Abstract
Poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple-membrane particles, termed intracellular enveloped virions (IEVs), are released from infected cells by fusion. Whereas the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to an RNAi screen directed against cellular membrane-trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of MVB-IEVs showed that they account for half of IEV production indicating that MVBs are a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread.
Highlights
All enveloped viruses must modulate cellular membrane systems throughout their lifecycles, from hijacking endosomes for entry and diverting cellular membrane compartments for replication, to taking over cellular secretion systems for viral egress and immune evasion (Welsch et al, 2007; Mercer et al, 2010; Glingston et al, 2019)
To identify novel cell factors used by vaccinia virus (VACV) to facilitate virus spread, we developed a fluorescent plaque formation assay compatible with RNAi screening (Fig 1A)
Quantification of the number of primary and secondary infected cells allowed us to differentiate between defects in primary infection by intracellular mature virions (IMVs) and defects in virus spread which could be caused by attenuated virion formation or entry of cell-associated enveloped virions (CEVs) or enveloped virions (EEVs) into surround cells
Summary
All enveloped viruses must modulate cellular membrane systems throughout their lifecycles, from hijacking endosomes for entry and diverting cellular membrane compartments for replication, to taking over cellular secretion systems for viral egress and immune evasion (Welsch et al, 2007; Mercer et al, 2010; Glingston et al, 2019). As exclusively cytoplasmic replicating viruses, poxviruses are no exception to this rule (Moss, 2007) As these viruses produce two distinct infectious virion forms—with multiple membranes sequestered from different cellular membrane compartments—poxviruses are perhaps more reliant on modulating cellular membrane compartments than most other enveloped viruses. Once formed, a subset of IMVs is transported to designated wrapping sites where they acquire two additional virus-modified membranes (Smith et al, 2002; Condit et al, 2006; Moss, 2007; Roberts & Smith, 2008). These triplemembrane virions termed intracellular enveloped virions (IEVs) are transported to the cell surface where they undergo fusion. Leaving behind their outermost membrane, these double-membrane virions remain as cell-associated enveloped virions (CEVs) or are released to become EEVs
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