Emerman, MichaelItell, Hannah L.2024-02-122024-02-122024-02-122023Itell_washington_0250E_26302.pdfhttp://hdl.handle.net/1773/51216Thesis (Ph.D.)--University of Washington, 2023Cellular antiviral factors comprise one of the first lines of defense against viral infection. In the case of HIV-1, it is well established that the early stages of natural infection are inefficient, characterized by infrequent transmission, a severe bottleneck in viral genetic diversity during transmission, and variable viral loads between individuals in the first weeks of infection. Because HIV-1 transmission and acute infection occur prior to the onset of adaptive immune responses, cell-intrinsic factors that inhibit HIV-1 replication may contribute to the inefficiency of early HIV-1 infection.Many studies have identified cellular factors with potent antiviral activity against HIV-1, which work has shed light on the cell types permissive to HIV-1 infection, the host range of HIV- 1, and the role of HIV accessory proteins. Though these prior reports emphasize the significance of restriction factors, they do not provide direct insight into the genes that impact natural HIV-1 transmission and infection, as nearly all were performed in cell lines with lab-adapted viruses. This caveat is largely due to the fact that the main target cells of HIV-1 infection, primary CD4+ T cells, were not amenable to reliable experimental manipulation until recently. To understand which host factors are relevant during the early stages of HIV-1 infection in vivo, we adapted the high throughput HIV-CRISPR screening approach for use in primary CD4+ T cells. This development allowed us to comprehensively interrogate hundreds of genes upregulated by the innate interferon response (Chapter 2) as well as endogenously expressed genes (Chapter 3) for inhibitory activity against a primary HIV-1 virus in a physiologically relevant cell type. In Chapter 2, we identified novel, previously undescribed HIV-restricting interferon- stimulated genes (HM13, IGFBP2, LAP3). We also found that two factors characterized in other HIV-1 infection models (IFI16 and UBE2L6) mediated interferon restriction in primary target cells, although several others (MX2, IFITM1, TRIM5a) did not impact the primary HIV-1 strain in this cell type. Moreover, our results from inactivating single and combinations of antiviral interferon- stimulated genes suggest that interferon restriction of HIV-1 is multifaceted, resulting from several effectors functioning collectively as opposed to one potent gene. In Chapter 3, I specifically explored host factors that influence the HIV-1 transmission bottleneck, which results in the preferential transmission of CCR5-tropic viruses as opposed to CXCR4-tropic variants. We identified SLC35A2, a constitutively expressed gene involved in glycosylation, as an CXCR4- tropic-specific restriction factor. Inactivation of SLC35A2 impacted HIV-1 in a tropism-dependent manner by truncating host cell glycans, underscoring a previously unappreciated role for host cell glycosylation on HIV-1 that may contribute to the bottleneck during HIV-1 transmission. Together, these projects shed light on the cellular factors that likely influence HIV-1 transmission and acute infection and demonstrate the value of a high throughput screening approach for host genes that impact HIV-1 in primary target cells. Future studies should investigate the mechanisms of the restriction factors described here and leverage HIV-CRISPR screens in primary cells to study host genes that influence HIV-1 in other contexts.application/pdfen-USCC BY-NC-NDAntiviral geneCD4+ T cellCRISPR screenHIV-1Interferon-stimulated geneRestriction factorVirologyMolecular biologyCellular biologyMolecular and cellular biologyThesis