The HIV-1 capsid protein makes up the core of the virion and plays a critical role in early steps of HIV replication. Due to its exposure in the cytoplasm after entry, HIV capsid is a target for host cell factors that act directly to block infection such as TRIM5α and MxB. Several host proteins also play a role in facilitating infection, including in the protection of HIV-1 capsid from recognition by host cell restriction factors. Through an unbiased screening approach, called HIV-CRISPR, we show that the CPSF6-binding deficient, N74D HIV-1 capsid mutant is sensitive to restriction mediated by human TRIM34, a close paralog of the wellcharacterized HIV restriction factor TRIM5α. This restriction occurs at the step of reverse transcription, is independent of interferon stimulation, and limits HIV-1 infection in key target cells of HIV infection including CD4+ T cells and monocyte-derived dendritic cells. TRIM34 can also restrict some SIV capsids. TRIM34 restriction requires TRIM5α as knockout or knockdown of TRIM5α results in a loss of antiviral activity. Through immunofluorescence studies, we show that TRIM34 and TRIM5α colocalize to cytoplasmic bodies and are more frequently observed to be associated with infecting N74D capsids than with WT HIV-1 capsids. Our results identify TRIM34 as an HIV-1 CA-targeting restriction factor and highlight the potential role for heteromultimeric TRIM interactions in contributing to restriction of HIV-1 infection in human cells.

With a small genome of ~9.2 kb that encodes 14 major proteins, HIV must hijack host cellular machinery to successfully establish infection. These host proteins necessary for HIV replication are called “dependency factors.” Whole-genome, and then targeted screens were done to try to comprehensively identify all dependency factors acting throughout the HIV replication cycle.

1The HIV-1 capsid protein makes up the core of the virion and plays a critical role in early 2 steps of HIV replication. Due to its exposure in the cytoplasm after entry, HIV capsid is a 3 target for host cell factors that act directly to block infection such as TRIM5 and MxB. 4 Several host proteins also play a role in facilitating infection, including in the protection 5 of HIV-1 capsid from recognition by host cell restriction factors. Through an unbiased 6 screening approach, called HIV-CRISPR, we show that the Cyclophilin A-binding 7 deficient P90A HIV-1 capsid mutant becomes highly-sensitized to TRIM5alpha restriction 8 in IFN-treated cells. Further, the CPSF6-binding deficient, N74D HIV-1 capsid mutant is 9 sensitive to restriction mediated by human TRIM34, a close paralog of the well-10 characterized HIV restriction factor TRIM5. This restriction occurs at the step of reverse 11 transcription, is independent of interferon stimulation and limits HIV-1 infection in key 12 target cells of HIV infection including CD4+ T cells and monocyte-derived dendritic cells. 13 TRIM34 restriction requires TRIM5alpha as knockout or knockdown of TRIM5alpha 14 results in a loss of antiviral activity. TRIM34 can also restrict some SIV capsids. Through 15 immunofluorescence studies, we show that TRIM34 and TRIM5alpha colocalize to 16 cytoplasmic bodies and are more frequently observed to be associated with infecting 17 N74D capsids than with WT capsids. Our results identify TRIM34 as an HIV-1 CA-18 targeting restriction factor and highlight the potential role for heteromultimeric TRIM 19 interactions in contributing restriction of HIV-1 infection in human cells.

At each stage of the HIV life cycle, host cellular proteins are hijacked by the virus to establish and enhance infection. We adapted the virus packageable HIV-CRISPR screening technology at a genome-wide scale to comprehensively identify host factors that affect HIV replication in a human T cell line. Using a smaller, targeted HIV Dependency Factor (HIVDEP) sub-library, we then performed screens across multiple HIV strains representing different clades and with different biological properties to define which T cell host factors are strain-specific versus which ones are important across all HIV strains and different clades. Nearly 90% genes selected across multiple host pathways validated in subsequent assays as bona fide host dependency factors including numerous proteins not previously reported to play role in HIV biology such as UBE2M, MBNL1, FBXW7, PELP1, SLC39A7, and others. Our ranked list of screen hits across multiple viral strains form a resource of HIV dependency factors for future investigation of host proteins involved in HIV biology.

Human immunodeficiency virus (HIV) and other lentiviruses adapt to new hosts by evolving to evade host-specific innate immune proteins that differ in sequence and often viral recognition between host species. Understanding how these host antiviral proteins, called restriction factors, constrain lentivirus replication and transmission is key to understanding the emergence of pandemic viruses like HIV-1. Human TRIM34, a paralogue of the well-characterized lentiviral restriction factor TRIM5α, was previously identified by our lab via CRISPR-Cas9 screening as a restriction factor of certain HIV and SIV capsids. Here, we show that diverse primate TRIM34 orthologues from non-human primates can restrict a range of Simian Immunodeficiency Virus (SIV) capsids including SIVAGM-SAB, SIVAGM-TAN and SIVMAC capsids, which infect sabaeus monkeys, tantalus monkeys, and rhesus macaques, respectively. All primate TRIM34 orthologues tested, regardless of species of origin, were able to restrict this same subset of viral capsids. However, in all cases, this restriction also required the presence of TRIM5α. We demonstrate that TRIM5α is necessary, but not sufficient, for restriction of these capsids, and that human TRIM5α functionally interacts with TRIM34 from different species. Finally, we find that both the TRIM5α SPRY v1 loop and the TRIM34 SPRY domain are essential for TRIM34-mediated restriction. These data support a model in which TRIM34 is a broadly-conserved primate lentiviral restriction factor that acts in tandem with TRIM5α, such that together, these proteins can restrict capsids that neither can restrict alone.