The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy, tomography and statistical analysis. We present evidence that suggests M can adopt two conformations and that membrane curvature is regulated by one M conformer. Elongated M protein is associated with rigidity, clusters of spikes and a relatively narrow range of membrane curvature. In contrast, compact M protein is associated with flexibility and low spike density. Analysis of several types of virus-like particles and virions revealed that S protein, N protein and genomic RNA each help to regulate virion size and variation, presumably through interactions with M. These findings provide insight into how M protein functions to promote virus assembly.

No abstract

Coronaviruses (CoV), like other positive-stranded RNA viruses, redirect and rearrange host cell membranes for use as part of the viral genome replication and transcription machinery. Specifically, coronaviruses induce the formation of double-membrane vesicles in infected cells. Although these double-membrane vesicles have been well characterized, the mechanism behind their formation remains unclear, including which viral proteins are responsible. Here, we use transfection of plasmid constructs encoding full-length versions of the three transmembrane-containing nonstructural proteins (nsps) of the severe acute respiratory syndrome (SARS) coronavirus to examine the ability of each to induce double-membrane vesicles in tissue culture. nsp3 has membrane disordering and proliferation ability, both in its full-length form and in a C-terminal-truncated form. nsp3 and nsp4 working together have the ability to pair membranes. nsp6 has membrane proliferation ability as well, inducing perinuclear vesicles localized around the microtubule organizing center. Together, nsp3, nsp4, and nsp6 have the ability to induce double-membrane vesicles that are similar to those observed in SARS coronavirus-infected cells. This activity appears to require the full-length form of nsp3 for action, as double-membrane vesicles were not seen in cells coexpressing the C-terminal truncation nsp3 with nsp4 and nsp6.

Coronavirus particles are enveloped and pleomorphic and are thus refractory to crystallization and symmetry-assisted reconstruction. A novel methodology of single-particle image analysis was applied to selected virus features to obtain a detailed model of the oligomeric state and spatial relationships among viral structural proteins. Two-dimensional images of the S, M, and N structural proteins of severe acute respiratory syndrome coronavirus and two other coronaviruses were refined to a resolution of ϳ4 nm. Proteins near the viral membrane were arranged in overlapping lattices surrounding a disordered core. Trimeric glycoprotein spikes were in register with four underlying ribonucleoprotein densities. However, the spikes were dispensable for ribonucleoprotein lattice formation. The ribonucleoprotein particles displayed coiled shapes when released from the viral membrane. Our results contribute to the understanding of the assembly pathway used by coronaviruses and other pleomorphic viruses and provide the first detailed view of coronavirus ultrastructure.

Infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in a demyelinating encephalomyelitis characterized by mononuclear cell infiltration and white matter destruction similar to the pathology of the human demyelinating disease multiple sclerosis. The contributions of CD4 ؉ and CD8 ؉ T cells in the pathogenesis of the disease were investigated. Significantly less severe inflammation and demyelination were observed in CD4؊/؊ mice than in CD8 ؊/؊ and C57BL/6 mice (P < 0.002 and P < 0.001, respectively). Immunophenotyping of central nervous system (CNS) infiltrates revealed that CD4؊/؊ mice had a significant reduction in numbers of activated macrophages/microglial cells in the brain compared to the numbers in CD8 ؊/؊ and C57BL/6 mice, indicating a role for these cells in myelin destruction. Furthermore, CD4 ؊/؊ mice displayed lower levels of RANTES (a C-C chemokine) mRNA transcripts and protein, suggesting a role for this molecule in the pathogenesis of MHV-induced neurologic disease. Administration of RANTES antisera to MHV-infected C57BL/6 mice resulted in a significant reduction in macrophage infiltration and demyelination (P < 0.001) compared to those in control mice. These data indicate that CD4 ؉ T cells have a pivotal role in accelerating CNS inflammation and demyelination within infected mice, possibly by regulating RANTES expression, which in turn coordinates the trafficking of macrophages into the CNS, leading to myelin destruction.Demyelination is a complex neuropathological process in which the myelin sheath that insulates and protects axons is damaged or destroyed. Several animal models of demyelination have been developed that have provided valuable contributions to the understanding of the immunopathological events that may drive human demyelinating diseases such as multiple sclerosis (MS) (22,31). Among these is the neurotropic mouse hepatitis virus (MHV) model of virus-induced demyelination (12,18). MHV is a positive-strand RNA virus that causes a variety of clinical diseases in susceptible strains of mice (23). Neurovirulent strains of MHV cause an acute encephalomyelitis that may ultimately progress to demyelinating disease characterized clinically by abnormal gait and hind-limb paralysis. Histologically, affected animals exhibit mononuclear cell infiltration and myelin destruction. Early studies suggested that the demyelination observed in MHV-infected mice was the result of virus-induced damage or destruction of oligodendrocytes (9, 36). However, more recent reports have indicated that MHV-induced demyelination is more complex and may also involve immunopathologic responses against viral antigens expressed in infected tissues (5, 35).As T cells are considered central to the development of demyelinating lesions in animal models of demyelination as well as MS, it is imperative to better understand the mechanisms by which these cells exert their pathological effect (24, 25). We sought to evaluate the contributions of CD4 ϩ and CD8 ϩ T cells in MHV-induced central nervous system (CNS) d...