Systematic identification of host factors influencing human cytomegalovirus replication
A high-throughput two-step siRNA screen was performed to identify novel host factors required during human cytomegalovirus (HCMV) replication. Almost 7,000 cellular genes were targeted by the siRNA library and 81 candidates, of which gene depletion led to a 50% decrease or 25% increase in virus production, were identified. Functional annotation clustering by STRING revealed functional clusters in mediator complex, proteasome, ubiquitin-dependent protein degradation and histone modification. Focused characterisation of asparagine synthetase (ASNS) demonstrated a strict requirement for asparagine for HCMV replication. Depletion of intracellular asparagine levels, as a result of ASNS knockdown, led to a reduced expression of viral immediate-early protein IE2 but not IE1. The reduction on protein expression was not due to a general hindrance of protein synthesis since replication of herpes simplex virus-1 or influenza A virus was not affected. Furthermore, mTOR signalling was maintained during ASNS knockdown, and asparagine supplementation during the course of infection can completely rescue virus replication, suggesting a potential signalling pathway regulated by asparagine required for HCMV replication. Detailed characterisation of another hit, SIN3A, showed a novel DNA damage and repair mechanism required by HCMV. SIN3A forms a complex with histone deacetylase 1 and 2 (HDAC1 and HDAC2), and the complex can exert a range of functions including DNA damage response. SIN3A knockdown had a moderate effect on viral immediate-early and early gene expression, however, production of infectious virions was substantially inhibited. Further studies showed that the expression of a DNA damage marker, γH2AX, is increased and deleterious DNA-RNA hybrids (R-loop) accumulate during HCMV replication when SIN3A is absent. This study represents the most comprehensive siRNA screen to identify host factors involved in HCMV replication and production to date, and identifies metabolism of non-essential amino acid asparagine and SIN3A/HDAC complex as critical host factors for HCMV.