Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA.
Antimicrobial Agents and Chemotherapy (Impact Factor: 4.48). 01/2012; 56(4):2048-61. DOI: 10.1128/AAC.06000-11
Source: PubMed


RNase H inhibitors (RNHIs) have gained attention as potential HIV-1 therapeutics. Although several RNHIs have been studied in the context of HIV-1 reverse transcriptase (RT) RNase H, there is no information on inhibitors that might affect the RNase H activity of other RTs. We performed biochemical, virological, crystallographic, and molecular modeling studies to compare the RNase H function and inhibition profiles of the gammaretroviral xenotropic murine leukemia virus-related virus (XMRV) and Moloney murine leukemia virus (MoMLV) RTs to those of HIV-1 RT. The RNase H activity of XMRV RT is significantly lower than that of HIV-1 RT and comparable to that of MoMLV RT. XMRV and MoMLV, but not HIV-1 RT, had optimal RNase H activities in the presence of Mn²⁺ and not Mg²⁺. Using hydroxyl-radical footprinting assays, we demonstrated that the distance between the polymerase and RNase H domains in the MoMLV and XMRV RTs is longer than that in the HIV-1 RT by ∼3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with 50% inhibitory concentrations ranging from ∼0.8 to 0.02 μM. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.

Download full-text


Available from: Stefan Sarafianos
  • Source
    • "However, in XMRV RNase H the consecutive basic residues (3 x Arg) are part of helix C [13], whereas in PFV RNase H four lysines (KKKPLK) are located in the adjacent basic loop (Figure 1). The slightly different position of the basic residues might cause different distances to the active site and might thus contribute to possible differences in cleavage activities of the two enzymes [5,13,22]. The orientation of helix C is determined by numerous hydrophobic contacts to helix D. This suggests that helix C acts as a molecular ruler to orient the basic loop in order to optimize substrate binding and specificity. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity. The solution structure of PFV RNase H shows that it contains a mixed five-stranded β-sheet, which is sandwiched by four α-helices (A-D), including the C-helix, on one side and one α-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding. The structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding.
    Full-text · Article · Sep 2012 · Retrovirology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The ribonuclease H (RNase H) domain of retroviral reverse transcriptase (RT) plays a critical role in the life cycle by degrading the RNA strands of DNA/RNA hybrids. In addition, RNase H activity is required to precisely remove the RNA primers from nascent (-) and (+) strand DNA. We report here three crystal structures of the RNase H domain of xenotropic murine leukemia virus-related virus (XMRV) RT, namely (i) the previously identified construct from which helix C was deleted, (ii) the intact domain, and (iii) the intact domain complexed with an active site α-hydroxytropolone inhibitor. Enzymatic assays showed that the intact RNase H domain retained catalytic activity, whereas the variant lacking helix C was only marginally active, corroborating the importance of this helix for enzymatic activity. Modeling of the enzyme-substrate complex elucidated the essential role of helix C in binding a DNA/RNA hybrid and its likely mode of recognition. The crystal structure of the RNase H domain complexed with β-thujaplicinol clearly showed that coordination by two divalent cations mediates recognition of the inhibitor.
    Full-text · Article · Feb 2012 · Journal of Structural Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: RNase H (retroviral ribonuclease H) cleaves the phosphate backbone of the RNA template within an RNA/DNA hybrid to complete the synthesis of double-stranded viral DNA. In the present study we have determined the complete structure of the RNase H domain from XMRV (xenotropic murine leukaemia virus-related virus) RT (reverse transcriptase). The basic protrusion motif of the XMRV RNase H domain is folded as a short helix and an adjacent highly bent loop. Structural superposition and subsequent mutagenesis experiments suggest that the basic protrusion motif plays a role in direct binding to the major groove in RNA/DNA hybrid, as well as in establishing the co-ordination among modules in RT necessary for proper function.
    Full-text · Article · Jun 2012 · Bioscience Reports
Show more