Identification of potential HIV restriction factors by combining evolutionary signatures with functional analyses

Abstract

Restrictions factors are host cell encoded proteins that interfere with multiple steps of human immunodeficiency virus (HIV) replication to inhibit viral spread. These antiretroviral factors are commonly under positive selection, upregulated during HIV-1 infection, and/or frequently interacting with viral proteins. Here, I combined evolutionary genomics, transcriptomics, and protein interaction data to identify thirty cellular genes sharing these characteristic features of known restriction factors. Cotransfection of a high proportion of candidate genes with HIV-1 proviruses impaired the production of infectious HIV-1 without affecting cell viability. Among them were members of the APOL and TNFR superfamilies, as well as CD164 that suppressed HIV-1 production by more than 90%. In the absence of the accessory viral proteins Vpr, Vpu and Nef, which are known to antagonize antiviral factors, additional candidate factors such as GBP5, IFI16, and SPN achieved more than 90% reduction. Notably, this screening approach also led to the identification of two genes (IL-1A and SP110) that enhanced HIV-1 production. Titration experiments for several factors confirmed their impact on HIV-1 and flow cytometric analyses, p24 capsid ELISA and Western blot revealed that many candidate genes influenced HIV-1 gene transcription and/or translation. Many factors exerted inhibitory effects on viral promoters not limited to the HIV-1 promoter. In addition, Western blot analyses demonstrated that CD164, CD1A, CD3G, GBP5, OAS1 and SPN might reduce virion infectivity by interfering with Env function. In summary, my results show that the number of human genes sharing the characteristics of known HIV restriction factors is very limited and are a first step towards clarifying how many of them display specific antiretroviral activity. Further analyses will reveal which of the newly identified candidate restriction factors inhibit HIV-1 replication in primary human cells.