Abstract
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.
Highlights
Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic life cycle
SMARTpool small interfering RNA (siRNA) targeting 6 719 genes were distributed into 384-well plates and reverse-transfected into HeLa cells
After 48 h, eGFP fluorescence was quantified as a measure of infection and compared to controls in order to determine the effect of individual gene depletion on VACV replication
Summary
Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic life cycle. It is the prototypical member of the orthopoxviridae genus of the Poxviridae family which includes Variola virus (the causative agent of smallpox), Monkeypox virus and Ectromelia virus. VACV, like all other viruses, harnesses the cell to enable its replication. It turns off or subverts multiple crucial anti-viral pathways including cytokine production, Toll-like receptor pathways, NF-kB activation and the dsRNA PKR response [2,3,4,5,6,7,8]. Modulation of so many different signalling pathways prevents viral-induced premature cell death and contributes to the ability of poxviruses to replicate in a wide range of cell types
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