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Jean-Yves Sgro
Inst. for Mol.Virology
731B Bock Labs
1525 Linden Drive Madison, WI 53706

Current Papers in Structure and Assembly (Journal of Virology)

Journal of Virology Structure and Assembly

  • Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production [Structure and Assembly]

  • The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at aalpha;2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the aalpha;2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles.

    IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

  • Antiangiogenic Vascular Endothelial Growth Factor-Blocking Peptides Displayed on the Capsid of an Infectious Oncolytic Parvovirus: Assembly and Immune Interactions [Structure and Assembly]

  • As many tumor cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, frontline cancer therapies often administer anti-VEGF (aalpha;-VEGF) antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumor vasculature, we studied the functional tolerance, evasion of neutralization, and induction of aalpha;-VEGF antibodies of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the 3-fold capsid spike was replaced by VEGF-blocking peptides: P6L (PQPRPL) and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture, exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti-spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an aalpha;-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the 3-fold axis to close up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture into a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow an infectious parvovirus with immune antineovascularization and evasion capacities by replacing an antibody footprint in the capsid 3-fold axis with VEGF-blocking peptides, and it also illustrates the evolutionary capacity of single-stranded DNA (ssDNA) viruses to overcome engineered capsid structural restrictions.

    IMPORTANCE Targeting the VEGF signaling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumors. VEGF-blocking peptides (VEbp) engineered in the capsid 3-fold axis endowed the infectious parvovirus MVM with the ability to induce aalpha;-VEGF antibodies without adjuvant and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restraints that the capsid imposes on the peptide configuration and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp resolved the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. These data show the promise of antineovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses but also raise safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.

  • Bile Salts Alter the Mouse Norovirus Capsid Conformation: Possible Implications for Cell Attachment and Immune Evasion [Structure and Assembly]

  • Caliciviruses are single-stranded RNA viruses with 180 copies of capsid protein comprising the T=3 icosahedral capsids. The main capsid feature is a pronounced protruding (P) domain dimer formed by adjacent subunits on the icosahedral surface while the shell domain forms a tight icosahedral sphere around the genome. While the P domain in the crystal structure of human Norwalk virus (genotype I.1) was tightly associated with the shell surface, the cryo-electron microscopy (cryo-EM) structures of several members of the Caliciviridae family (mouse norovirus [MNV], rabbit hemorrhagic disease virus, and human norovirus genotype II.10) revealed a "floating" P domain that hovers above the shell by nearly 10 to 15 AAring; in physiological buffers. Since this unusual feature is shared among, and unique to, the Caliciviridae, it suggests an important biological role. Recently, we demonstrated that bile salts enhance cell attachment to the target cell and increase the intrinsic affinity between the P domain and receptor. Presented here are the cryo-EM structures of MNV-1 in the presence of bile salts (~3 AAring;) and the receptor CD300lf (~8 AAring;). Surprisingly, bile salts cause the rotation and contraction of the P domain onto the shell surface. This both stabilizes the P domain and appears to allow for a higher degree of saturation of receptor onto the virus. Together, these results suggest that, as the virus moves into the gut and the associated high concentrations of bile, the entire capsid face undergoes a conformational change to optimize receptor avidity while the P domain itself undergoes smaller conformational changes to improve receptor affinity.

    IMPORTANCE Mouse norovirus and several other members of the Caliciviridae have been shown to have a highly unusual structure with the receptor binding protruding (P) domain only loosely tethered to the main capsid shell. Recent studies demonstrated that bile salts enhance the intrinsic P domain/receptor affinity and is necessary for cell attachment. Presented here are the high-resolution cryo-EM structures of apo MNV, MNV/bile salt, and MNV/bile salt/receptor. Bile salts cause a 90ddeg; rotation and collapse of the P domain onto the shell surface that may increase the number of available receptor binding sites. Therefore, bile salts appear to be having several effects on MNV. Bile salts shift the structural equilibrium of the P domain toward a form that binds the receptor and away from one that binds antibody. They may also cause the entire P domain to optimize receptor binding while burying a number of potential epitopes.

  • The Guanine Nucleotide Exchange Factor GBF1 Participates in Rotavirus Replication [Structure and Assembly]

  • Cellular and viral factors participate in the replication cycle of rotavirus. We report that the guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference or inhibiting its activity by treatment with brefeldin A (BFA) or Golgicide A (GCA) significantly reduces the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly, since in the presence of either BFA or GCA, the assembly of infectious mature triple-layered virions was significantly prevented and only double-layered particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface protein VP7, a step required for its incorporation into virus particles. Although a posttranslational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking down its expression reduces VP7 trimerization. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when cotransfected with NSP4.

    IMPORTANCE Rotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in children and young animals worldwide. Despite significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine nucleotide exchange factor GBF1, relevant for COPI/Arf1-mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4 and the assembly of the virus surface proteins VP7 and VP4.