Marie Chow

Structural factors that control conformational transitions and serotype specificity in type 3 poliovirus

The three‐dimensional structure of the Sabin strain of type 3 poliovirus has been determined at 2.4 A resolution. Significant structural differences with the Mahoney strain of type 1 poliovirus are confined to loops and terminal extensions of the capsid proteins, occur in all of the major antigenic sites of the virion and typically involve insertions, deletions or the replacement of prolines. Several newly identified components of the structure participate in assembly‐dependent interactions which are relevant to the biologically important processes of viral assembly and uncoating. These include two sites of lipid substitution, two putative nucleotides and a beta sheet formed by the N‐termini of capsid proteins VP4 and VP1. The structure provides an explanation for the temperature sensitive phenotype of the P3/Sabin strain. Amino acids that regulate temperature sensitivity in type 3 poliovirus are located in the interfaces between promoters, in the binding site for a lipid substituent and in an assembly‐dependent extended beta sheet that stabilizes the association of pentamers. Several lines of evidence indicate that these structural components also control conformational transitions at various stages of the viral life cycle.

Genome Delivery and Ion Channel Properties Are Altered in VP4 Mutants of Poliovirus

ABSTRACT During entry into host cells, poliovirus undergoes a receptor-mediated conformational transition to form 135S particles with irreversible exposure of VP4 capsid sequences and VP1 N termini. To understand the role of VP4 during virus entry, the fate of VP4 during infection by site-specific mutants at threonine-28 of VP4 (4028T) was compared with that of the parental Mahoney type 1 virus. Three virus mutants were studied: the entry-defective, nonviable mutant 4028T.G and the viable mutants 4028T.S and 4028T.V, in which residue threonine-28 was changed to glycine, serine, and valine, respectively. We show that mutant and wild-type (WT) VP4 proteins are localized to cellular membranes after the 135S conformational transition. Both WT and viable 4028T mutant particles interact with lipid bilayers to form ion channels, whereas the entry-defective 4028T.G particles do not. In addition, the electrical properties of the channels induced by the mutant viruses are different from each other and from those of WT Mahoney and Sabin type 3 viruses. Finally, uncoating and/or cytoplasmic delivery of the viral genome is altered in the 4028T mutants: the 4028T.G lethal mutant does not release its genome into the cytoplasm, and genome delivery is slower during infection by mutant 4028T.V 135S particles than by mutant 4028T.S or WT 135S particles. The distinctive electrical characteristics of the different 4028T mutant channels indicate that VP4 sequences might form part of the channel structure. The different entry phenotypes of these VP4 mutants suggest that the ion channels may be related to VP4s role during genome uncoating and/or delivery.

Cholesterol Removal by Methyl-β-Cyclodextrin Inhibits Poliovirus Entry

ABSTRACT Upon binding to the poliovirus receptor (PVR), the poliovirus 160S particles undergo a conformational transition to generate 135S particles, which are believed to be intermediates in the virus entry process. The 135S particles interact with host cell membranes through exposure of the N termini of VP1 and the myristylated VP4 protein, and successful cytoplasmic delivery of the genomic RNA requires the interaction of these domains with cellular membranes whose identity is unknown. Because detergent-insoluble microdomains (DIMs) in the plasma membrane have been shown to be important in the entry of other picornaviruses, it was of interest to determine if poliovirus similarly required DIMs during virus entry. We show here that methyl-β-cyclodextrin (MβCD), which disrupts DIMs by depleting cells of cholesterol, inhibits virus infection and that this inhibition was partially reversed by partially restoring cholesterol levels in cells, suggesting that MβCD inhibition of virus infection was mediated by removal of cellular cholesterol. However, fractionation of cellular membranes into DIMs and detergent-soluble membrane fractions showed that both PVR and poliovirus capsid proteins localize not to DIMs but to detergent-soluble membrane fractions during entry into the cells, and their localization was unaffected by treatment with MβCD. We further demonstrate that treatment with MβCD inhibits RNA delivery after formation of the 135S particles. These data indicate that the cholesterol status of the cell is important during the process of genome delivery and that these entry pathways are distinct from those requiring DIM integrity.

Is the 135S Poliovirus Particle an Intermediate during Cell Entry

ABSTRACT Poliovirus binding to its receptor (PVR) on the cell surface induces a conformational transition which generates an altered particle with a sedimentation value of 135S versus the 160S of the native virion. A number of lines of evidence suggest that the 135S particle is a cell entry intermediate. However, the low infection efficiencies of the 135S particle and the absence of detectable 135S particles during infection at 26°C by the cold-adapted mutants argue against a role for the 135S particle during the cell entry process. We show here that binding of 135S-antibody complexes to the Fc receptor (CDw32) increases the infectivity of these particles by 2 to 3 orders of magnitude. Thus, the low efficiency of infection by 135S particles is due in part to the low binding affinity of these particles. In addition, we show that there is an additional stage in the entry process that is associated with RNA release. This stage occurs after formation of the 135S particle, is rate limiting during infection at 37°C, but not at 26°C, and is PVR independent. The data also demonstrate that during infection at 26°C, the rate-limiting step is the PVR-mediated conversion of wild-type 160S particles to 135S particles. This suggests that during infection at 26°C by the cold-adapted viruses, 135S particles are formed, but they fail to accumulate to detectable levels because the subsequent post-135S particle events occur at a significantly faster rate than the initial conversion of 160S to 135S particles. These data support a model in which the 135S particle is an intermediate during poliovirus entry.

An Externalized Polypeptide Partitions between Two Distinct Sites on Genome-Released Poliovirus Particles

ABSTRACT During cell entry, native poliovirus (160S) converts to a cell-entry intermediate (135S) particle, resulting in the externalization of capsid proteins VP4 and the amino terminus of VP1 (residues 1 to 53). Externalization of these entities is followed by release of the RNA genome (uncoating), leaving an empty (80S) particle. The antigen-binding fragment (Fab) of a monospecific peptide 1 (P1) antibody, which was raised against a peptide corresponding to amino-terminal residues 24 to 40 of VP1, was utilized to track the location of the amino terminus of VP1 in the 135S and 80S states of poliovirus particles via cryogenic electron microscopy (cryo-EM) and three-dimensional image reconstruction. On 135S, P1 Fabs bind to a prominent feature on the external surface known as the “propeller tip.” In contrast, our initial 80S-P1 reconstruction showed P1 Fabs also binding to a second site, at least 50 Å distant, at the icosahedral 2-fold axes. Further analysis showed that the overall population of 80S-P1 particles consisted of three kinds of capsids: those with P1 Fabs bound only at the propeller tips, P1 Fabs bound only at the 2-fold axes, or P1 Fabs simultaneously bound at both positions. Our results indicate that, in 80S particles, a significant fraction of VP1 can deviate from icosahedral symmetry. Hence, this portion of VP1 does not change conformation synchronously when switching from the 135S state. These conclusions are compatible with previous observations of multiple conformations of the 80S state and suggest that movement of the amino terminus of VP1 has a role in uncoating. Similar deviations from icosahedral symmetry may be biologically significant during other viral transitions.

Poliovirus: new insights from an old paradigm

A combination of structural and genetic studies of poliovirus suggests that the final stages of viral assembly lock the virus in a metastable structure primed to undergo the receptor-catalyzed conformational changes required for cell entry. Future studies promise to provide detailed insights into the conformational dynamics of the virion during its life cycle.

The Three-dimensional Structure of Poliovirus: Implications for Virus Evolution, Assembly, and Immune Recognition

In the past year, the structures of two animal viruses have been determined at near atomic resolution [1,2]. The viruses, poliomyelitis virus type 1 (Mahoney strain) and human rhino virus 14, are both members of the Picornaviridae family, a large family of small spherical RNA viruses that also includes hepatitis A virus, the coxsackieviruses, and foot-and-mouth disease virus. Although poliovirus and rhinovirus are the first animal viruses to be mapped at high resolution, these studies have their roots in the earlier determinations of several plant virus structures [3–6]. The application of x-ray crystallographic techniques to animal viruses is particularly exciting because the animal viruses in general, and poliovirus in particular, are far better characterized biologically. These structures thus provide for the first time the opportunity to study the structural basis for immune recognition (and neutralization), host and tissue specificity, and viral pathogenesis. In the future, this work will also facilitate structural studies of other picornaviruses (several of which have already been crystallized) and the extension of the techniques to other families of animal viruses.