Assembly of virus particles is an essential step for a productive viral replication cycle. The intracellular sites of virus assembly vary among different viruses (35,43). Assembly of enveloped viruses requires complex interactions between the lipid envelope, envelope proteins, and internal viral components. Budding of enveloped viruses, through cellular membranes, involves the process of envelopment of the viral nucleocapsid. The interaction of the viral nucleocapsid with envelope proteins is believed to drive the incorporation of the nucleocapsid in enveloped viruses (41). Indeed, interactions between viral envelope protein and nucleocapsid protein are required for the formation of alphaviruses (25,45). In other enveloped viruses, such as rhabdovirus and paramyxovirus, a matrix protein mediates the interaction between the viral envelope, envelope proteins, and the nucleocapsid (6, 36). Studies of viral assembly mechanisms not only provide an excellent model system for understanding the macromolecular interactions in cells, but also offer valuable information for the development of preventive and therapeutic agents against viral infection.Coronavirus is an enveloped virus containing a large, positive-stranded RNA genome. The prototypic coronavirus, mouse hepatitis virus (MHV), contains three envelope proteins, M, E, and S. S protein forms 180/90-kDa peplomers that bind to receptors (9) on coronavirus-susceptible cells and induce cell fusion (7, 12). M protein, the most abundant glycoprotein in the virus particle and in infected cells, is characterized as having three domains: a short N terminal ectodomain, a triple-spanning transmembrane domain, and a C-terminal endodomain (1). E protein is present only in minute amounts in infected cells and in the virus envelope (13,23,37,47,51), yet it is an essential protein for coronavirus envelope formation; coronavirus-like particles (VLPs) are assembled and released from cells that express both E and M proteins (4, 49). Furthermore, expression of E protein alone results in the production of membrane vesicles, which contain E protein (27). E protein also affects coronavirus morphogenesis, as it was shown that MHV mutants, encoding mutated E protein, are morphologically aberrant compared to wild-type MHV (10). Viral genomic RNA and N protein are found inside the viral envelope (44). A generally accepted model of coronavirus structure proposes that viral genomic RNA and N protein form a helical nucleocapsid (44).In coronavirus-infected cells, genomic-size RNA, mRNA 1, and six to eight species of subgenomic mRNAs are produced. These virus-specific mRNAs comprise a nested set with common 3Ј cotermini (20,22) and a common leader sequence of approximately 60 to 80 nucleotides at the 5Ј end (19,42). Each of the coronavirus-specific proteins is translated from only one of these mRNAs. Among the mRNAs, only mRNA 1 is efficiently packaged into coronavirus particles, while subgenomic mRNAs either are not incorporated into virus particles (21,30,32) or are incorporated at a low effic...
The viral replication cycle concludes with the assembly of viral components to form progeny virions. For influenza A viruses, the matrix M1 protein and two membrane integral glycoproteins, hemagglutinin and neuraminidase, function cooperatively in this process. Here, we asked whether another membrane protein, the M2 protein, plays a role in virus assembly. The M2 protein, comprising 97 amino acids, possesses the longest cytoplasmic tail (54 residues) of the three transmembrane proteins of influenza A viruses. We therefore generated a series of deletion mutants of the M2 cytoplasmic tail by reverse genetics. We found that mutants in which more than 22 amino acids were deleted from the carboxyl terminus of the M2 tail were viable but grew less efficiently than did the wild-type virus. An analysis of the virions suggested that viruses with M2 tail deletions of more than 22 carboxy-terminal residues apparently contained less viral ribonucleoprotein complex than did the wild-type virus. These M2 tail mutants also differ from the wild-type virus in their morphology: while the wild-type virus is spherical, some of the mutants were filamentous. Alanine-scanning experiments further indicated that amino acids at positions 74 to 79 of the M2 tail play a role in virion morphogenesis and affect viral infectivity. We conclude that the M2 cytoplasmic domain of influenza A viruses plays an important role in viral assembly and morphogenesis.
For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into virus particles. In this report we show that expressed coronavirus envelope protein M specifically interacted with coexpressed noncoronavirus RNA transcripts containing the short viral packaging signal in the absence of coronavirus N protein. Furthermore, this M protein-packaging signal interaction led to specific packaging of the packaging signal-containing RNA transcripts into coronavirus-like particles in the absence of N protein. These findings not only highlight a novel RNA packaging mechanism for an enveloped virus, where the specific RNA packaging can occur without the core or N protein, but also point to a new, biologically important general model of precise and selective interaction between transmembrane proteins and specific RNA elements.The process of packaging the viral genome is a critical step in the assembly of infectious viruses. For any of the enveloped RNA viruses studied to date, the association of an intracellular form of viral genomic RNA with the nucleocapsid protein is the first step in the process of selective genome packaging into virus particles. A specific RNA element(s), usually referred to as a packaging signal (PS) or an encapsidation signal, which is present in intracellular viral genomic RNA, determines the selective and specific binding of viral nucleocapsid protein to the viral genomic RNA. Subsequently, the viral ribonucleoprotein (RNP) complex containing viral RNA and the nucleocapsid protein binds to a viral envelope protein(s) at the virus budding site, which leads to the budding of virus particles containing the viral RNP complex. In some enveloped viruses, an interaction between the viral RNP complex and envelope proteins drives the budding of virus particles (21,31,34,41), while in other enveloped viruses, the viral RNP complex is dispensable for viral envelope formation and production of virus particles. A typical example of the latter phenomenon is observed in coronavirus envelope formation; coronavirus-like particles (VLPs) that are morphologically similar to infectious virus particles are produced in the absence of viral RNP complex (37).The murine coronavirus mouse hepatitis virus (MHV) contains three envelope proteins, S, M, and E, and a helical nucleocapsid consisting of N protein and a large single-stranded, positive-stranded RNA genome (19, 33). S protein is dispensable for viral nucleocapsid packaging and viral assembly (13,15,30). M protein and E protein are essential for viral envelope formation and release of virus particles; VLPs are released from cells that express both M...
Coronavirus E protein is a small viral envelope protein that plays an essential role in coronavirus assembly; coexpression of coronavirus M and E proteins results in the production of virus-like particles. The present study demonstrated that mouse hepatitis virus (MHV) E protein was released as an integral membrane protein in lipid vesicles from E-protein-expressing mammalian cells, in the absence of other MHV proteins. Furthermore, our data indicated that the E-protein-containing vesicles, which had a slightly lighter buoyant density than that of MHV, were released from MHV-infected cells. These data implied that E protein alone can drive the production and release of coronavirus envelope in the absence of M protein.
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