A Mutant Tat Protein Inhibits HIV-1 Reverse Transcription by Targeting the Reverse Transcription Complex

Min-Hsuan Lin, Ann Apolloni, Vincent Cutillas, Haran Sivakumaran, Sally Martin, Dongsheng Li, Ting Wei, Rui Wang, Hongping Jin, Kirsten Spann, David Harrich

Previously we reported that a mutant of Tat referred to as Nullbasic inhibits HIV-1 reverse transcription although the mechanism of action is unknown. Here we show that Nullbasic is a reverse transcriptase (RT) binding protein that targets the reverse transcription complex rather than directly inhibiting RT activity. An interaction between Nullbasic and RT was observed by co-immunoprecipitation, pull-down assays, and a direct interaction was measured by biolayer interferometry (BLI) assay. Mixtures of recombinant 6×-His-RT and Nullbasic-FLAG-V5-6×His at molar ratios of up to 1:20,000 did not inhibit RT activity in standard homopolymer primer template assays. An analysis of virus made by cells that co-expressed Nullbasic showed that Nullbasic co-purified with virus particles indicating it was a virion protein. In addition, analysis of reverse transcription complexes (RTCs) isolated from cells infected with wild type or Nullbasic-treated HIV-1 showed that Nullbasic reduced levels of viral DNA in RTC fractions. In addition, a shift in the distribution of viral DNA and CAp24 to less dense non-RTC fractions was observed, indicating that RTC activity from Nullbasic treated virus was impaired. Further analysis showed that viral cores isolated from Nullbasic treated HIV undergo increased disassembly in vitro compared to untreated HIV-1. To our knowledge this is the first description of an antiviral protein that inhibits reverse transcription by targeting the RTC and affecting core stability.