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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

  • A Single Amino Acid Substitution in Poliovirus Nonstructural Protein 2CATPase Causes Conditional Defects in Encapsidation and Uncoating [Structure and Assembly]

  • The specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2CATPase. In particular, residue N252 of poliovirus 2CATPase interacts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2CATPase has important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2CATPase, near N252, that are required for encapsidation. Accordingly, segments adjacent to N252 were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2CATPase, caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive (ts) growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K259A mutation, mapping to an aalpha; helix in the predicted structure of 2CATPase, resulted in a cold-sensitive virus. In vivo protein synthesis and virus production were strikingly delayed at 33ddeg;C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33ddeg;C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33ddeg;C relative to the wt. In conclusion, we have for the first time linked a cold-sensitive encapsidation defect in 2CATPase (K259A) to a subsequent delay in uncoating of the virus particle at 33ddeg;C during the next cycle of infection.

    IMPORTANCE Enterovirus morphogenesis, which involves the encapsidation of newly made virion RNA, is a process still poorly understood. Elucidation of this process is important for future drug development for a large variety of diseases caused by these agents. We have previously shown that the specificity of encapsidation of poliovirus and of C-cluster coxsackieviruses, which are prototypes of enteroviruses, is dependent on an interaction of capsid proteins with the multifunctional nonstructural protein 2CATPase. In this study, we have searched for residues in poliovirus 2CATPase, near a presumed capsid-interacting site, important for encapsidation. An unusual cold-sensitive mutant of 2CATPase possessed a defect in encapsidation at 37ddeg;C and subsequently in uncoating during the next cycle of infection at 33ddeg;C. These studies not only reveal a new site in 2CATPase that is involved in encapsidation but also identify a link between encapsidation and uncoating.

  • Membrane Anchors of the Structural Flavivirus Proteins and Their Role in Virus Assembly [Structure and Assembly]

  • The structural proteins of flaviviruses carry a unique set of transmembrane domains (TMDs) at their C termini that are derived from the mode of viral polyprotein processing. They function as internal signal and stop-transfer sequences during protein translation, but possible additional roles in protein interactions required during assembly and maturation of viral particles are ill defined. To shed light on the role of TMDs in these processes, we engineered a set of tick-borne encephalitis virus mutants in which these structural elements were replaced in different combinations by the homologous sequences of a distantly related flavivirus (Japanese encephalitis virus). The effects of these modifications were analyzed with respect to protein synthesis, viral particle secretion, specific infectivity, and acidic-pH-induced maturation processes. We provide evidence that interactions involving the double-membrane anchor of the envelope protein E (a unique feature compared to other viral fusion proteins) contribute substantially to particle assembly, stability, and maturation. Disturbances of the inter- and intra-TMD interactions of E resulted in the secretion of a larger proportion of capsidless subviral particles at the expense of whole virions, suggesting a possible role in the still incompletely understood mechanism of capsid integration during virus budding. In contrast, the TMD initially anchoring the C protein to the endoplasmic reticulum membrane does not appear to take part in envelope protein interactions. We also show that E TMDs are involved in the envelope protein rearrangements that are triggered by acidic pH in the trans-Golgi network and represent a hallmark of virus maturation.

    IMPORTANCE The assembly of flaviviruses occurs in the endoplasmic reticulum and leads to the formation of immature, noninfectious particles composed of an RNA-containing capsid surrounded by a lipid membrane, with the two integrated envelope proteins, prM and E, arranged in an icosahedral lattice. The mechanism by which the capsid is formed and integrated into the budding viral envelope is currently unknown. We provide evidence that the transmembrane domains (TMDs) of E are essential for the formation of capsid-containing particles and that disturbances of these interactions lead to the preferential formation of capsidless subviral particles at the expense of whole virions. E TMD interactions also appear to be essential for the envelope protein rearrangements required for virus maturation and for the generation of infectious virions. Our data thus provide new insights into the biological functions of E TMDs and extend their role during viral polyprotein processing to additional functions in particle assembly and maturation.

  • In Vivo Analysis of Infectivity, Fusogenicity, and Incorporation of a Mutagenic Viral Glycoprotein Library Reveals Determinants for Virus Incorporation [Structure and Assembly]

  • Enveloped viruses utilize transmembrane surface glycoproteins to gain entry into target cells. Glycoproteins from diverse viral families can be incorporated into nonnative viral particles in a process termed pseudotyping; however, the molecular mechanisms governing acquisition of these glycoproteins are poorly understood. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles has been shown to be an active process, but it does not appear to be caused by direct interactions among viral proteins. In this study, we coupled in vivo selection systems with Illumina next-generation sequencing (NGS) to test hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles and to perform other glycoprotein functions. NGS analyses on a subset of these mutants predicted that the residues important for incorporation are in the membrane-proximal external region (MPER), particularly W127 and W137, and the residues in the membrane-spanning domain (MSD) and also immediately flanking it (T140 to L163). These predictions were validated by directly measuring the impact of mutations in these regions on fusogenicity, infectivity, and incorporation. We suggest that these two regions dictate pseudotyping through interactions with specific lipid environments formed during viral assembly.

    IMPORTANCE Researchers from numerous fields routinely exploit the ability to manipulate viral tropism by swapping viral surface proteins. However, this process, termed pseudotyping, is poorly understood at the molecular level. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles is an active process, but it does not appear to occur through direct viral protein-protein interactions. In this study, we tested hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles as well as perform other glycoprotein functions. Our analyses on a subset of these mutants predict that the glycoprotein regions embedded in and immediately flanking the viral membrane dictate active incorporation into viral particles. We suggest that pseudotyping occurs through specific lipid-protein interactions at the viral assembly site.