Explorer Identification of host factors involved in human cytomegalovirus replication, assembly and egress using a two-step siRNA screen

As obligate intracellular parasites, viruses are completely dependent on host factors for replication. Assembly and egress of complex virus particles, such as human cytomegalovirus (HCMV), are likely to require many host factors. Despite this, relatively few have been identiﬁed and characterized. This study describes a novel high-throughput, two-step small interfering RNA (siRNA) screen, which indepen-dently measures virus replication and virus production. By combining data from replication and virus production, multiple candidate genes were identiﬁed in which knockdown resulted in substantial loss of virus production with limited effect on primary replication, suggesting roles in later stages such as virus assembly and egress. Knockdown of the top candidates, ERC1, RAB4B, COPA, and COPB2, caused profound loss of virus production. Despite COPA and COPB2 being reported to function in the same complex, knockdown of these genes produced distinct phenotypes. Further-more, knockdown of COPA caused increased expression of viral late genes despite substantial inhibition of viral DNA replication. This suggests that efﬁcient viral genome replication is not required for late gene expression. Finally, we show that RAB4B relocates to the viral assembly compartment following infection with HCMV and knockdown of RAB4B reduces the release of intact virion particles, suggesting that it plays a role in virion assembly and egress. This study demonstrates a powerful high-throughput screen for identiﬁcation of host-virus interactions, identiﬁes multiple host genes associated with HCMV assembly and egress, and uncovers potentially independent functions for coatomer components COPA and COPB2 during infection. individuals with heart disease, and recipients of solid organ and bone marrow transplants. HCMV is also the leading cause of infectious congenital birth defects. The majority of antivirals in clinical use target components of the virus to speciﬁcally inhibit replication. However, a major drawback of this approach is the emergence of resistance. An alternative approach is to target host factors that the virus requires for successful infection. In this study, multiple host factors were identiﬁed that were found to be essential for the production of newly infectious human cytomegalovirus. Identifying which host genes are necessary for virus replication extends our understanding of how viruses replicate and how cells function and provides potential targets for novel antivirals. ﬂuores- cence intensity (MFI) range of negative-control (uninfected) and positive-control (infected) cells was used to deﬁne the MFI range of GFP-negative and GFP-positive cells. The fold change of GFP-positive cells following siCOPA, siCOPB2, siERC1, and siRAB4B treatment was determined relative to GFP-positive cells from siNeg-treated wells at each MOI and time point. Western blot analysis. Following transfection, cells were harvested daily at 1 to 7 dpi in RIPA buffer. Protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo Fisher) according to the manufacturer’s instructions. Proteins were separated on 4 to 12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes by either wet transfer (20% methanol [MeOH]) or Trans-Blot SD semidry transfer (mixed molecular weight). Membranes were probed with antibodies to viral proteins IE1 (Merck Millipore; MAB8131), IE2 (Merck Millipore; MAB8131), pp52 (Santa Cruz Biotech- nology; sc-56971), and pp28 (Santa Cruz Biotechnology; sc-69749) and with antibodies to host proteins and of correlations. Two-tailed ho- moscedastic Student’s t test was applied to assess whether primary replication and virus production results differed signiﬁcantly ( P (cid:3) 0.05, NS; P (cid:2) 0.05, * ; P (cid:2) 0.01, ** ; P (cid:2) 0.001, *** ; P (cid:2) 0.0001, **** ).