Abstract
ABSTRACTPoxviruses, such as vaccinia virus (VACV), undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms and includes a crucial wrapping step whereby intracellular mature virions (IMVs) are wrapped in two additional membranes to form intracellular enveloped virions (IEVs). To determine if cellular retrograde transport pathways are required for this wrapping step, we examined VACV morphogenesis in cells with reduced expression of the tetrameric tethering factor known as the GARP (Golgi-associated retrograde pathway), a central component of retrograde transport. VACV multistep replication was significantly impaired in cells transfected with small interfering RNA targeting the GARP complex and in cells with a mutated GARP complex. Detailed analysis revealed that depletion of the GARP complex resulted in a reduction in the number of IEVs, thereby linking retrograde transport with the wrapping of IMVs. In addition, foci of viral wrapping membrane proteins without an associated internal core accumulated in cells with a mutated GARP complex, suggesting that impaired retrograde transport uncouples nascent IMVs from the IEV membranes at the site of wrapping. Finally, small-molecule inhibitors of retrograde transport strongly suppressed VACV multistep growth in vitro and reduced weight loss and clinical signs in an in vivo murine model of systemic poxviral disease. This work links cellular retrograde transport pathways with the morphogenesis of poxviruses and identifies a panel of novel inhibitors of poxvirus replication.IMPORTANCE Cellular retrograde transport pathways traffic cargo from endosomes to the trans-Golgi network and are a key part of the intracellular membrane network. This work reveals that the prototypic poxvirus vaccinia virus (VACV) exploits cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions and therefore promote the production of extracellular virus. Inhibition of retrograde transport by small-molecule inhibitors reduced the replication of VACV in cell culture and alleviated disease in mice experimentally infected with VACV. This research provides fundamental new knowledge about the wrapping step of poxvirus morphogenesis, furthers our knowledge of the complex cellular retrograde pathways, and identifies a new group of antipoxvirus drugs.
Highlights
Cellular retrograde transport pathways traffic cargo from endosomes to the trans-Golgi network and are a key part of the intracellular membrane network
This work reveals that the prototypic poxvirus vaccinia virus (VACV) exploits cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions and promote the production of extracellular virus
In cells transfected with VPS52-targeting small interfering RNA (siRNA), the level of VPS52 was consistently reduced to approximately 60% of the level in cells treated with nontargeting control siRNA (Fig. 1A and B), confirming the efficacy of the SMARTpool
Summary
Cellular retrograde transport pathways traffic cargo from endosomes to the trans-Golgi network and are a key part of the intracellular membrane network. Endosome-to-Golgi retrograde transport pathway (EGRTP) proteins have been identified to be proviral host factors in two independent high-throughput small interfering RNA (siRNA) screens of VACV replication [13, 14]. This evidence collectively supports the utilization of EGRTP by VACV; direct evidence is lacking. The pathway can begin at early, late, or recycling endosomes, where cargo-sorting complexes, such as retromer, recognize and select cargo and sort it into vesicles, which are transported to the TGN [15] Tethering factors, such as golgin-97 and the GARP (Golgi-associated retrograde protein) complex, tether the transport intermediates to the TGN and enable SNARE (soluble N-ethylmaleimide-sensitive fusion factor attachment receptor) complexes on the transport vesicles (vSNAREs) and target membrane (tSNAREs) to promote membrane fusion. Endosome-toGolgi retrograde pathways are key physiological transport routes that are implicated in a number of pathogenic mechanisms
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