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Structural Basis for HIV-1 Reverse Transcriptase Maturation

Slack, Ryan L (2020) Structural Basis for HIV-1 Reverse Transcriptase Maturation. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Reverse transcriptase (RT) is the viral enzyme responsible for generating a double stranded DNA copy of the viral RNA genome, which is an essential step in the replication process of Human Immunodeficiency Virus-1. Mature RT consists of a 66 kDa subunit (p66) and a smaller 51 kDa subunit. The smaller subunit, p51, is formed from proteolytic cleavage between residues F440 and Y441 in the ribonuclease H domain (RNH) of p66 by HIV-1 protease (PR). Evidence suggests that the RT heterodimer is formed via processing of a p66/p66 homodimer intermediate, but a structure of this homodimer has yet to be reported. The p51-RNH processing site is located between residues 440/441 and lies in the β-sheet region of the RNH domain. According to available structures of the RNH domain, this site would be inaccessible to PR. Because the available structural data fails to explain the molecular basis for this processing event, the mechanism of RT maturation remains unclear. In an effort to clarify the mechanism of RT maturation, we initially studied structural integrity of the RNH domain by introducing mutations at the p51-RNH processing site. We proceeded to characterize the conformational symmetry and long-term stability of p66/p66 homodimer using principal component analysis of solution NMR spectra. We then studied in vitro maturation of RT, and the effect of nucleic acids on the kinetics and specificity of this process. Finally, we investigated the conformational change required for the enhancement of the RT maturation.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Slack, Ryan Lrls128@pitt.edurls1280000-0002-5928-6205
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairIshima, Riekoishima@pitt.eduIshima0000-0002-3418-0922
Ahn, Jinwoojia12@pitt.eduJIA12
Ambrose, Zandreazaa4@pitt.eduZAA40000-0002-8610-2766
Tang, Peiptang@pitt.eduPTANG
Date: 5 February 2020
Date Type: Publication
Defense Date: 14 December 2019
Approval Date: 5 February 2020
Submission Date: 17 December 2019
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 155
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Structural Biology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: NMR, HIV-1, Reverse Transcriptase, Maturation, Structure
Date Deposited: 05 Feb 2020 15:07
Last Modified: 05 Feb 2020 15:07
URI: http://d-scholarship.pitt.edu/id/eprint/38054

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