Quinones-Molina, A. A. | bioRxiv Science

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↗

bioRxiv2023

Incorporation of 5 methylcytidine alleviates innate immune response to self-amplifying RNA vaccine

In order to improve vaccine effectiveness and safety profile of existing synthetic RNA-based vaccines, we have developed a self-amplifying RNA (saRNA)-based vaccine expressing membrane-anchored receptor binding domain (RBD) of SARS-CoV-2 S protein (S-RBD) and have demonstrated that a minimal dose of this saRNA vaccine elicits robust immune responses. Results from a recent clinical trial with 5-methylcytidine (5mC) incorporating saRNA vaccine demonstrated reduced vaccine-induced adverse effects while maintaining robust humoral responses. In this study, we investigate the mechanisms accounting for induction of efficient innate and adaptive immune responses and attenuated adverse effects induced by the 5mC-incorporated saRNA. We show that the 5mC-incorporating saRNA platform leads to prolonged and robust expression of antigen, while induction of type-I interferon (IFN-I), a key driver of reactogenicity, is attenuated in peripheral blood mononuclear cells (PBMCs), but not in macrophages and dendritic cells. Interestingly, we find that the major cellular source of IFN-I production in PBMCs is plasmacytoid dendritic cells (pDCs), which is attenuated upon 5mC incorporation in saRNA. In addition, we demonstrate that monocytes also play an important role in amplifying proinflammatory responses. Furthermore, we show that the detection of saRNA is mediated by a host cytosolic RNA sensor, RIG-I. Importantly, 5mC-incorporating saRNA vaccine candidate produced robust IgG responses against S-RBD upon injection in mice, thus providing strong support for the potential clinical use of 5mC-incorporating saRNA vaccines.

immunology↗