HIV-1 Reverse Transcriptase Dissociates during Strand Transfer

JOURNAL OF MOLECULAR BIOLOGY

卷:412

Article

Muchiri, John M.^2,3  Rigby, Sean T.^1,4,5  Nguyen, Laura A.⁶  Kim, Baek²  Bambara, Robert A.^1,2,4 

[1] Univ Rochester, Sch Med & Dent, Dept Biochem & Biophys, Rochester, NY 14642 USA

[2] Univ Rochester, Sch Med & Dent, Dept Microbiol & Immunol, Rochester, NY 14642 USA

[3] Kenya Methodist Univ, Basic & Appl Sci Dept, Meru 26760200, Kenya

[4] Univ Rochester, Sch Med & Dent, Ctr RNA Biol, Rochester, NY 14642 USA

[5] BASF Plant Sci Corp, Houston, TX 77002 USA

[6] Univ Rochester, Sch Med & Dent, Dept Pathol, Rochester, NY 14642 USA

关键词: HIV-1;    reverse transcription;    reverse transcriptase;    RNase H;    strand transfer;   

DOI  :  10.1016/j.jmb.2011.07.055

来源: Elsevier

【 摘 要 】

Steps in the replication of human immunodeficiency virus type 1 (HIV-1) occurring in the virus but not in the host are preferred targets of antiretroviral therapy. Strand transfer is unique; the DNA strand being made by viral reverse transcriptase (RT) is moved from one RNA template position to another. Understanding the mechanism requires knowing whether the RT directly mediates the template exchange or dissociates during the exchange, so that it occurs by polymer dynamics. Earlier work in vitro showed that the presence of an RT-trapping polymer would allow synthesis on the original or donor template but completely block transfer and subsequent synthesis on the second or acceptor template. One interpretation is that the RT must dissociate during transfer, but an alternative is that sequestration of non-polymerizing RTs prevents polymerization-independent ribonuclease H (RNase H) cleavages of the donor template necessary for strand exchange. To resolve this ambiguity, we designed a primer template system that allows strand transfer without RNase H activity. Using an RNase H negative mutant RT, we showed that a polymer trap still prevented strand transfer. This confirms that RT dissociates during strand transfer. The presence of HIV-1 nucleocapsid protein, which promotes strand exchange, had little effect on this outcome. Additional assays showed that both the wild-type RI and a multiple nucleoside RT inhibitor-resistant HIV-1 RT containing an extended fingers domain, which is characterized by its enhanced primer-template binding affinity, were unable to transfer with the trapping polymer. This implies that common sequence variations among RTs are unlikely to alter dissociation feature. (C) 2011 Elsevier Ltd. All rights reserved.

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