Structural Analysis of Respiratory Syncytial Virus Reveals the Position of M2-1 between the Matrix Protein and the Ribonucleoprotein Complex

Kiss, Gergely; Holl, Jens; Williams, Grant M.; Alonas, Eric; Vanover, Daryll; Lifland, Aaron W.; Gudheti, Manasa V.; Guerrero-Ferreira, Ricardo C.; Nair, Vinod; Yi, Hong; Graham, Barney S.; Santangelo, Philip J.; Wright, Elizabeth

doi:10.1128/jvi.00256-14

Cited by 116 publications

(145 citation statements)

References 82 publications

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“…Our two crystal structures show that the dimerization interface itself also shows plasticity. The slightly different opening angle of the dimers, if borne out in vivo, would result in different degrees of curvature in the assembled virus and help explain the structural heterogeneity observed in RSV virions (14,18). Atomic-resolution structural information is needed to propose a model of the fully assembled virion.…”

Section: Discussionmentioning

confidence: 99%

“…The minimal RSV protein requirement for filament formation and budding of virus-like particles (VLPs) is F, N, P, and M (11,12). M, a key structural protein, directs assembly and budding on the plasma membrane, presumably by interacting with the cytoplasmic tails of the glycoproteins and with the RNP complex in the cytoplasm (7,(13)(14)(15). At the start of RSV assembly, M localizes into viral IBs (5,16).…”

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confidence: 99%

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Dimerization of Matrix Protein Is Required for Budding of Respiratory Syncytial Virus

Förster

Maertens

Farrell

et al. 2015

J Virol

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Respiratory syncytial virus (RSV) infects epithelial cells of the respiratory tract and is a major cause of bronchiolitis and pneumonia in children and the elderly. The virus assembles and buds through the plasma membrane, forming elongated membrane filaments, but details of how this happens remain obscure. Oligomerization of the matrix protein (M) is a key step in the process of assembly and infectious virus production. In addition, it was suggested to affect the conformation of the fusion protein, the major current target for RSV antivirals, in the mature virus. The structure and assembly of M are thus key parameters in the RSV antiviral development strategy. The structure of RSV M was previously published as a monomer. Other paramyxovirus M proteins have been shown to dimerize, and biochemical data suggest that RSV M also dimerizes. Here, using size exclusion chromatography-multiangle laser light scattering, we show that the protein is dimeric in solution. We also crystallized M in two crystal forms and show that it assembles into equivalent dimers in both lattices. Dimerization interface mutations destabilize the M dimer in vitro. To assess the biological relevance of dimerization, we used confocal imaging to show that dimerization interface mutants of M fail to assemble into viral filaments on the plasma membrane. Additionally, budding and release of virus-like particles are prevented in M mutants that fail to form filaments. Importantly, we show that M is biologically active as a dimer and that the switch from M dimers to higher-order oligomers triggers viral filament assembly and virus production. IMPORTANCEHuman respiratory syncytial virus (RSV) is the most frequent cause of infantile bronchiolitis and pneumonia. The enormous burden of RSV makes it a major unmet target for a vaccine and antiviral drug therapy. Oligomerization of the matrix protein is a key step in the process of assembly and production of infectious virus, but the molecular mechanism of RSV assembly is still poorly understood. Here we show that the RSV matrix protein forms dimers in solution and in crystals; the dimer is essential for formation of higher-order oligomers. Destabilizing the dimer interface resulted in the loss of RSV filament formation and a lack of budding of virus-like particles. Importantly, our findings can potentially lead to new structure-based RSV inhibitors targeting the assembly process.H uman respiratory syncytial virus (RSV), an enveloped, nonsegmented negative-strand RNA paramyxovirus, is the major worldwide cause of bronchiolitis and pneumonia in infants and of morbidity and mortality in the elderly. Despite the large global impact of RSV infection, no specific antivirals or licensed vaccine is yet available (1, 2). Designing new antivirals requires detailed molecular and structural understanding of key steps in RSV replication.RSV encodes 11 proteins, with the 3 glycoproteins, the fusion protein (F), glycoprotein (G), and small hydrophobic protein (SH), being present in the viral envelope. The virion ...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Dimerization of Matrix Protein Is Required for Budding of Respiratory Syncytial Virus

Förster

Maertens

Farrell

et al. 2015

J Virol

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“…The hRSV F glycoprotein, when labeled for direct stochastic optical reconstruction microscopy (dSTORM) imaging, was present along the length of viral filaments , which is consistent with our results. However, at EM-level resolution, where we are able to resolve the individual glycoproteins (Kiss et al 2014;Liljeroos et al 2013), we expected to find the distribution of the immunolabeled F glycoproteins to be impacted by: (1) the ratio and organization of the F and G glycoproteins on the virus, and (2) the steric hindrance/accessibility associated with the primary and 6-nm gold-conjugated secondary antibody with the F glycoprotein antigenic site II (McLellan et al 2011). The native immunolabeling of the hRSV F glycoprotein underscores this level of glycoprotein organization (Figs.…”

Section: Targeted Labeling Of Cellular and Viral Proteinsmentioning

confidence: 99%

“…4; Supplementary Fig. 2) (Kiss et al 2014;Liljeroos et al 2013). Native immunolabeling of the hRSV F glycoprotein also proved useful for identifying the location of an early stage of virus assembly ( Fig.…”

Section: Three-dimensional Structure and Localization Informationmentioning

confidence: 99%

Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications

Strauss

et al. 2015

J Histochem Cytochem.

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SummaryNumerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells. (J Histochem Cytochem 63:780-792, 2015) Keywords cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), immuno-electron microscopy (immuno-EM), transmission electron microscopy (TEM)

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“…This may be due to the differences in RNP assembly into virions. For RSV, a transcription antiterminator, M2-1, mediates the association of RNPs with the M protein and is required for the incorporation of RNPs into virions (17), and further structural analysis showed that M2-1 is located between the RNP and M in isolated viral particles (18). However, for viruses belonging to Paramyxovirinae subfamilies and some other enveloped viruses, such as retroviruses and filoviruses, N has been described to be a mediator of virion assembly and budding (6,14,(19)(20)(21).…”

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Interaction of Human Parainfluenza Virus Type 3 Nucleoprotein with Matrix Protein Mediates Internal Viral Protein Assembly

Zhang

Zhong

Qin

et al. 2016

J Virol

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Human parainfluenza virus type 3 (HPIV3) belongs to the Paramyxoviridae family. Its three internal viral proteins, the nucleoprotein (N), the phosphoprotein (P), and the polymerase (L), form the ribonucleoprotein (RNP) complex, which encapsidates the viral genome and associates with the matrix protein (M) for virion assembly. We previously showed that the M protein expressed alone is sufficient to assemble and release virus-like particles (VLPs) and a mutant with the L305A point mutation in the M protein (M L305A ) has a VLP formation ability similar to that of wild-type M protein. In addition, recombinant HPIV3 (rHPIV3) containing the M L305A mutation (rHPIV3-M L305A ) could be successfully recovered. In the present study, we found that the titer of rHPIV3-M L305A was at least 10-fold lower than the titer of rHPIV3. Using VLP incorporation and coimmunoprecipitation assays, we found that VLPs expressing the M protein (M-VLPs) can efficiently incorporate N and P via an N-M or P-M interaction and M L305A -VLPs had an ability to incorporate P via a P-M interaction similar to that of M-VLPs but were unable to incorporate N and no longer interacted with N. Furthermore, we found that the incorporation of P into M L305A -VLPs but not M-VLPs was inhibited in the presence of N. In addition, we provide evidence that the C-terminal region of P is involved in its interaction with both N and M and N binding to the C-terminal region of P inhibits the incorporation of P into M L305A -VLPs. Our findings provide new molecular details to support the idea that the N-M interaction and not the P-M interaction is critical for packaging N and P into infectious viral particles. IMPORTANCEHuman parainfluenza virus type 3 (HPIV3) is a nonsegmented, negative-sense, single-stranded RNA virus that belongs to the Paramyxoviridae family and can cause lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. However, no effective vaccine has been developed or licensed. We used virus-like particle (VLP) incorporation and coimmunoprecipitation assays to determine how the M protein assembles internal viral proteins. We demonstrate that both nucleoprotein (N) and phosphoprotein (P) can incorporate into M-VLPs and N inhibits the M-P interaction via the binding of N to the C terminus of P. We also provide additional evidence that the N-M interaction but not the P-M interaction is critical for the regulation of HPIV3 assembly. Our studies provide a more complete characterization of HPIV3 virion assembly and substantiation that N interaction with M regulates internal viral organization. Human parainfluenza virus type 3 (HPIV3) is a negativestrand RNA virus (NSV) that belongs to the Paramyxoviridae family and often causes lower respiratory tract infections in infants and young children. The HPIV3 genome consists of 6 open reading frames that encode 6 structural proteins: the nucleoprotein (N), phosphoprotein (P), RNA-dependent RNA polymerase (L), matrix protein (M), and two glycoprotei...

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Structural Analysis of Respiratory Syncytial Virus Reveals the Position of M2-1 between the Matrix Protein and the Ribonucleoprotein Complex

Cited by 116 publications

References 82 publications

Dimerization of Matrix Protein Is Required for Budding of Respiratory Syncytial Virus

Dimerization of Matrix Protein Is Required for Budding of Respiratory Syncytial Virus

Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications

Interaction of Human Parainfluenza Virus Type 3 Nucleoprotein with Matrix Protein Mediates Internal Viral Protein Assembly

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