The RNase H activity of retroviral RTs is critical for successful replication of the viral genome. In addition to the polymerase activity of RTs, which has historically been a popular target for antiretrovirals, the RNase H domain has also become an attractive target for antiviral therapies. A sound understanding of the function and structure of not only lentiviral but also gammaretroviral enzymes will aid in the design of novel, specific, and more potent inhibitors against retroviral RNase H function.
Our detailed analysis of the functional properties of gammaretroviral RNases H highlights similarities to and differences from the related lentiviral HIV-1 counterpart. XMRV and MoMLV RNases H are considerably less active than HIV-1 RNase H. This may be due to the weaker binding affinity of XMRV RT for the nucleic acid substrate (XMRV RT Kd
> HIV-1 RT Kd
), due to a rate of dissociation (koff
) that is higher than that of HIV-1 RT (61
). Structural differences may also play a role in the observed decrease in RNase H activity for the gammaretroviral enzymes. Both the MoMLV and XMRV RTs exist as monomers, unlike the heterodimeric HIV-1 RT, which benefits from the additional support provided by the p51 subunit (17
). An additional difference in nucleic acid binding includes the longer distance between the polymerase and RNase H active sites in XMRV RT than in HIV-1 RT (1 bp or approximately 3.4 Å) (28
). Such differences may affect the trajectory of the nucleic acid at the RNase H active site and the coordination of active site metals with the scissile phosphate group, thus explaining the difference in metal preference between the gammaretroviral (Mn2+
) and lentiviral (Mg2+
) RNases H.
Unlike the gammaretroviral RTs, HIV-1 RT lacks α-helix C in the RNase H domain, which is thought to be important for substrate binding (5
), as the p15 RNase H domain of HIV-1 RT has no enzymatic activity (81
). Addition of a highly basic loop from E. coli
RNase H to the p15 HIV-1 RNase H fragment has been shown to enhance the RNase H activity of HIV-1 (41
RNHIs may offer expanded therapeutic options by complementing existing treatments. Some of these inhibitors were initially discovered as HIV-1 integrase inhibitors (85
) and were designed as active-site-directed compounds that would bind divalent metals at the integrase or RNase H active site (2
). Acylhydrazone-based (11
), hydroxyisoquinolinedione-based (8
), and naphthyridinone-based (84
) compounds are three classes of HIV-1 RNHIs. Like most RNHIs, these compounds are based on metal-chelating pharmacophore scaffolds that have been optimized for potent inhibition of HIV-1 RNase H. Our data are consistent with previous results suggesting that naphthyridinone NAPHRHI binds to the RNase H active site of HIV-1 RT and extend these findings for XMRV RNase H (95
). Interestingly, unlike the hydroxytropolone β-thujaplicinol, which can inhibit HIV-1 RNase H only when added to the reaction mixture before the nucleic acid substrate (6
), NAPHRHI can access the RNase H active site even in the presence of RNA-DNA, especially in the presence of Mn2+
(B and C). Furthermore, in the presence of Mg2+
, NAPHRHI is less effective against both XMRV RT and isolated RNase H. This suggests that Mn2+
binds NAPHRHI more tightly than Mg2+
, thus making NAPHRHI a more effective inhibitor in the presence of Mn2+
. Additional data show that, unlike the potency of β-thujaplicinol, the concentration of the RNA-DNA substrate does not decrease that of NAPHRHI (), and therefore, the inhibitor does not compete with the nucleic acid substrate for binding to the enzyme. Our data also demonstrate that NAPHRHI inhibited the DNA polymerase activities of XMRV and HIV-1 RTs significantly less efficiently (>50-fold) than the corresponding RNase H activities. Moreover, the inhibitor efficiently blocked the RNase H function of the XMRV and p15-Ec HIV-1 RNase H fragments. Therefore, the primary mechanism of RNase H inhibition by NAPHRHI is targeting of the RNase H active site (95
The hydroxyisoquinolinedione compound YLC2-155 was a potent RNHI of both HIV-1 RT and p15-Ec RNase H and a considerably less efficient inhibitor of the DNA polymerase activity of the RT. Interestingly, while YLC2-155 was also a potent RNHI of XMRV RT, it did not inhibit the DNA polymerase of XMRV RT and was also ineffective against the isolated XMRV RNase H domain. These data suggest that this compound may not bind at the RNase H or polymerase active site of XMRV RT. Instead, it may target a distinct binding site that allows inhibition of the RNase H and not of the DNA polymerase function. Collectively, our data demonstrate that RNHIs can specifically block RNase H function and could thus be further modified to enhance binding potency and selectivity.
Many RNHIs have been found to be potent against HIV-1 RT in vitro
but exhibit little to no anti-HIV activity in cell-based assays (47
). Similar to previous investigators, we found that NAPHRHI inhibits the RNase H function of HIV-1 RT (95
). We also found that this compound inhibits the RNase H activity of the p15-Ec RNase H fragment. In addition, we showed that NAPHRHI blocks the replication of HIV in both pseudotype- and cell-based assays. The compound also appeared to have some antiviral activity against XMRV and MoMLV, but because the respective EC50
s were close to the CC50
(4.1 μM), it was not possible to accurately determine its potency against these viruses. These results suggest that the naphthyridinone scaffold is a promising candidate for future studies that will focus on identifying potent inhibitors of RNase H activity that are less toxic. Similarly, the acylhydrazone (BHMP07 and THBNH) and hydroxyisoquinolinedione (YLC2-155) compounds exhibit antiviral activity in cell-based assays but demonstrate low cytotoxic effects. Hence, further development of potent RNHIs based on the acylhydrazone and hydroxyisoquinolinedione scaffolds should be pursued.
In order to determine how the structure of XMRV RNase H might affect its function and inhibition, we determined the crystal structure of the XMRV ΔC RNase H fragment. Our data show extensive similarity between the XMRV and MoMLV ΔC RNase H structures (RMSD, 0.39 Å), consistent with their high sequence identity (61
). The active site of XMRV RNase H is also similar to that of HIV-1 RNase H (RMSD, 1.6 Å). The catalytic residues (Asp524, Glu562, and Asp583) of XMRV RNase H correspond to residues Asp443, Glu478, and Asp498 of HIV-1 RNase H (B). An additional HIV-1 RNase H residue, Asp549 (Asp653 in XMRV RNase H), also participates in RNase H cleavage by helping to stabilize one of the catalytic metals (5
). Many crystal structures of HIV-1 RT and RNase H show a single Mg2+
ion in the RNase H active site (37
). However, recent structures of HIV-1 RT and isolated RNase H with active-site-directed RNHIs show two Mn2+
ions in the RNase H active site (47
). In addition, crystal structures of B. halodurans
and human RNases H have also revealed two active-site Mg2+
). Biochemical studies have also suggested a two-metal mechanism for the RNase H activity of RTs (44
). Interestingly, the XMRV and MoMLV ΔC RNase H structures contain a single metal at the active site. However, given the degree of similarity with the HIV-1 RT, it is likely that RNase H cleavage or binding of RNHI would also involve two divalent metals.
In conclusion, we have determined the differences in RNase H activity and the effectiveness of acylhydrazone-, naphthyridinone-, and hydroxyisoquinolinedione-based RNHIs against gammaretroviral and lentiviral RTs. The crystal structure of the isolated XMRV RNase H domain provides a structural framework for better understanding the function and inhibition of gammaretroviral RNase H activity. This information may be useful in the design of next-generation RNHIs with increased potency against retroviral RTs.