Abstract
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus causing outbreaks of severe disease in humans, with a fatality rate approaching 30%. There are no widely accepted therapeutics available to prevent or treat the disease. CCHFV enters host cells through clathrin-mediated endocytosis and is subsequently transported to an acidified compartment where the fusion of virus envelope with cellular membranes takes place. To better understand the uptake pathway, we sought to identify host factors controlling CCHFV transport through the cell. We demonstrate that after passing through early endosomes in a Rab5-dependent manner, CCHFV is delivered to multivesicular bodies (MVBs). Virus particles localized to MVBs approximately 1 hour after infection and affected the distribution of the organelle within cells. Interestingly, blocking Rab7 activity had no effect on association of the virus with MVBs. Productive virus infection depended on phosphatidylinositol 3-kinase (PI3K) activity, which meditates the formation of functional MVBs. Silencing Tsg101, Vps24, Vps4B, or Alix/Aip1, components of the endosomal sorting complex required for transport (ESCRT) pathway controlling MVB biogenesis, inhibited infection of wild-type virus as well as a novel pseudotyped vesicular stomatitis virus (VSV) bearing CCHFV glycoprotein, supporting a role for the MVB pathway in CCHFV entry. We further demonstrate that blocking transport out of MVBs still allowed virus entry while preventing vesicular acidification, required for membrane fusion, trapped virions in the MVBs. These findings suggest that MVBs are necessary for infection and are the sites of virus-endosome membrane fusion.
Highlights
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tickborne virus causing outbreaks of severe hemorrhagic disease in humans, with a fatality rate approaching 30%
While it has been demonstrated that CCHFV cell entry depends on clathrin-mediated endocytosis, low pH, and early endosomes, the identity of the endosomes where virus penetrates into cell cytoplasm to initiate genome replication is unknown
We showed that CCHFV was transported through early endosomes to multivesicular bodies (MVBs)
Summary
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tickborne virus causing outbreaks of severe hemorrhagic disease in humans, with a fatality rate approaching 30%. Despite the high mortality and global distribution of CCHFV, there are presently no licensed therapeutics to prevent or treat the disease. It is an enveloped, pleomorphic virus, possessing a tripartite single-stranded RNA genome in negative orientation. The small segment, S, encodes the nucleocapsid protein N, whose role is to encapsidate viral RNA during transcription and genome replication. The large segment, L, encodes the RNA-dependent RNA polymerase, which associates with N to form the viral polymerase complex [5]. The Gc and Gn form complexes on the virion surface and are responsible for binding to the cellular receptors and subsequent fusion of the viral envelope with host membranes [10,11]
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