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

Abstract

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 using coimmunoprecipitation and pulldown assays, and a direct interaction was measured by using a biolayer interferometry 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 coexpressed Nullbasic showed that Nullbasic copurified with virus particles, indicating that 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 the 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. Like all retroviruses, HIV-1 has a single positive-sense strand of RNA genome that is converted into double-strand proviral DNA by a hallmark process called reverse transcription. Proviral DNA is subsequently integrated into the host chromosomes and is transcribed by RNA polymerase II producing viral mRNA. The mechanisms regulating reverse HIV-1 transcription have been described in detail elsewhere (1). Briefly, the viral mRNA genome annealed to host cell tRNA Lys3 form a ribonucleoprotein complex with viral proteins, including reverse transcriptase (RT), integrase (IN), and nucleocapsid to form a prototypical reverse transcription complex (RTC) (2). The initiation of reverse transcription by the RTC begins shortly after cell infection after cytoplasmic nucleotides become available. Using tRNA Lys3 as a primer, DNA synthesis by RT produces a short strand of DNA called negativestrand strong stop DNA (ϪsssDNA). Degradation of the viral RNA strand by RT RNase H activity liberates ϪsssDNA that is transferred to the 3= end of the viral RNA by annealing of complementary nucleotide sequences, a step called first-strand transfer. The synthesis of the remaining negative-strand DNA can then be completed by RT. The complete synthesis of double-strand proviral DNA follows additional DNA synthesis following additional priming reactions and strand displacement DNA synthesis by RT. Cellular factors, including eEF1A, associate with the RTC and play an important role in the reverse transcription process (3, 4).Many virion ...

Combating human immunodeficiency virus replication by gene therapy

“…However in primary CD4 + T cells, the virus is able to replicate if Nullbasic expression is too low 14 . This observation is in agreement with the report that Nullbasic inhibits HIV-1 in a dose dependent manner 14,15,18 . So, in this study we used an improved SIN gammaretroviral vector that is able to express genes using the SFFV promoter.…”

“…In binding to HIV-1 reverse transcriptase in the virion, leading to premature uncoating and defective reverse transcription in newly infected cells 18 . Given that Nullbasic inhibits HIV-1 by binding to both cellular (PTEFb and DDX1) and viral RT targets, we found that NB fusion proteins had antiviral activity against all strains tested, although some differences were observed.…”

“…We selected cells that expressed high levels of fluorescent protein (Fig. 4-7), because previous studies indicated that Nullbasic antiviral activity is dose dependent 14,15,18 . Therefore, we selected cells that …”

“…Nullbasic is packaged in VLP and studies showed that Nullbasic downregulates the VLP infectivity by inhibiting HIV-1 reverse transcription 14,18 . Nullbasic directly binds reverse transcriptase and this leads to instability of the reverse transcription complex and decreased viral DNA synthesis 14,18 , and therefore resulted in inefficient transduction of cells by NB-EGFP VLPs.…”

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

“…An HIV-1 Tat mutant called nullbasic, whose entire arginine-rich domain is replaced by either glycine or alanine, has also been proven to interact with RT by using coimmunoprecipitation assays, pulldown assays, and biolayer interferometry assays (140). As an antiviral protein, nullbasic reduces viral core stability to prevent HIV-1 reverse transcription (140). Although Tat is dispensable for reverse transcription, Tat in complex with RT stimulates viral DNA synthesis (139).…”