Role of the vacuolar ATPase in the Alphavirus replication cycle

Ryan M Schuchman,Ricardo Vancini,Amanda Piper,Denitra Breuer,Mariana Ribeiro,Davis Ferreira,Joseph Magliocca,Veronica Emmerich,Raquel Hernandez,Dennis T Brown

doi:10.1016/j.heliyon.2018.e00701

Ryan M Schuchman, Ricardo Vancini + Show 8 more

Open Access

https://doi.org/10.1016/j.heliyon.2018.e00701

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Abstract

We have shown that Alphaviruses can enter cells by direct penetration at the plasma membrane (R. Vancini, G. Wang, D. Ferreira, R. Hernandez, and D. Brown, J Virol, 87:4352–4359, 2013). Direct penetration removes the requirement for receptor-mediated endocytosis exposure to low pH and membrane fusion in the process of RNA entry. Endosomal pH as well as the pH of the cell cytoplasm is maintained by the activity of the vacuolar ATPase (V-ATPase). Bafilomycin is a specific inhibitor of V-ATPase. To characterize the roll of the V-ATPase in viral replication we generated a Bafilomycin A1(BAF) resistant mutant of Sindbis virus (BRSV). BRSV produced mature virus and virus RNA in greater amounts than parent virus in BAF-treated cells. Sequence analysis revealed mutations in the E2 glycoprotein, T15I/Y18H, were responsible for the phenotype. These results show that a functional V-ATPase is required for efficient virus RNA synthesis and virus maturation in Alphavirus infection.

Highlights

Sindbis virus is the prototype Alphavirus in the Togaviridae family
We have investigated the role that V-ATPase plays in the Sindbis virus replication cycle by producing a virus mutant of SVHR, resistant to the effects of bafilomycin A1 (BAF)
The goal of this study is to identify what the effect of an inhibited V-ATPase has on alphavirus replication by examining entry, RNA production, virus maturation, and egress of SVHR compared to a BAF-resistant Sindbis virus mutant (BRSV)

Summary

Introduction

The proteins of the outer shell provide the tools by which the virus attaches to the cell surface (E2) and a mechanism that facilitates the entry of the viral genome into the cell (E1) [3]. The model suggesting that receptor-mediated endocytosis is the mechanism for virus entry was challenged by the development of an electron microscopy protocol that directly visualized the entry process [5, 6]. This protocol uses immunogold-labeling to facilitate the identification of empty virus particles on the surface of the cell that would have otherwise been overlooked after entry took place (Fig. 1). Once the genome has entered the host cell, the viral polycistronic RNA is translated into 4 non-structural proteins, nsP1-4. nsP1 functions as a methyltransferase to add a methylguanosine cap to newly synthesized viral RNA [11, 12, 13, 14]. nsP2 is the protease that cleaves the nonstructural polypeptide and is the viral helicase [15, 16, 17, 18, 19]. nsP3 is an accessory in the synthesis of the negative RNA strand [20,

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