Volume 10 Supplement 1

Frontiers of Retrovirology: Complex retroviruses, retroelements and their hosts

Open Access

Novel insights from structural analysis of lentiviral and gammaretroviral reverse transcriptases in complex with RNA/DNA hybrids

  • Stuart Le Grice1,
  • Mikalai Lapkouski2,
  • Lan Tian2,
  • Jennifer Miller1,
  • Elzbieta Nowak3,
  • Wojciech Potrzebowski3,
  • Peter Konarev4,
  • Jason Rausch1,
  • Marion Bona1,
  • Dmitri Svergun4,
  • Janusz Bujnicki3,
  • Marcin Nowotny3 and
  • Wei Yang2
Retrovirology201310(Suppl 1):P46

https://doi.org/10.1186/1742-4690-10-S1-P46

Published: 19 September 2013

Structures of HIV-1 reverse transcriptase (RT) have been reported in several forms, but only one contains an RNA/DNA hybrid, the conformation of which has been controversial. We have been successful in obtaining three structures of HIV-1 RT complexed with a non-nucleoside RT inhibitor (NNRTI) and an RNA/DNA hybrid [1]. In the presence of an NNRTI, our RNA/DNA structure differs from all prior nucleic acid bound to RT including the previously-reported RNA/DNA hybrid derived froom the polypurine tract. The enzyme structure observed in our cocrystals also differs from all previous RT-DNA complexes. As a result, the hybrid has ready access to the ribonuclease H (RNase H) active site. These observations collectively reinforce previous proposals that an RT-nucleic acid complex may be required to adopt independent structural states competent for DNA synthesis and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites map to the unique RT-hybrid interface that undergoes conformational changes between two catalytic states. Structural features of the nucleoprotein complex, including drug resistance mutations, have been verified by site-directed mutagenesis, and will be presented.

Although the single-subunit RT of Moloney murine leukemia virus (Mo-MLV) has been extensively characterized biochemically, structural information is lacking that describes the substrate binding mechanism for this RT species. We also present data on the first crystal structure of a complex between an RNA/DNA hybrid and the 72 kDa single-subunit RT from the related xenotropic murine leukemia virus-related virus (XMRV) [2]. A comparison of this structure with its HIV-1 counterpart shows that substrate binding around the DNA polymerase active site is conserved but differs between the two enzymes in their thumb and connection subdomains. Small-angle X-ray scattering (SAXS) was used to model full-length XMRV RT, demonstrating its flexible RNase H domain becomes ordered in the presence of substrate, a key difference between monomeric and dimeric RTs.

Authors’ Affiliations

(1)
HIV DRP, National Cancer Institute
(2)
NIDDK, National Institutes of Health
(3)
International Institute of Molecular & Cell Biology
(4)
European Molecular Biology Laboratory

References

  1. Lapkouski M, Tian L, Miller JT, Le Grice SFJ, Yang W: Complexes of HIV-1 RT,an NNRTI and an RNA/DNA hybrid reveal a structure compatible with RNA degradation. Nat Struct Mol Biol. 2013, 20: 230-236. 10.1038/nsmb.2485.PubMed CentralView ArticlePubMedGoogle Scholar
  2. Nowak E, Potrzebowski W, Konarev PV, Rausch JW, Bona MK, Svergun DI, Bujnicki JM, Le Grice SFJ, Nowotny M: Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid. Nucleic Acids Res. 2013, 41: 3874-3887. 10.1093/nar/gkt053.PubMed CentralView ArticlePubMedGoogle Scholar

Copyright

© Le Grice et al; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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