Cell-free assays reveal that the HIV-1 capsid protects reverse transcripts from cGAS immune sensing.

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect viral genomes from cGAS even after completing reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, the capsid inhibitor lenacapavir also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling. Author summary: Human Immunodeficiency Virus type 1 (HIV-1) remains a global health threat. Drug therapies can help manage HIV-1 infection and prevent its spread, but without treatment, the immune system fails to control the virus. In part, this is because HIV-1 is thought to avoid detection by immune cells during entry. Once inside a target cell, the virus copies its RNA genome into double-stranded DNA within a viral protein shell called the capsid. In this study, we developed a novel cell-free method to study how the HIV-1 capsid shields viral DNA from detection. We found that the capsid normally functions as a molecular cloak that hides the viral DNA from innate immune sensors. We also identified experimental conditions and drug treatments that can "break" the capsid and allow the viral DNA to be detected, both in vitro and in cells. We anticipate that future work using cell-free systems will reveal how host factors regulate innate detection of HIV-1, possibly guiding new therapeutic strategies that block infection and engage antiviral immune responses simultaneously. [ABSTRACT FROM AUTHOR]