An increase in glycoprotein concentration on extracellular virions dramatically alters vaccinia virus infectivity and pathogenesis without impacting immunogenicity.

Stephanie R Monticelli,Matthew G Brewer,Christopher C Norbury,Peter Bryk,Brian M Ward,Hector C Aguilar,David C Tscharke

doi:10.1371/journal.ppat.1010177

Stephanie R Monticelli, Matthew G Brewer + Show 5 more

Open Access

https://doi.org/10.1371/journal.ppat.1010177

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Abstract

The extracellular virion (EV) form of Orthopoxviruses is required for cell-to-cell spread and pathogenesis, and is the target of neutralizing antibodies in the protective immune response. EV have a double envelope that contains several unique proteins that are involved in its intracellular envelopment and/or subsequent infectivity. One of these, F13, is involved in both EV formation and infectivity. Here, we report that replacement of vaccinia virus F13L with the molluscum contagiosum virus homolog, MC021L, results in the production of EV particles with significantly increased levels of EV glycoproteins, which correlate with a small plaque phenotype. Using a novel fluorescence-activated virion sorting assay to isolate EV populations based on glycoprotein content we determine that EV containing either higher or lower levels of glycoproteins are less infectious, suggesting that there is an optimal concentration of glycoproteins in the outer envelope that is required for maximal infectivity of EV. This optimal glycoprotein concentration was required for lethality and induction of pathology in a cutaneous model of animal infection, but was not required for induction of a protective immune response. Therefore, our results demonstrate that there is a sensitive balance between glycoprotein incorporation, infectivity, and pathogenesis, and that manipulation of EV glycoprotein levels can produce vaccine vectors in which pathologic side effects are attenuated without a marked diminution in induction of protective immunity.

Highlights

Viral glycoproteins are a major component of the outermost envelope of viruses, actively participating in critical aspects of the viral lifecycle [1,2]
A previous report from our lab determined that vMC021L-HA had similar replication kinetics as vF13L-HA, but produced a small plaque phenotype and extracellular virion (EV) that incorporated more MC021-HA compared to its homolog, F13-HA [28]
Considering that there are differences in the levels of F13/MC021, it seemed likely that vMC021-HA altered the glycoprotein content of EV, leading to their decreased infectivity

Summary

Introduction

Viral glycoproteins are a major component of the outermost envelope of viruses, actively participating in critical aspects of the viral lifecycle [1,2]. IEV are a transient form that are transported to the cell surface, where fusion with the plasma membrane releases the EV form of the virus, which is critical for cell-to-cell spread and long-range dissemination [11,12,13,14,15] Both IMV and EV are surrounded by lipid envelopes, but the EV surface proteins are glycosylated whereas the IMV surface proteins are not. Four proteins found in EV and not IMV (A33, A34, B5 and F13), are highly conserved between members of the Orthopoxvirus genus [16,17,18,19,20,21] These proteins play critical roles in the formation of infectious EV as deletion of any one of them results in a small plaque phenotype [22,23,24,25]. Deletion of F13 has the most profound effect on EV production due to defects in both EV production and infectivity [26,27]

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