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

Viral Mediated Redirection of NEMO/IKKγ to Autophagosomes Curtails the Inflammatory Cascade

The early host response to viral infections involves transient activation of pattern recognition receptors leading to an induction of inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα). Subsequent activation of cytokine receptors in an autocrine and paracrine manner results in an inflammatory cascade. The precise mechanisms by which viruses avert an inflammatory cascade are incompletely understood. Nuclear factor (NF)-κB is a central regulator of the inflammatory signaling cascade that is controlled by inhibitor of NF-κB (IκB) proteins and the IκB kinase (IKK) complex. In this study we show that murine cytomegalovirus inhibits the inflammatory cascade by blocking Toll-like receptor (TLR) and IL-1 receptor-dependent NF-κB activation. Inhibition occurs through an interaction of the viral M45 protein with the NF-κB essential modulator (NEMO), the regulatory subunit of the IKK complex. M45 induces proteasome-independent degradation of NEMO by targeting NEMO to autophagosomes for subsequent degradation in lysosomes. We propose that the selective and irreversible degradation of a central regulatory protein by autophagy represents a new viral strategy to dampen the inflammatory response

Transcriptional modulation of <i>Vaccinia virus</i> HFs.

<p>Plot of seven VACV HFs identified in the RNAi screen that are also strongly transcriptionally regulated in VACV infected cells. The x-axis represents the level of fluorescence in the RNAi screen (viral replication) expressed as a z-score with pro-viral genes to the left and anti-viral genes to the right. The y-axis represents the relative expression of the seven genes in VACV infected cells.</p

Validation of <i>Vaccinia virus</i> HFs.

<p>(a) Validation of primary screen hits using plaque assays. siRNA SMARTpools targeting five genes identified in the primary RNAi screen as modulating VACV growth (one anti-viral factor MAP3K14 and four pro-viral factors TRIP, PPAP2A, VPS52 and CCT7), and one non-specific SMARTpool (VP16) were transfected into HeLa cells and, after 48 h, infected at low MOI (0.05) with VACV-A5eGFP. At 12 h intervals, cells were collected and the amount of virus present calculated using a plaque assay. Results obtained in the primary RNAi screen are plotted on the right hand axis for comparison.</p

Identification of anti and pro-viral HFs common to multiple RNAi viral screens.

<p>Venn diagram showing the (a) pro-viral and (b) anti-viral hits common to at least two VACV RNAi screens and (c) hits common to the VACV screen reported in this study and three published influenza A RNAi screens with a total of 662 hits <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098431#pone.0098431-Brass1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098431#pone.0098431-Karlas1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098431#pone.0098431-Konig2" target="_blank">[47]</a> and three published HIV RNAi screens with a total of 826 hits <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098431#pone.0098431-Brass2" target="_blank">[41]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098431#pone.0098431-Zhou1" target="_blank">[43]</a>.</p