Tafrate, S. | Search bioRxiv

bioRxiv2026

An Aurora Kinase-Dependent Role for Vif in Regulating HIV-1-Induced Cell-Cell Fusion

Viral infectivity factor (Vif) is an HIV-1 accessory protein best known for its counteraction of APOBEC3 enzymes, interferon-inducible host defenses against viral infection, as well as PPP2R5A-E, which are regulatory subunits of the PP2A cellular phosphatase holoenzyme, resulting in striking Vif-dependent phosphoproteome remodeling. One reported consequence of this remodeling is hyperphosphorylation of several Aurora kinase substrates in HIV-1 infected cells, which is reversed when Vif-deficient virus is used. We previously showed that infection of T cells with Vif-deficient HIV-1 results in significantly accelerated formation of syncytia compared to wild-type HIV-1 infection. More recently, others have shown that application of Aurora kinase B inhibitors during HIV-1 infection in T cells also results in a similar hyperfusogenic phenotype. Both effects were specific to Env-driven cell-cell fusion, and did not influence virus infectivity. We thus hypothesized that Vifs influence on the rate of HIV-1-induced cell-cell fusion was mediated by Aurora kinase activity. To start testing this hypothesis, we have evaluated the effects of a small panel of Aurora kinase inhibitors on HIV-1-induced cell-cell fusion in the presence or absence of Vif. Our results replicate the previously documented increase in cell-cell fusion in the absence of Vif, as well as the increase in cell-cell fusion observed upon inhibition of Aurora kinase B in the presence of Vif. Critically, we now present evidence that Vif deletion significantly blunts the impact of Aurora kinase inhibition on cell-cell fusion, supporting our hypothesis that Vif-mediated regulation of cell-cell fusion depends on Aurora kinase signaling dysregulation, likely because of PPP2R5A-E degradation. Further, we document that the cell-cell fusion regulator downstream of Aurora kinase signaling is likely Ezrin, which we have previously shown to prevent excess HIV-1-induced syncytium formation when in its phosphorylated (activated) state. Taken together, these findings establish a Vif, Aurora kinase, and Ezrin-dependent mechanistic framework for the regulation of HIV-1-induced cell-cell fusion in infected T cells which likely helps preserve optimal cell-to-cell virus transmission.

Cell Biology↗

bioRxiv2026

Bloom syndrome helicase is required for efficient HIV-1 reverse transcription in macrophages

The induction of DNA damage by HIV-1 prior to integration suggests a function for DNA damage responses (DDR) during early infection, however what this role is remains incompletely understood. Initial experiments, using specific inhibitors for DDR pathways demonstrate that both ATM and ATR are necessary for efficient HIV-1 infection of macrophages with ATM acting at the late reverse transcription step. To identify DDR factors associated with ATM/ATR pathways that influence HIV-1 infection, a CRISPR knockout screen using a DDR-focused sgRNA library was performed. Approximately 30 DDR genes that impacted HIV-1 infection were identified with 13 factors that facilitated HIV-1 infection and 17 DDR factors that restrict HIV-1 infection. Several hits were factors associated with the Fanconi anemia pathway, such as BTR complex proteins, including the RecQ helicase Bloom syndrome helicase. BLM was specifically demonstrated to enhance HIV-1 infection and replication with knockdown of BLM expression diminishing integration and the establishment of intact HIV-1 proviruses in macrophages by 50%. BLM is associated with HIV-1 late reverse transcription intermediates, the step that proceeds HIV-1 integration. These findings identify BLM as a DDR host factor that promotes early HIV-1 infection by facilitating completion of reverse transcription and subsequent integration. Significance StatementHIV-1 infection elicits cellular DNA damage responses although the role of DNA damage in HIV-1 infection has not been fully characterized. Our study identifies specific DNA Damage factors that facilitate or restrict HIV infection. In particular, we show the RecQ helicase Bloom syndrome helicase (BLM) is a mediator of HIV-1 reverse transcription and integration in macrophages. This work highlights a functional interface between DNA damage repair pathways and HIV-1 integration suggesting that targeting select host DNA damage response factors can limit HIV-1 infection and persistence.

microbiology↗