Role of novel pro-viral cellular proteins in the replication of Vaccinia virus

Abstract

Vaccinia virus (VACV), the prototypic poxvirus, undergoes a complex life cycle, with multiple stages that are not yet fully understood. This work studied two cellular proteins which had previously been identified by siRNA screens as playing proviral roles in the replication cycle of VACV: the dual specificity mitogen-activated protein kinase kinase 3 (MKK3) and vacuolar protein sorting 52 (Vps52). MKK3 is an upstream regulator in the p38 pathway which, along with MKK6, phosphorylates and therefore activates p38. In HeLa cell cultures, siRNA depletion experiments confirmed that MKK3 supported VACV replication. MKK3 knockdown reduced production of both early and late-class VACV proteins, suggesting that it facilitates viral gene expression. However, this difference did not translate to an in vivo model, as comparison between wild type and MKK3 knockout mice infected with VACV revealed no significant differences in virus replication or overall disease. The Golgi-associated retrograde protein complex (GARP) is composed of four large heteromeric proteins: Vps51, Vps52, Vps53 and Vps54, and plays a key role in retrograde transport from endosomes to the TGN. The effects of loss of GARP function were investigated using three techniques: mouse embryonic fibroblasts (MEFs) containing the hypomorphic Vps54 “wobbler” mutation, Vps52-targetting siRNA in HeLa cells and pharmacological inhibition of retrograde transport using the drug Retro-2. GARP loss resulted in a marked reduction in VACV spread due to a reduction specifically in “double wrapped” extracellular enveloped virion (EEV) production. Investigation of the mechanism by which GARP facilitates EEV production revealed a disruption of the VACV morphogenesis pathway prior to the double wrapping event, resulting in mislocalisation and aggregation of the viral membrane protein B5 within the cytoplasm. The effects of GARP loss translated to an in vivo model, as mice infected with VACV and treated with Retro-2 exhibited reduced viral replication and overall disease. These results identify GARP as a pro-viral host complex required for EEV production, and suggest that cellular retrograde transport pathways are required for double-wrapping of VACV virions. Overall, the study illustrates both the potential pitfalls of carrying out genetic screens in a transformed cell line and the power of such studies to nevertheless identify novel features of virus biology as well as druggable targets for antiviral intervention.