Biology subjects

Hogle, J. M.

Publications and source records attributed to Hogle, J. M..

3 recordsLinked to original sources

bioRxiv2024

Mechanism of enterovirus VP0 maturation cleavage based on the structure of a stabilised assembly intermediate.

Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0. We have demonstrated an essential role of VP0 E096 in VP0 cleavage independent of RNA encapsidation and generated a new model of capsid maturation, supported by bioinformatic analysis. This provides a molecular basis for RNA-dependence, where RNA induces conformational changes required for VP0 maturation, but that RNA packaging itself is not sufficient to induce maturation. These data have implications for understanding production of infectious virions and potential relevance for future vaccine and antiviral drug design.

microbiology↗

bioRxiv2023

Production of antigenically stable enterovirus A71 virus-like particles in Pichia pastoris as a vaccine candidate.

Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are produced during the viral lifecycle. While initially antigenically indistinguishable from virions, ECs readily convert to an expanded conformation at moderate temperatures. In the closely related poliovirus, these conformational changes result in loss of antigenic sites required to elicit protective immune responses. Whether this is true for EVA71 remains to be determined and is the subject of this investigation. We previously reported the selection of a thermally resistant EVA71 genogroup B2 population using successive rounds of heating and passage. The mutations found in the structural protein-coding region of the selected population conferred increased thermal stability to both virions and naturally produced ECs. Here, we introduced these mutations into a recombinant expression system to produce stabilised virus-like particles (VLPs) in Pichia pastoris. The stabilised VLPs retain the native virion-like antigenic conformation as determined by reactivity with a specific antibody. Structural studies suggest multiple potential mechanisms of antigenic stabilisation, however, unlike poliovirus, both native and expanded EVA71 particles elicited antibodies able to directly neutralise virus in vitro. Therefore, the anti-EVA71 neutralising antibodies are elicited by sites which are not canonically associated with the native conformation, but whether antigenic sites specific to the native conformation provide additional protective responses in vivo remains unclear. VLPs are likely to provide cheaper and safer alternatives for vaccine production and these data show that VLP vaccines are comparable with inactivated virus vaccines at inducing neutralising antibodies.

immunology↗

bioRxiv2020

Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate.

The virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the "late" 135S particles have detectable levels of the VP1 N-terminus trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously. AUTHOR SUMMARYNonenveloped viruses need to provide mechanisms that allow their genomes to be delivered across membrane. This process remains poorly understood. For enterovirus such as poliovirus, genome delivery involves a program of conformational changes that include expansion of the particle and externalization of two normal internal peptides, VP4 and the VP1 N-terminus, which then insert into the cell membrane, triggering endocytosis and the creation of pores that facilitate the transfer of the viral RNA genome across the endosomal membrane. This manuscript describes five high-resolution cryo-EM structures of altered poliovirus particles that represent a number of intermediates along this pathway. The structures reveal several surprising findings, including the discovery of a new intermediate that is expanded but has not yet externalized the membrane interactive peptides, the clear identification of a unique exit site VP1 N-terminus, the demonstration that the externalized VP1 N-terminus partitions between two different sites in a temperature-dependent fashion, direct visualization of an amphipathic helix at the N-terminus of VP1 in an ideal position for interaction with cellular membranes, and the observation that a significant portion of VP4 remains inside the particle and accounts for a feature that had been previously ascribed to part of the viral RNA. These findings represent significant additions to our understanding of the cell entry process of an important class of human pathogens.

biophysics↗