Role of ZNF proteins in the establishment and maintenance of HIV latency - Title: Role of ZNF proteins in the establishment and maintenance of HIV latency Abstract Antiretroviral combination therapy against the human immunodeficiency virus (HIV) infection has significantly prolonged the life span of people living with HIV, transforming viral infection into a chronic condition with undetectable viral loads. However, a complete cure against HIV is still out of reach, as a pool of infected reservoir cells carrying a transcriptionally silent, yet replication-competent virus persists. Within these reservoirs, infected cells do not express viral proteins, and were until recently regarded to be protected from host immune responses. However, new single-cell analytic techniques revealed that this pool of infected cells displays discrete footprints of immune selection, implying that human immune responses effectively recognize at least some of them. In particular, genome-intact HIV preferentially persists in ZNF genes that impose proviral transcriptional silencing through the recruitment of transcriptional repressors that induce epigenetic heterochromatin markers. The central hypothesis of this study is that ZNF proteins have pivotal functions in establishing and maintaining the HIV-1 reservoir by silencing proviral transcriptional activity and protecting HIV-1 genomes from antiviral immune recognition. To validate this hypothesis, Aim 1 proposes to evaluate the role of ZNF proteins in changing the epigenetic landscape in primary CD4+ T cells. By employing CRISPR-CAS9, we will modulate the expression of selected ZNF genes in primary CD4+ T cells, and use well-established tools to assess the genome-wide epigenetic changes in the context of ZNF depletion. We expect to demonstrate that HIV-1 selectively persists at chromosomal locations that are susceptible to ZNF protein-dependent gene silencing through recruitment of heterochromatin markers. Aim 2 will then explore mechanisms of HIV-1 latency reversal through inhibition of ZNF proteins in CD4 T cells from participants living with HIV. We will use single-cell assays to investigate changes in proviral gene transcription following depletion of selected ZNF genes, applying tools permitting simultaneous assessments of HIV-1 transcription and integration. Based on these findings, specific pharmacological approaches that will target ZNF proteins may be developed that might help to eliminate the viral reservoir by increasing its immunological vulnerability. 1
