Abstract
Venezuelan Equine Encephalitis Virus (VEEV) is a major biothreat agent that naturally causes outbreaks in humans and horses particularly in tropical areas of the western hemisphere, for which no antiviral therapy is currently available. The host response to VEEV and the cellular factors this alphavirus hijacks to support its effective replication or evade cellular immune responses are largely uncharacterized. We have previously demonstrated tremendous cell-to-cell heterogeneity in viral RNA (vRNA) and cellular transcript levels during flaviviral infection using a novel virus-inclusive single-cell RNA-Seq approach. Here, we used this unbiased, genome-wide approach to simultaneously profile the host transcriptome and vRNA in thousands of single cells during infection of human astrocytes with the live-attenuated vaccine strain of VEEV (TC-83). Host transcription was profoundly suppressed, yet "superproducer cells" with extremely high vRNA abundance emerged during the first viral life cycle and demonstrated an altered transcriptome relative to both uninfected cells and cells with high vRNA abundance harvested at later time points. Additionally, cells with increased structural-to-nonstructural transcript ratio exhibited upregulation of intracellular membrane trafficking genes at later time points. Loss- and gain-of-function experiments confirmed pro- and antiviral activities in both vaccine and virulent VEEV infections among the products of transcripts that positively or negatively correlated with vRNA abundance, respectively. Lastly, comparison with single cell transcriptomic data from other viruses highlighted common and unique pathways perturbed by infection across evolutionary scales. This study provides a high-resolution characterization of the VEEV (TC-83)-host interplay, identifies candidate targets for antivirals, and establishes a comparative single-cell approach to study the evolution of virus-host interactions.
Highlights
For more than a century, Venezuelan Equine Encephalitis Virus (VEEV), a member of the Alphavirus genus, has been the causative agent of outbreaks of febrile neurological disease in both animals and humans in Central and South America [1,2]
Little is known about the host response to Venezuelan Equine Encephalitis Virus (VEEV) and the cellular factors this alphavirus hijacks to support effective replication or evade cellular immune responses
A single-cell RNA sequencing technology that detects host and viral RNA was used to investigate the interactions between TC-83, the vaccine strain of VEEV, and the human host during the course of infection of U-87 MG cells
Summary
For more than a century, Venezuelan Equine Encephalitis Virus (VEEV), a member of the Alphavirus genus, has been the causative agent of outbreaks of febrile neurological disease in both animals and humans in Central and South America [1,2]. VEEV TC-83 replicates rapidly: viral protein production is observed as early as 6 hours postinfection (hpi) in human astrocytoma cells (U-87 MG) at multiplicity of infection (MOI) of 2, and over 1010 copies of intracellular viral RNA (vRNA) can be detected by 24 hpi [5]. It remains unknown, whether a large number of cells, each producing a small number of virions, or a few “superproducer” cells drive this effective virus production. Since the virus relies on cellular machineries, it is important to identify which host factors are “spared” from this shutdown, as they may represent essential factors for effective viral replication
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