A viral replication level-dependent continuum of transcriptional and microRNA reprogramming in HIV-1-infected microglia links productive infection to neurocognitive-associated gene networks

Microglia are major targets of HIV-1 in the brain. Despite effective antiviral therapy, infected microglia, including those containing a replication-defective provirus, can express viral products that contribute to neuroinflammation, yet how productive infection shapes the host response remains poorly defined. Using single-cell RNA-seq of HIV-1-infected C20 microglia, we stratified cells by HIV-1 splice junction-supporting reads into low- and high-infection groups, resolving a graded rather than binary host transcriptional response. The computational pipeline developed for this quantification is made publicly available. A core set of immune and cell cycle pathways was induced, regardless of infection level, but several programs reversed direction with increasing viral burden: interferon-α/γ and EMT /hypoxia signatures peaked at low infection, while high infection featured suppression of these signatures alongside induction of mTORC1 / MYC signaling, ribosome biogenesis, RNA processing, and mitotic activity, with progressive loss of cytoskeletal, motility, and developmental programs. This pattern, except for cell cycle/mitotic signatures, was reproduced in iPSC-derived microglia, supporting its generalizability beyond an immortalized cell line. To identify potential regulatory mechanisms underlying these transcriptional programs, we integrated differentially expressed miRNAs and genes from the low- and high-infection groups, filtered for the canonical inverse miRNA-target relationship, revealing miRNA/mRNA networks enriched for miRNAs previously implicated in HIV-1 replication (e.g., miR-33a-5p and miR-3065-3p ) and in neurodegenerative and psychiatric disease (e.g., miR-107 and let-7c-5p ). These findings define a productive infection-level continuum in microglial transcription and miRNA reprogramming and identify candidate miRNA-mediated mechanisms, complementary to the canonical Env / Nef / Tat pathway by which HIV-1-infected microglia may contribute to neuroinflammation and neurocognitive impairment in people with HIV-1. IMPORTANCE HIV-1 persists in the brain despite antiviral therapy in part because infected microglia continue to express viral RNA and proteins that promote neuroinflammation, contributing to the cognitive impairment seen in many people with HIV-1. How the level of active viral replication within a microglial cell shapes its response has been unclear since most infection models capture only an averaged, post-replication snapshot rather than the transition from low to advanced infection. By ranking microglia by HIV-1 splice junction-supporting reads, we show that the host transcriptional response is graded rather than binary, and that several pathways, including interferon and growth/proliferative signaling, reverse direction as viral load increases. We further identify a network of HIV-1-dysregulated miRNAs, several previously linked to viral replication or to neurodegenerative and neurocognitive disease, pointing to a mechanism by which infected microglia may influence neuronal health, complementary to the well-studied viral Env , Nef , and Tat proteins.