scholarly journals Crystal Structure of the Moloney Murine Leukemia Virus RNase H Domain

2006 ◽  
Vol 80 (17) ◽  
pp. 8379-8389 ◽  
Author(s):  
David Lim ◽  
G. Glenn Gregorio ◽  
Craig Bingman ◽  
Erik Martinez-Hackert ◽  
Wayne A. Hendrickson ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Bacillus Halodurans ◽  
Catalytic Core ◽  
E Coli ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT A crystallographic study of the Moloney murine leukemia virus (Mo-MLV) RNase H domain was performed to provide information about its structure and mechanism of action. These efforts resulted in the crystallization of a mutant Mo-MLV RNase H lacking the putative helix C (ΔC). The 1.6-Å resolution structure resembles the known structures of the human immunodeficiency virus type 1 (HIV-1) and Escherichia coli RNase H. The structure revealed the coordination of a magnesium ion within the catalytic core comprised of the highly conserved acidic residues D524, E562, and D583. Surface charge mapping of the Mo-MLV structure revealed a high density of basic charges on one side of the enzyme. Using a model of the Mo-MLV structure superimposed upon a structure of HIV-1 reverse transcriptase bound to an RNA/DNA hybrid substrate, Mo-MLV RNase H secondary structures and individual amino acids were examined for their potential roles in binding substrate. Identified regions included Mo-MLV RNase H β1-β2, αA, and αB and residues from αB to αD and its following loop. Most of the identified substrate-binding residues corresponded with residues directly binding nucleotides in an RNase H from Bacillus halodurans as observed in a cocrystal structure with RNA/DNA. Finally, superimposition of RNases H of Mo-MLV, E. coli, and HIV-1 revealed that a loop of the HIV-1 connection domain resides within the same region of the Mo-MLV and E. coli C-helix. The HIV-1 connection domain may serve to recognize and bind the RNA/DNA substrate major groove.

scholarly journals Small Dumbbell Oligonucleotides Inhibitors of RNase H Activity of MOMULV Reverse Transcriptase

2011 ◽  
Vol 8 (2) ◽  
pp. 629-634
Author(s):  
Ajay Kumar
Keyword(s):  
Reverse Transcriptase ◽  
Murine Leukemia Virus ◽  
Dissociation Constants ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Mobility Shift ◽  
Hiv 1 ◽  
Murine Leukemia

The small dumbbell oligonucleotides containing loops of phosphodiester (OL-1), two trimethylene, C3moieties in each loop (OL-2) and phosphorothioate (OL-3) linkages were synthesized. Incubation of OL-1 and OL-2 with S-1 nuclease generated break down products whereas incubation of OL-3 did not result in significant cleavage. Their binding to moloney murine leukemia virus reverse transcriptase was evaluated by PAGE band mobility shift assays. The OL-3 bound more strongly to the reverse transcriptase than OL-1 and OL-2. The dissociation constants evaluated using PAGE band mobility shift assays were of the order of 10-7. Investigation of inhibition of RNase H activity of reverse transcriptase showed that the OL-3 is a better inhibitor of the retroviral RNase H activity than both OL-1 and OL-2. Thus OL-3 may be used as RNase H inhibitor. Our studies demonstrated that this particularly designed oligonucleotide (OL-3) displays an IC50of 25 nM in its inhibition on the reverse transcriptase RNase H activity, a magnitude lower than that of first nucleotide reverse transcriptase of HIV-1, tenofovir, introduced by Gilead Science in the market.

scholarly journals The Retroviruses Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Adopt Radically Different Strategies To Regulate Promoter-Proximal Polyadenylation

2001 ◽  
Vol 75 (23) ◽  
pp. 11735-11746 ◽  
Author(s):  
Andre Furger ◽  
Joan Monks ◽  
Nick J. Proudfoot
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Immunodeficiency Virus ◽  
A Site ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT Maximal gene expression in retroviruses requires that polyadenylation in the 5′ long terminal repeat (LTR) is suppressed. In human immunodeficiency virus type 1 (HIV-1) the promoter-proximal poly(A) site is blocked by interaction of U1 snRNP with the closely positioned major splice donor site (MSD) 200 nucleotides downstream. Here we investigated whether the same mechanism applies to down-regulate 5′ LTR polyadenylation in Moloney murine leukemia virus (MoMLV). Although the same molecular architecture is present in both viruses, the MoMLV poly(A) signal in the 5′ LTR is active whether or not the MSD is mutated. This surprising difference between the two retroviruses is not due to their actual poly(A) signals or MSD sequences, since exchange of either element between the two viral sequences does not alter their ability to regulate 5′ LTR poly(A) site use. Instead we demonstrate that sequence between the cap and AAUAAA is required for MSD-dependent poly(A) regulation in HIV-1, indicating a key role for this part of the LTR in poly(A) site suppression. We also show that the MoMLV poly(A) signal is an intrinsically weak RNA-processing signal. This suggests that in the absence of a poly(A) site suppression mechanism, MoMLV is forced to use a weak poly(A) signal.

scholarly journals Revealing Domain Structure through Linker-Scanning Analysis of the Murine Leukemia Virus (MuLV) RNase H and MuLV and Human Immunodeficiency Virus Type 1 Integrase Proteins

2006 ◽  
Vol 80 (19) ◽  
pp. 9497-9510 ◽  
Author(s):  
Jennifer Puglia ◽  
Tan Wang ◽  
Christine Smith-Snyder ◽  
Marie Cote ◽  
Michael Scher ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
C Terminus ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT Linker-scanning libraries were generated within the 3′ terminus of the Moloney murine leukemia virus (M-MuLV) pol gene encoding the connection-RNase H domains of reverse transcriptase (RT) as well as the structurally related M-MuLV and human immunodeficiency virus type 1 (HIV-1) integrase (IN) proteins. Mutations within the M-MuLV proviral vectors were Tn7 based and resulted in 15-bp insertions. Mutations within an HIV-1 IN bacterial expression vector were based on Tn5 and resulted in 57-bp insertions. The effects of the insertions were examined in vivo (M-MuLV) and in vitro (HIV-1). A total of 178 individual M-MuLV constructs were analyzed; 40 in-frame insertions within RT connection-RNase H, 108 in-frame insertions within IN, 13 insertions encoding stop codons within RNase H, and 17 insertions encoding stop codons within IN. For HIV-1 IN, 56 mutants were analyzed. In both M-MuLV and HIV-1 IN, regions are identified which functionally tolerate multiple-linker insertions. For MuLV, these correspond to the RT-IN proteolytic junction, the junction between the IN core and C terminus, and the C terminus of IN. For HIV-1 IN, in addition to the junction between the IN core and C terminus and the C terminus of IN, insertions between the N terminus and core domains maintained integration and disintegration activity. Of the 40 in-frame insertions within the M-MuLV RT connection-RNase H domains, only the three C-terminal insertions mapping to the RT-IN proteolytic junction were viable. These results correlate with deletion studies mapping the domain and subdomain boundaries of RT and IN. Importantly, these genetic footprints provide a means to identify nonessential regions within RT and IN for targeted gene therapy applications.

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

2012 ◽  
Vol 56 (4) ◽  
pp. 2048-2061 ◽  
Author(s):  
Karen A. Kirby ◽  
Bruno Marchand ◽  
Yee Tsuey Ong ◽  
Tanyaradzwa P. Ndongwe ◽  
Atsuko Hachiya ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Xenotropic Murine Leukemia ◽  
Related Virus ◽  
Hydroxyl Radical Footprinting ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACTRNase 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.

scholarly journals Ubiquitin Is Covalently Attached to the p6GagProteins of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus and to the p12Gag Protein of Moloney Murine Leukemia Virus

1998 ◽  
Vol 72 (4) ◽  
pp. 2962-2968 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Terry D. Copeland ◽  
Bradley P. Kane ◽  
Donald G. Johnson ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Simian Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.

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

2015 ◽  
Vol 113 ◽  
pp. 44-50 ◽  
Author(s):  
Kosaku Nishimura ◽  
Kanta Yokokawa ◽  
Tetsuro Hisayoshi ◽  
Kosuke Fukatsu ◽  
Ikumi Kuze ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Murine Leukemia
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