As partial validation, Evans et al.
] compared their predictions with the findings of several genome-wide RNAi screens, with varying results. In those surveys, almost the entire human genome was screened by individual gene silencing in HIV infection or transfection systems. The RNAi screens rest on the assumption that any reduction in HIV production resulting from the knockdown of an individual gene implies a role for that gene in virus replication.
Using a HeLa-CD4 cell line, Brass et al.
] and Zhou et al.
] both reported more than 200 host-dependency factors. Following transfection with small interfering RNAs (siRNAs), Brass et al.
] implicated cellular factors on the basis of their effects on two indicators: the production of viral Gag p24 antigen during early viral replication; and the infectivity of culture supernatant from siRNA-transfected cells. The screen by Zhou et al.
] was carried out in a similar manner, but with infectivity monitored over a longer period of time. A similar screen by König et al.
] was carried out with a vesicular stomatitis virus glyco-protein (VSV-G)-pseudotyped virus in 293T cells; while substituting the HIV envelope with VSV-G allowed for the incorporation of the HIV core into the 293T cells, the experimental design precluded the identification of host factors that participate in viral entry mediated by fusion of the HIV envelope with the cellular membrane. As a result, only events from uncoating of the viral core to viral gene expression were detectable by that siRNA screen.
Despite the differences in experimental designs, the three screens [4
] reported similar findings at the level of cellular functions. In the two screens performed in HeLa-CD4 cells that captured the production of infectious virions, both groups [4
] identified large numbers of cellular factors involved in Tat-mediated transcription as part of the Mediator complex, and also cellular factors involved in energy metabolism regulated by the Akt kinase. Similarly, Brass et al.
] and König et al.
] both identified constituents of the nuclear pore complex, presumably related to the nuclear entry of the viral pre-integration complex. However, although all three screens [4
] each reported between 200 and 300 cellular genes, together they reported a total of 842 unique genes as contributing to HIV replication. Only three genes were commonly reported by all three screens.
Perhaps more significantly, as revealed in a recent meta-analysis of the genome-wide screens [7
], several known cellular cofactors of HIV replication were not identified by any of the siRNA screens [4
]. Missing were the cell-surface antigens HLA-B57 and HLA-C, both of which regulate the immune response to HIV and have known effects on viral loads and disease progression; also missing was the integration cofactor LEDGF/p75. Although components of the ubiquitylation machinery were identified, members of the Tsg101/ESCRT pathway, which is specifically co-opted for the virus to leave the cell, were also missing. In addition, proteins such as the Gag-binding protein cyclophilin A and the HIV long terminal repeat binding partner Sp1 were identified only in the 293T-based screen [5
]. The false negatives arising from siRNA screens can be attributed to the fact that cellular proteins that are particularly abundant and stable cannot be easily knocked down by siRNA transfection in the time available in these experiments. Furthermore, RNAi specificity requirements and siRNA off-target effects can introduce false positives and negatives into the analysis. Considering the various gaps in identification by the siRNA screens, it should come as no surprise that Evans et al.
] reported a moderate overlap between findings from the siRNA screens [4
] and those that they made on the basis of conserved ELMs and counter domains.