Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid

Abstract

A key step in proliferation of retroviruses is the conversion of their RNA genome to double-stranded DNA, a process catalysed by multifunctional reverse transcriptases (RTs). Dimeric and monomeric RTs have been described, the latter exemplified by the enzyme of Moloney murine leukaemia virus. However, structural information is lacking that describes the substrate binding mechanism for a monomeric RT. We report here the first crystal structure of a complex between an RNA/DNA hybrid substrate and polymerase-connection fragment of the single-subunit RT from xenotropic murine leukaemia virus-related virus, a close relative of Moloney murine leukaemia virus. A comparison with p66/p51 human immunodeficiency virus-1 RT shows that substrate binding around the polymerase active site is conserved but differs in the thumb and connection subdomains. Small-angle X-ray scattering was used to model full-length xenotropic murine leukaemia virus-related virus RT, demonstrating that its mobile RNase H domain becomes ordered in the presence of a substrate—a key difference between monomeric and dimeric RTs.

Reverse Transcription in the Saccharomyces cerevisiae Long-Terminal Repeat Retrotransposon Ty3

“…However, the observation that (a) Ty1 and Ty3 RTs use a bipartite primer binding site (PBS); (b) the Schizosaccharomyces pombe element Tf1 uses a tRNA-independent mechanism; and (c) a “half-tRNA” is employed by Drosophila melanogaster copia to initiate (−) strand DNA synthesis [4] suggests their respective polymerases might also not share the topological features of HIV-1 RT. This issue is highlighted by structural data for several monomeric retroviral and retrotransposon RTs such as the gammaretroviruses xenotropic murine leukemia virus-related virus (XMRV) and Moloney murine leukemia viruses, mouse mammary tumor virus, simian foamy virus, bovine leukemia virus, and the Tf1 element [5,6,7,8,9,10]. As the third RNA-dependent DNA polymerase to be crystallized in the presence of an RNA/DNA hybrid, the goal of data presented in this review is to illustrate the unique topological complexity of Ty3 RT and point out to the reader that our understanding of reverse transcription should be the consequence of comparative studies and not simply those from a single enzyme.…”

Preparation and characterization of the RNase H domain of Moloney murine leukemia virus reverse transcriptase

“…From the crystal of full-length MMLV RT (Thr24-Leu671), the structures of the fingers, palm, thumb, and connection subdomains (Thr24-Asn479) have been determined, but that of the RNase H domain has not [1,9]. On the other hand, from the crystal of the isolated RNase H domain, its whole structure has been determined [10]. In that study, the RNase H domain variant lacking the polypeptide Ile593-Leu603 was successfully crystallized, while the wild-type RNase H domain was not [10].…”

“…On the other hand, from the crystal of the isolated RNase H domain, its whole structure has been determined [10]. In that study, the RNase H domain variant lacking the polypeptide Ile593-Leu603 was successfully crystallized, while the wild-type RNase H domain was not [10]. The deleted polypeptide region, Ile593-Leu603, contains a positively charged a-helix called the C helix (H 594 GEIYRRR 601 ).…”

Conservation of antiviral activity and improved selectivity in PMEO-DAPym upon pyrimidine to triazine scaffold hopping

“…Given that the viral DNA polymerases represent the eventual targets for the antireplicative action of these compounds, we tried to account for the selectivity profile displayed by 27 and 8 by focusing on the 3D structures of these viral enzymes. The well characterized dimeric HIV-1 and HIV-2 RTs and monomeric MSV RT are known to share a common active site architecture (Nowak et al, 2013) which, on the basis of multiple sequence alignments and functional data (Menendez-Arias et al, 2014), can be confidently extended to that of the still unresolved HBV DNA polymerase (Voros et al, 2014). Therefore, the same binding mode that was experimentally determined for the diphosphate of 3 (3-pp) in the active site of HIV-1 RT in complex with a templateprimer double helix (Tuske et al, 2004) can be safely assumed for 27-pp, as previously reported for 8-pp (Herman et al, 2010), and not only for HIV-1 and HIV-2 RTs but also for MSV RT and HBV DNA polymerase ( Fig.…”