDV, SARS-CoV-2, IBV, and PDCoV can all interact with Aurora A (AurA) and histone deacetylase 6 (HDAC6) in host cells via their M proteins, thereby inducing ciliary disassembly during the early infection (Zhuang et al., 2025). This finding suggests that AurA and HDAC6 may serve as broad-spectrum therapeutic targets for multiple CoV infections.

At present, numerous studies have identified a variety of compounds with potential activity against HCoVs (Table 5). CIM-834, an M-targeting molecule, exerts its late-stage mechanism of action by impeding the conformations interconversion from the M-short form to the M-long form, thereby inhibiting viral particle assembly (Laporte et al., 2025). Meanwhile, JNJ-9676, a small-molecule inhibitor targeting the M protein, exerts its antiviral effect by inducing the formation of a binding pocket and stabilizing a novel conformational state of the M protein (Van Damme et al., 2025). It exhibits potent antiviral activity in vitro against zoonotic strains of SARS-CoV and SARS-CoV-2 originating from bats and pangolins. Caffeic acid and ferulic acid are considered to have the potential to inhibit the SARS-CoV-2 M protein, which provides new insights for the development of anti-COVID-19 drugs (Bhowmik et al., 2020). Both compounds exert their effects by binding to Lys50 of the SARS-CoV-2 M protein. Similarly, colchicine, remdesivir, bafilomycin A1, and temozolomide can also bind to the M protein, thereby exerting an inhibitory effect on it (Peele et al., 2021). Additionally, ARF1 small-molecule inhibitors brefeldin A (BFA) and golgicide A (GCA), as well as the synthesized mimic peptide PEP17, can disrupt the localization of ARF1 and M protein on the ERGIC, thereby weakening virion assembly and inhibiting SARS-CoV-2 replication (Zhang et al., 2025). However, the effectiveness of these drugs still needs to be further verified through research.

As one of the viral structural proteins, the biochemical and immunological properties of the M protein indicate that it is highly suitable as a specific antigen for detecting CoV infection (Wu et al., 2023). The M protein can be used to develop immunoassay methods, such as enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assays. In CCoV detection, ELISA based on recombinant M protein is regarded as an effective method for detecting CCoV-specific antibodies in canine serum (Elia et al., 2003). Similarly, an ELISA based on the M protein is highly significant for detecting PEDV and distinguishing its infection from other related porcine viral infections (Ren et al., 2011). In the research of TGEV, the M protein has been expressed in Escherichia coli as a GST fusion protein. Using the phage clone phTGEV-M7 as the antigen, a phage-based immunosorbent assay (PHAGE-ELISA) was established, which not only distinguishes TGEV from other CoVs., but also exhibits higher sensitivity than the conventional antibody-based ELISA (Zou et al., 2013). The M gene sequences of different PDCoV strains share 99% nucleotide identity, and are therefore commonly used as target genes for establishing nucleic acid-based diagnostic assays (Zhang et al., 2016). Monoclonal antibodies (mAbs) are laboratory-produced molecules that target specific viral antigens on the surface of pathogen. They are valuable and indispensable tools for investigating the antigenic properties of the M protein (Dong et al., 2016). Moreover, the establishment of a monoclonal antibody-producing hybridoma cell line specific for the CoV. M protein has laid a solid foundation for CoV identification and the development of related diagnostic methods. When developing molecular diagnostic methods (such as RT-PCR) for CoVs, researchers typically design specific primers and probes targeting the hydrophilic N-terminal coding region of the M gene (Xin et al., 2024). Compared with the high variability of the S protein, using the highly conserved M protein as a diagnostic antigen can theoretically improve the stability and accuracy of detection. However, this approach remains in the early stage of research and is far from clinical application.

The M protein is not only the most abundant viral structural protein but also exhibits high conservation of its amino acid sequence. Specific antibodies recognize its extracellular domain, and this high degree of conservative makes it a promising candidate for a broad-spectrum vaccine. Vaccines based on the M protein therefore hold significant advantages. First, its high degree of conservation may enable cross-protective immunity against diverse CoVs, potentially even across species barriers. Second, in CoV, membrane fusion is mediated by the S protein, whereas the M protein does not participate in this process (Wang Q. et al., 2020). Vaccines developed based on the S protein carry certain risks (Amanat and Krammer, 2020), whereas the distinction between the S and M proteins suggests that vaccines targeting the M protein may be associated with a lower risk of vaccine-associated antibody-dependent enhancement (ADE). Additionally, the M protein can simultaneously stimulate antibody production and a robust T-cell immune response in the host, which may contribute to a more balanced and potentially long-lasting immunity. The antibody response to the extracellular domain of M protein likely confers broad protective benefits against diverse SARS-CoV-2 variants in the population, exerting this effect through cross-protection both in vitro and in vivo (Tang et al., 2024). This finding highlights the value of the M protein as a promising and important antigenic target for designing effective CoV vaccines. The majority of recent vaccines are based on the S protein as the immunogen to stimulate protective immunity against SARS-CoV-2 in humans (Corbett et al., 2020; Liu et al., 2021). In individuals who have recovered from COVID-19, the proportion of CD8 + T cells specific for the M protein exceeds that specific for the S protein (Peng et al., 2020). This finding highlights the need to include M protein epitopes in future vaccine designs (Mattoo and Myoung, 2022). Recently, researchers have identified a cluster of polyfunctional, CD4-restricted T-cell epitopes in a highly conserved region of the SARS-CoV-2 M protein. The epitopes that elicit polyfunctional T-cell responses are promising targets for developing effective vaccines and T-cell-based therapies, as they are designed to induce broad, protective immunity (Grifoni et al., 2020; Keller et al., 2020; Khadri et al., 2024). Currently, in clinical practice, there is no vaccine that uses the M protein as the primary immunogen. However, in models such as that of PDCoV, multi-antigen vaccines based on VLPs (containing S, M, and E proteins) have demonstrated superior efficacy (Liu et al., 2023a). The VLPs induce high levels of serum-specific IgG and virus-neutralizing (VN) antibodies in mice. Furthermore, incorporating both the N and M proteins into the vaccine can elicit a stronger cellular immune response, promoting CD8 + T-cell expansion and IFN-γ production (Yu et al., 2024). Compared with vaccines that express only the S protein, the multi-antigen vaccine can elicit a stronger and broader immune response in the host.

The M protein is indispensable for the process of virus assembly and release. Studying the structure, function, and interactions of the M protein with other viral or host cell proteins provides insights into the fundamental biological processes of CoVs, including infection pathogenesis, the viral replication cycle, and the molecular mechanisms underlying essential steps in virus assembly. Furthermore, the high-level expression of the M protein in infected cells makes it an ideal model system for studying glycosylation and ER-to-Golgi vesicular trafficking (Fung and Liu, 2018).