CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

WT SIVmac 239 and a mutant lacking vpr . To determine whether RhoA impacts HIV-1 in human cells, we knocked out RHOA in primary CD4 + T cells by sgRNA/ Cas9 electroporation. To examine the interplay between Vpr and RhoA, we performed proximity ligation assays (PLAs), western blot, immunoprecipitation and analyses of type I IFN signaling activity using reporter cell lines and qRT-PCR. Results: Lack of Vpr associated with significantly increased levels of RhoA signaling pathway in SIVmac 239 infected macaques (n=3 animals, p value 0.0447). Similarly, vpr -deficient HIV-1 was more susceptible to inhibition by RhoA in primary human CD4 + T cells than WT HIV-1. PLA revealed that Vpr and RhoA are in close proximity. Our data further indicate that RhoA promotes IFN signalling by increasing pSTAT-1 levels and may be degraded by Vpr. Conclusions: Altogether, we identified RhoA as novel antiviral factor that restricts HIV-1 by promoting IFN signaling and is counteracted by Vpr. Background: Primate lentiviruses (SIVs) have been evolving with their hosts for millions of years. Capsid-interacting dependency and restriction factors are known to play a key role in HIV replication. Small changes in viral capsid sequence can alter these interactions for the benefit of the virus (e.g. to escape a restriction factor). Previous work has focused primarily on interactions between only a few primate lentiviruses and host proteins. We do not understand what host factors are important for lentiviral replication more broadly. Further we lack understanding of how differences in capsid sequence impact these interactions to influence outcomes of lentiviral infection more broadly. Methods: I created a panel of capsid chimeric lentiviruses by synthesizing capsid sequences from 10 different SIV strains selected to represent much of the diversity of known SIV sequences and constructing chimeric molecular clones in an HIV-1 NL4-3∆envGFP proviral backbone. I infected HeLa and Jurkat cell lines with chimeric viruses pseudotyped with VSV-G and measured infectivity 2 days post infection. To identify unknown restriction factors, I used an unbiased screening approach, HIV-CRISPR in Jurkat cells. Results: Of these chimeric viruses, six infect both Jurkat and HeLa cell lines but to varying degrees. All chimeric viruses have reduced infectivity compared to the wild type HIV-1 virus. Three chimeric viruses, SIVmac, SIVver, and SIVgrv, show reduced infectivity in Jurkat cells compared to HeLa cells. The restriction phenotype of SIVmac CA in Jurkat cells known as “Lv4” was previously described to be the result of a dominant-acting restriction factor. HIV-CRISPR screening in Jurkat cells with the SIVmac capsid chimeric virus revealed NFKBIA as a putative restriction factor specific to SIVmac CA compared to NL4-3 WT. NFKBIA is an inhibitor of NFKB, an immune transcription factor that also drives proviral transcription. This suggests that NFKBIA or NFKB differentially affect infection or transcription of the SIVmac capsid chimera. Conclusions: We see reduced replication of SIVmac, SIVver, and SIVgrv capsid chimeric viruses in Jurkat cells. Using HIV-CRISPR screening of the SIVmac chimeric virus we identified a candidate gene, NFKBIA, that may be responsible for the restriction of SIVmac capsids in Jurkat cells. This demonstrates that HIV CRISPR screening using capsid chimeric viruses can identify unique capsid-host factor interactions, that reveal new insights into HIV and SIV biology. Capsid Mutant HIV-CRISPR Screens Reveal Mechanisms of TRIM5 Restriction Background: Upon entry into a cell, the HIV core plays a pivotal part in many of the interactions that lead to successful viral replication. The HIV core has evolved to interact with proteins necessary for replication and evade proteins that inhibit replication. We aim to characterize the mechanisms of capsid (CA) recognition by known and novel antiviral proteins. Methods: Here we use poorly adapted CA mutant viruses to learn more about the mechanisms of capsid recognition by host antiviral proteins. N57A, A77V, and A105T CA mutant viruses that have altered interactions with host factors including CPSF6 and nuclear pore proteins. To identify host genes that contribute to reduced fitness of these mutant viruses we use HIV-CRISPR screening, a screening approach that allows us to uncover proviral and antiviral genes. Isaiah Grant, Laura Gong, Molly Ohainle University of California Berkeley, Berkeley, CA, USA The Role of Capsid-Host Interaction on Lentiviral Evolution Clare Gill, Molly Ohainle University of California Berkeley, Berkeley, CA, USA

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The Effects of Accessory Genes on T/F HIV Infection of Primary CD4+ T Cells From Tissues Harnoor Virk 1 , Guorui Xie 1 , Xiaoyu Luo 1 , Kailin Yin 1 , Jason Neidleman 1 , Ifeanyi Ezeonwumelu 2 , Julie Frouard 1 , Ashley F. George 1 , Daniel Sauter 3 , Frank Kirchhoff 4 , Nadia R. Roan 1 1 Gladstone Institutes, San Francisco, CA, USA, 2 Gladstone Institute of Virology and Immunology, San Francisco, CA, USA, 3 Tübingen University Hospital, Tübingen, Germany, 4 Ulm University Medical Center, Ulm, Germany Background: HIV transmission typically results from infection by a single a transmitted/founder (T/F) virus. Accessory genes of T/F HIV often exhibit functions lost in cell culture-adapted HIV strains. Understanding the effects of accessory proteins from T/F HIV on infected cells is important for understanding HIV pathogenesis. Previous work has established that accessory proteins Nef, Vpu, and Vpr are important for HIV fitness. Both Nef and Vpu are known to downregulate a variety of host proteins from the cell surface, including CD4. However, prior accessory gene studies were largely performed in cell lines or PBMCs, whereas in vivo HIV primarily replicates in tissues. Here, we compared by CyTOF primary tissue CD4+ T cells infected with wildtype T/F HIV CH077, to those infected with CH077 deleted individually for nef , vpu , or vpr . Methods: A 34-parameter CyTOF panel was implemented on unstimulated tonsils from 9 donors, that each were mock-treated, infected with wildtype CH077, or infected with CH077 deleted for nef , vpu , or vpr (dNef, dVpu, and dVpr). HIV-infected cells were identified as p24+ CD3+CD8- cells. Results: CH077 infection rates were similar between wildtype and accessory gene mutants. Overall, dVpu-infected cells exhibited the most phenotypic changes as compared to wildtype-infected cells. As expected, dNef and dVpu infected cells exhibited less cell-surface CD4 downregulation than wildtype infected cells. Cells infected with wildtype CH077 also downregulated a4b7 and CD62L when compared to those infected with dNef and dVpu. Relative to dVpu-infected cells, those infected with wildtype CH077 additionally exhibited diminished expression of CD38, CXCR4, and CD127. No antigens were identified that were preferentially downregulated on cells infected with wildtype as compared to dVpr virus. However, dVpr-infected cells had diminished CCR4 and TIGIT relative to wildtype-infected cells. Conclusions: We identify a4b7, CD62L, CD38, CXCR4, and CD127 as potential antigens downregulated by Vpu in the context of T/F HIV infection of primary tonsillar CD4+ T cells. Of these, a4b7 and CD62L may also be downregulated by T/F Nef. We did not find evidence for Vpr-mediated downregulation of cell-surface proteins; however, dVpr-infected cells expressed low levels of chemokine receptor CCR4 and checkpoint molecule TIGIT relative to their wildtype infected counterparts, suggesting a potential role for Vpr in regulating cell-surface expression levels of receptors commonly expressed on CD4+ T cells. RhoA Restricts HIV-1 by Modulating IFN Signaling and Is Counteracted by Vpr Caterina Prelli Bozzo 1 , Alexandre Laliberté 2 , Aurora De Luna 2 , Meta Volcic 2 , Helene Hönigsperger 2 , Maximilian Hirschenberger 2 , Denise Clesle 2 , Annika Betzler 2 , Konstantin M. J. Sparrer 2 , Frank Kirchhoff 2 1 Yale University, New Haven, CT, USA, 2 Ulm University Medical Center, Ulm, Germany Background: Cellular innate defense mechanisms govern the outcome of pathogen exposure. Numerous restriction factors (RFs) that may inhibit HIV-1 and other viral pathogens have been reported. However, it has also become clear that we are still missing important RFs. To uncover them, we recently established a virus-driven CRISPR/Cas9 system using replication-competent HIV-1 expressing sgRNAs to identify novel RFs as well as antiviral mechanisms (Prelli Bozzo, Laliberté et al. 2024). These studies revealed that targeting RHOA conferred replication advantage to primary transmitted-founder HIV-1. Methods: To assess the role of RhoA and its putative counteraction by Vpr in vivo , we analyzed the RhoA signaling activity in rhesus macaques infected with

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CROI 2025