title: 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.
  
  
  OBJECTIVE
  HIV-1 infection is treated by using combinations of antiretroviral drugs that target independent steps of virus replication. A newly described antiviral protein called Nullbasic can also inhibit a combination of different steps in virus replication (transcription, reverse transcription, and Rev-mediated viral mRNA transport), although the precise mechanism of action is unknown. This study shows that Nullbasic can inhibit reverse transcription by binding to the viral enzyme called reverse transcriptase, which results in accelerated uncoating of the viral core and instability of the viral apparatus called the reverse transcription complex (RTC). This unique antiviral activity may inform development of other RTC inhibitors, as well as providing a unique investigative tool for dissecting the RTC cellular composition.
authors: QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors: Min-Hsuan
authors: MH
authors: Lin
authors: QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors: Ann
authors: A
authors: Apolloni
authors: QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors: Vincent
authors: V
authors: Cutillas
authors: QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors: Haran
authors: H
authors: Sivakumaran
authors: Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, St. Lucia, Queensland, Australia.
authors: Sally
authors: S
authors: Martin
authors: QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors: Dongsheng
authors: D
authors: Li
authors: QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors: Ting
authors: T
authors: Wei
authors: QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors: Rui
authors: R
authors: Wang
authors: QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
authors: Hongping
authors: H
authors: Jin
authors: School of Biomedical Science, Queensland University of Technology, Brisbane, Queensland, Australia.
authors: Kirsten
authors: K
authors: Spann
authors: QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia Australian Infectious Disease Research Centre at the University of Queensland, St. Lucia, Queensland, Queensland, Australia david.harrich@qimrberghofer.edu.au.
authors: David
authors: D
authors: Harrich
mesh_terms: D002498:Centrifugation; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D047468:Immunoprecipitation; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D048348:Reverse Transcription; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase