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Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids.

ABSTRACT: Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell electron cryotomography, we monitored morphological changes in virus-infected host cells. Subtomogram averaging reveals the VAP structure. By heterologous expression of PVAP in cells from all three domains of life, we demonstrate that the protein integrates indiscriminately into virtually any biological membrane, where it forms sevenfold pyramids. We identify the protein domains essential for VAP formation in PVAP truncation mutants by their ability to remodel the cell membrane. Self-assembly of PVAP into pyramids requires at least two different, in-plane and out-of-plane, protein interactions. Our findings allow us to propose a model describing how PVAP arranges to form sevenfold pyramids and suggest how this small, robust protein may be used as a general membrane-remodeling system.

SUBMITTER: Daum B

PROVIDER: S-EPMC3956195 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids.

Daum Bertram B Quax Tessa E F TE Sachse Martin M Mills Deryck J DJ Reimann Julia J Yildiz Özkan Ö Häder Sabine S Saveanu Cosmin C Forterre Patrick P Albers Sonja-Verena SV Kühlbrandt Werner W Prangishvili David D

Proceedings of the National Academy of Sciences of the United States of America 20140224 10

Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell ele ...[more]

PMID: 24567401

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Project description:BackgroundRecently, three-dimensional (3D) imaging techniques have been used to detect viral invasion and the appearance of specialized structures established in virus-infected cells. These methods have had a positive impact in the field of virology and helped to further our knowledge of how viruses invade cells. Nearly all positive-strand RNA viruses propagate their viral genomes in part through intracellular membranes. Porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus, accumulates viral RNA that forms replication complexes (RCs) in infected cells. In this study, using immunofluorescence and electron microscopy (EM), we dissected PRRSV-induced membrane structures in infected cells and determined the correlations between PRRSV particles and vesicles stimulated by PRRSV to understand the structural and dynamic aspects of PRRSV infection.MethodsWe identified the appropriate time point by determining the 50% tissue culture infectious dose (TCID50) and using qRT-PCR and Western blotting. The co-localization of viruses and organelles was determined by immunofluorescence and immune-electron microscopy (IEM). The ultrastructure of cells infected by PRRSV was observed using EM and electron tomography (ET).ResultsIn our study, we found that PRRSV dsRNA was located at the endoplasmic reticulum (ER) and autophagosomes; in addition, the N protein was located at the mitochondria, ER and autophagosomes. Vesicles induced by PRRSV appeared at 16 hours post-infection (h.p.i.) and increased in size with time during the infection period. In addition, our findings demonstrated that the virus vesicles originated from the ER, and these two organelle structures connected with each other to form a reticulovesicular network (RVN) that provided a site for virus replication and assembly.ConclusionOur results revealed that membrane vesicles induced by PRRSV were derived from the ER. The vesicles may provide a location for PRRSV replication and assembly.

Project description:Newcastle disease virus (NDV) is an enveloped paramyxovirus. The matrix protein of the virus (M-NDV) has an innate propensity to produce virus-like particles budding from the plasma membrane of the expressing cell without recruiting other viral proteins. The virus predominantly infects the host cell via fusion with the host plasma membrane or, alternatively, can use receptor-mediated endocytic pathways. The question arises as to what are the mechanisms supporting such diversity, especially concerning the assembling and membrane binding properties of the virus protein scaffold under both neutral and acidic pH conditions. Here, we suggest a novel method of M-NDV isolation in physiological ionic strength and employ a combination of small-angle X-ray scattering, atomic force microscopy with complementary structural techniques, and membrane interaction measurements to characterize the solution behavior/structure of the protein as well as its binding to lipid membranes at pH 4.0 and pH 7.0. We demonstrate that the minimal structural unit of the protein in solution is a dimer that spontaneously assembles in a neutral milieu into hollow helical oligomers by repeating the protein tetramers. Acidic pH conditions decrease the protein oligomerization state to the individual dimers, tetramers, and octamers without changing the density of the protein layer and lipid membrane affinity, thus indicating that the endocytic pathway is a possible facilitator of NDV entry into a host cell through enhanced scaffold disintegration.IMPORTANCE The matrix protein of the Newcastle disease virus (NDV) is one of the most abundant viral proteins that regulates the formation of progeny virions. NDV is an avian pathogen that impacts the economics of bird husbandry due to its resulting morbidity and high mortality rates. Moreover, it belongs to the Avulavirus subfamily of the Paramyxoviridae family of Mononegavirales that include dangerous representatives such as respiratory syncytial virus, human parainfluenza virus, and measles virus. Here, we investigate the solution structure and membrane binding properties of this protein at both acidic and neutral pH to distinguish between possible virus entry pathways and propose a mechanism of assembly of the viral matrix scaffold. This work is fundamental for understanding the mechanisms of viral entry as well as to inform subsequent proposals for the possible use of the virus as an adequate template for future drug or vaccine delivery.

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Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids.

Publications

Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids.

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