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Host translation shutoff mediated by non-structural protein 2 is a critical factor in the antiviral state resistance of Venezuelan equine encephalitis virus
Text by Nishank Balla
Alphaviruses are positive-sense, single-stranded RNA viruses belonging to the Togaviridae family of viruses, and are classified as Old World (SINV, CHIKV) and New World (VEEV, EEEV) alphaviruses based on geographic distribution and disease type. Infection of mouse models and humans with SINV and VEEV can induce IFN-α/β that upregulates antiviral mechanisms in infected and uninfected areas of the body. VEEV can replicate more efficiently in the presence of IFN than other tested alphaviruses which are inhibited to different degrees due to the antiviral effects of IFN treatment. In this study we show that inhibition of host translation by VEEV nsP2 in cells already primed with IFN is critical for the virus to resist host antiviral mechanisms and cause disease. We have also identified the viral proteins responsible for inhibition of host transcription and translation, which we show are activities induced independently during infection.
We had previously shown that VEEV could replicate in IFN primed cells very efficiently, as if there was minimal effect of IFN on the virus. In most previous studies, virus-induced transcription and translation inhibition were studied without taking into account that these activities are localized to the same protein in Old world alphaviruses. Our current study attempts to resolve discrepancies in published data and address, separately, the role of transcription and translation inhibition in the antiviral state resistance of alphaviruses.
A major difficulty we encountered was to separate macromolecular synthesis inhibition phenotypes from virus replication efficiency. In our studies and previously demonstrated by others, particular mutations in nsP2 and/or capsid proteins in full-length viruses abrogate this inhibition but also reduce replication rates in cells without functional IFN responses, confounding analyses. We chose to study nsP2 and capsid independently of replication by expressing these proteins from a DNA plasmid. We exerted considerable effort selecting an appropriate plasmid vector that would give high enough expression of nsP2 and/or capsid to observe transcription or translation inhibition, and then to determine measurement times such that levels of viral proteins were high but not directly cytopathic, and the effects of auto-inhibitory activity on expressed protein levels would not give a false-negative result. The breakthrough was demonstrating significantly greater translation inhibition in IFN-primed cells expressing VEEV nsP2 over those expressing SINV, nsP2. This was achieved by manipulating the IFN dose and time of measurement and minimizing the effect of IFN priming on transfection efficiency and expression levels of nsP2.
Figure legend
VEEV nsP2 can shut off global host macromolecular synthesis and downregulate the antiviral state. (A and B) Cells primed with IFN and expressing nsP2 were labeled with [35S] Cys/Met to measure translation levels. VEEV nsP2 expression significantly reduced levels of host translation in primed cells. (C and D) Levels of representative ISGs (IFIT1 and TGTP) were measured in IFN primed cells expressing nsP2. Cells expressing VEEV nsP2 had lower ISG levels due to shutoff of translation. (E) Translation shutoff in IFN primed cells can support the replication of an IFN sensitive virus. Replication of Yellow fever 17-D virus in IFN primed cells expressing VEEV nsP2 was comparable to replication in unprimed cells.
Introducing the authors
Pictured left: Nishank Bhalla; right William B. Klimstra. Both from the Center for Vaccine Research, University of Pittsburgh.
About the research
Host translation shutoff mediated by non-structural protein 2 is a critical factor in the antiviral state resistance of Venezuelan equine encephalitis virus
Nishank Bhalla, Chengqun Sun, L.K. Metthew Lam, Christina L. Gardner, Kate D. Ryman, William B. Klimstra
Virology, Volume 496, September 2016, Pages 147–165
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