Drug Resistant HIV Reverse Transcriptase

Project: Research project

Project Details

Description

DESCRIPTION (provided by the applicant): HIV-1 RT continues to be a key target
for anti-HIV therapy. The long term goal of our project is to comprehensively
define HIV RT function and the mechanism of drug resistance. There is an
increasing realization that proper positioning of template-primer is central to
several key aspects of RT function. We intend to delineate the functional role
of three specific interactions between HIV-1 RT and the template-primer. These
contact points lie along the template-primer cleft, in front of the active site
(contacting the template 5'-overhang), behind the active site (contacting the
template at penultimate basepair), or further away in the minor groove
(contacting the primer at 3rd to 6th basepairs) respectively. Three specific
aims are listed. First, we will study the fingers subdomain-template contacts
near the active site via nested deletions of 33-[M loop of HIV-1 RT created to
disrupt the proposed interactions with the template overhang and/or the dNTP.
In addition to testing these mutants for dNTP-binding, accuracy of dNTP
insertion, processivity and strand transfer, we will also test the role of
J33-j34 loop as a determinant of the natural high susceptibility of HIV-1 RT to
2',3'-dideoxyNTPs. The mutation rate of multi-dideoxynucleoside analog
resistant RT variants and whether a proofreading-like activity contributes to
the overall fidelity will be tested. Second, we will investigate the role of
the template grip 135a, which forms a close contact with the penultimate
basepair of the template-primer, in RT function. We will both attempt to
delineate the mechanism of frameshift mutagenesis by HIV-1 RT, the precise role
of template grip 135a in frameshift fidelity and the mechanistic basis for its
global influence on fidelity. Altered template grip mutants of HIV-1 RT will be
tested for effects on affinity to the template-primer duplex, processivity,
RNase H activity and strand transfer. Lastly, we will test whether the thumb
helix clamp involving aH and cxl helices indeed serves a critical function in
enzyme translocation subsequent to each dNTP addition cycle. We will also test
its role in processive polymerization and in frameshift fidelity.
StatusFinished
Effective start/end date9/1/913/31/05

Funding

  • National Institute of Allergy and Infectious Diseases: $482,132.00
  • National Institute of Allergy and Infectious Diseases: $468,323.00
  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases

ASJC

  • Infectious Diseases
  • Molecular Biology

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