Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions

Bianca S Heinrich,David K Cureton,Sean P J Whelan,Amal A Rahmeh,Karla Kirkegaard

doi:10.1371/journal.ppat.1000958

Abstract

Positive-strand and double-strand RNA viruses typically compartmentalize their replication machinery in infected cells. This is thought to shield viral RNA from detection by innate immune sensors and favor RNA synthesis. The picture for the non-segmented negative-strand (NNS) RNA viruses, however, is less clear. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we examined the location of the viral replication machinery and RNA synthesis in cells. By short-term labeling of viral RNA with 5′-bromouridine 5′-triphosphate (BrUTP), we demonstrate that primary mRNA synthesis occurs throughout the host cell cytoplasm. Protein synthesis results in the formation of inclusions that contain the viral RNA synthesis machinery and become the predominant sites of mRNA synthesis in the cell. Disruption of the microtubule network by treatment of cells with nocodazole leads to the accumulation of viral mRNA in discrete structures that decorate the surface of the inclusions. By pulse-chase analysis of the mRNA, we find that viral transcripts synthesized at the inclusions are transported away from the inclusions in a microtubule-dependent manner. Metabolic labeling of viral proteins revealed that inhibiting this transport step diminished the rate of translation. Collectively those data suggest that microtubule-dependent transport of viral mRNAs from inclusions facilitates their translation. Our experiments also show that during a VSV infection, protein synthesis is required to redirect viral RNA synthesis to intracytoplasmic inclusions. As viral RNA synthesis is initially unrestricted, we speculate that its subsequent confinement to inclusions might reflect a cellular response to infection.

Highlights

RNA viruses that replicate within the cytoplasm often form specialized structures that are the sites of RNA replication [1]
Positive-strand and double-strand RNA viruses compartmentalize their replication machinery in infected cells. This compartmentalization is thought to favor the catalysis of RNA synthesis, and sequester viral RNA molecules from detection by innate immune sensors
Our work shows that prior to viral protein synthesis the invading viral cores synthesize mRNA throughout the host cell cytoplasm

Summary

Introduction

RNA viruses that replicate within the cytoplasm often form specialized structures that are the sites of RNA replication [1]. Experiments with poliovirus and with flock house virus (FHV) have provided compelling evidence that the viral RNA and the non-structural proteins required for RNA replication are localized to such sites. Double-strand RNA viruses form phase-dense inclusions or ‘‘viral factories’’ to which transcription competent viral cores and the machinery required for RNA synthesis are localized [7]. In contrast to the structures formed by positive-strand RNA viruses, the double-strand RNA virus factories are not membrane bound [8,9,10]. The formation of such specialized replication compartments is thought to concentrate the viral machinery necessary for RNA synthesis and thereby favor catalysis. Compartmentalization of the replication machinery might shield the viral RNA from detection by cytosolic innate immune sensors

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Results

Discussion

Conclusion