Vaccinia Virus Uses Retromer-Independent Cellular Retrograde Transport Pathways To Facilitate the Wrapping of Intracellular Mature Virions during Virus Morphogenesis.

Poxviruses, 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.

RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions.

Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor.

A loss of function analysis of host factors influencing Vaccinia virus replication by RNA interference.

Vaccinia virus (VACV) is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA) screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF) influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics.

TRAF2 facilitates vaccinia virus replication by promoting rapid virus entry.

UNLABELLED: Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) is a pivotal intracellular mediator of signaling pathways downstream of TNFR1 and -2 with known pro- and antiviral effects. We investigated its role in the replication of the prototype poxvirus vaccinia virus (VACV). Loss of TRAF2 expression, either through small interfering RNA treatment of HeLa cells or through genetic knockout in murine embryonic fibroblasts (MEFs), led to significant reductions in VACV growth following low-multiplicity infection. In single-cycle infections, there was delayed production of both early and late VACV proteins as well as accelerated virus-induced alterations to cell morphology, indicating that TRAF2 influences early stages of virus replication. Consistent with an early role, uncoating assays showed normal virus attachment but delayed virus entry in the absence of TRAF2. Although alterations to c-Jun N-terminal kinase (JNK) signaling were apparent in VACV-infected TRAF2(-/-) MEFs, treatment of wild-type cells with a JNK inhibitor did not affect virus entry. Instead, treatment with an inhibitor of endosomal acidification greatly reduced virus entry into TRAF2(-/-) MEFs, suggesting that VACV is reliant on the endosomal route of entry in the absence of TRAF2. Thus, TRAF2 is a proviral factor for VACV that plays a role in promoting efficient viral entry, most likely via the plasma membrane. IMPORTANCE: Tumor necrosis factor receptor-associated factors (TRAFs) are key facilitators of intracellular signaling with roles in innate and adaptive immunity and stress responses. We have discovered that TRAF2 is a proviral factor in vaccinia virus replication in both HeLa cells and mouse embryonic fibroblasts and that its influence is exercised through promotion of efficient virus entry.