NSUN2-mediated m5C modification of HIV-1 RNA enables evasion of RIG‑I‑dependent innate immunity.

Recently, 5-methylcytosine (m5C) modification has been identified in HIV-1 genomic RNA. However, the functional role of this RNA modification in the antiviral innate immune response remains unclear. Here, we demonstrate that m5C modification of HIV-1 genomic RNA enables the virus to evade the type I interferon (IFN-I)-mediated antiviral response, thereby promoting viral replication. Depletion of NSUN2 in viral-producing cells significantly reduced m5C modification of HIV-1 RNA, leading to progeny viruses that are more susceptible to innate immune detection and consequently displaying attenuated replication. Furthermore, in vitro-transcribed m5C-modified RNA exhibited a reduced ability to induce IFN-I production relative to unmodified RNA. Additionally, m5C-modified RNA displayed markedly impaired binding to RIG-I compared with unmodified RNA. Collectively, our findings reveal that HIV-1 utilizes m5C modification of viral genomic RNA as a strategy to escape RIG-I-mediated immune recognition, thereby facilitating efficient viral replication.