Altered error specificity of RNase H-deficient HIV-1 reverse transcriptases during DNA-dependent DNA synthesis

ABSTRACT The RNase H activity of retroviral reverse transcriptases (RTs) degrades viral genomic RNA after it has been copied into DNA, removes the tRNA used to initiate negative-strand DNA synthesis, and generates and removes the polypurine tract (PPT) primer used to initiate positive-strand DNA synthesis. The cleavages that remove the tRNA and that generate and remove the PPT primer must be specific to generate linear viral DNAs with ends that are appropriate for integration into the host cell genome. The crystal structure of human immunodeficiency virus type 1 (HIV-1) RT in a complex with an RNA/DNA duplex derived from the PPT revealed that the 5′ end of the PPT deviates from traditional Watson-Crick base pairing. This unusual structure may play a role in the proper recognition of the PPT by HIV-1 RT. We made substitution mutations in the 5′ end of the PPT and determined their effects on virus titer. The results indicated that single and double mutations in the 5′ end of the PPT had modest effects on virus replication in a single-cycle assay. More complex mutations had stronger effects on virus titer. Analysis of the two-long-terminal-repeat circle junctions derived from infecting cells with the mutant viruses indicated that the mutations affected RNase H activity, resulting in the retention of PPT sequences on viral DNA. The mutants tested preferentially retained specific segments of the PPT, suggesting an effect on cleavage specificity. These results suggest that structural features of the PPT are important for its recognition and cleavage in vivo.

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Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization

Biochemical Journal ◽

10.1042/bj3290579 ◽

1998 ◽

Vol 329 (3) ◽

pp. 579-587 ◽

Cited By ~ 21

Author(s):

Ran TAUBE ◽

Shoshana LOYA ◽

Orna AVIDAN ◽

Michal PERACH ◽

Amnon HIZI

Keyword(s):

Reverse Transcriptase ◽

Dna Synthesis ◽

Biochemical Characterization ◽

Ph Dependence ◽

Rnase H ◽

Mammary Tumour ◽

Nucleoside Triphosphate ◽

Mouse Mammary Tumour Virus ◽

Dna Primers ◽

Hiv 1

We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the -1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMTV RT were compared with those of the well-studied RT of HIV-1, the causative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivity to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs), as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV-1 RT.

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Specific Cleavages by RNase H Facilitate Initiation of Plus-Strand RNA Synthesis by Moloney Murine Leukemia Virus

Journal of Virology ◽

10.1128/jvi.77.9.5275-5285.2003 ◽

2003 ◽

Vol 77 (9) ◽

pp. 5275-5285 ◽

Cited By ~ 15

Author(s):

Sharon J. Schultz ◽

Miaohua Zhang ◽

James J. Champoux

Keyword(s):

Dna Synthesis ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Rnase H ◽

Primer Extension ◽

Moloney Murine Leukemia Virus ◽

Cleavage Sites ◽

Reverse Transcriptases ◽

Hybrid Substrates ◽

Murine Leukemia

ABSTRACT Successful generation, extension, and removal of the plus-strand primer is integral to reverse transcription. For Moloney murine leukemia virus, primer removal at the RNA/DNA junction leaves the 3′ terminus of the plus-strand primer abutting the downstream plus-strand DNA, but this 3′ terminus is not efficiently reutilized for another round of extension. The RNase H cleavage to create the plus-strand primer might similarly result in the 3′ terminus of this primer abutting downstream RNA, yet efficient initiation must occur to synthesize the plus-strand DNA. We hypothesized that displacement synthesis, RNase H activity, or both must participate to initiate plus-strand DNA synthesis. Using model hybrid substrates and RNase H-deficient reverse transcriptases, we found that displacement synthesis alone did not efficiently extend the plus-strand primer at a nick with downstream RNA. However, specific cleavage sites for RNase H were identified in the sequence immediately following the 3′ end of the plus-strand primer. During generation of the plus-strand primer, cleavage at these sites generated a gap. When representative gaps separated the 3′ terminus of the plus-strand primer from downstream RNA, primer extension significantly improved. The contribution of RNase H to the initiation of plus-strand DNA synthesis was confirmed by comparing the effects of downstream RNA versus DNA on plus-strand primer extension by wild-type reverse transcriptase. These data suggest a model in which efficient initiation of plus-strand synthesis requires the generation of a gap immediately following the plus-strand primer 3′ terminus.

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Fidelity of Mutant HIV-1 Reverse Transcriptases: Interaction with the Single-Stranded Template Influences the Accuracy of DNA Synthesis†

Biochemistry ◽

10.1021/bi972672g ◽

1998 ◽

Vol 37 (17) ◽

pp. 5831-5839 ◽

Cited By ~ 33

Author(s):

Baek Kim ◽

Tanya R. Hathaway ◽

Lawrence A. Loeb

Keyword(s):

Dna Synthesis ◽

Reverse Transcriptases ◽

Hiv 1

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Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.142123199 ◽

2002 ◽

Vol 99 (14) ◽

pp. 9515-9520 ◽

Cited By ~ 66

Author(s):

J. G. Julias ◽

M. J. McWilliams ◽

S. G. Sarafianos ◽

E. Arnold ◽

S. H. Hughes

Keyword(s):

Reverse Transcriptase ◽

Dna Synthesis ◽

Rnase H ◽

Hiv 1

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In Vitro Analysis of Human Immunodeficiency Virus Type 1 Minus-Strand Strong-Stop DNA Synthesis and Genomic RNA Processing

Journal of Virology ◽

10.1128/jvi.75.2.672-686.2001 ◽

2001 ◽

Vol 75 (2) ◽

pp. 672-686 ◽

Cited By ~ 31

Author(s):

Mark D. Driscoll ◽

Marie-Pierre Golinelli ◽

Stephen H. Hughes

Keyword(s):

Human Immunodeficiency Virus ◽

Dna Synthesis ◽

Viral Genome ◽

Rnase H ◽

Minus Strand ◽

Genomic Rna ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Strong Stop

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), nucleocapsid protein (NC), genomic RNA, and the growing DNA strand all influence the copying of the HIV-1 RNA genome into DNA. A detailed understanding of these activities is required to understand the process of reverse transcription. HIV-1 viral DNA is initiated from a tRNA3 Lys primer bound to the viral genome at the primer binding site. The U3 and R regions of the RNA genome are the first sequences to be copied. The TAR hairpin, a structure found within the R region of the viral genome, is the site of increased RT pausing, RNase H activity, and RT dissociation. Template RNA was digested approximately 17 bases behind the site where polymerase paused at the base of TAR. In most template RNAs, this was the only cleavage made by the RT responsible for initiating polymerization. If the RT that initiated DNA synthesis dissociated from the base of the TAR hairpin and an RT rebound at the end of the primer, there was competition between the polymerase and RNase H activities. After the complete heteroduplex was formed, there were additional RNase H cleavages that did not involve polymerization. Levels of NC that prevented TAR DNA self-priming did not protect genomic RNA from RNase H digestion. RNase H digestion of the 100-bp heteroduplex produced a 14-base RNA from the 5′ end of the RNA that remained annealed to the 3′ end of the minus-strand strong-stop DNA only if NC was present in the reaction.

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Inhibition of the integrases of human immunodeficiency viruses type 1 and type 2 by reverse transcriptases

Biochemical Journal ◽

10.1042/bj3610557 ◽

2002 ◽

Vol 361 (3) ◽

pp. 557-566 ◽

Cited By ~ 20

Author(s):

Iris OZ ◽

Orna AVIDAN ◽

Amnon HIZI

Keyword(s):

Protein Structures ◽

Three Dimensional ◽

Rnase H ◽

Strand Transfer ◽

Leukaemia Virus ◽

Reverse Transcriptases ◽

Show Evidence ◽

Human Immunodeficiency Viruses ◽

Hiv 1

We present evidence that the integrases (INs) of HIV types 1 and 2 are inhibited in vitro by the reverse transcriptases (RTs) of HIV-1, HIV-2 and murine leukaemia virus. Both 3′-end processing and 3′-end joining (strand transfer) activities of IN were affected by the RTs. Full inhibitions were accomplished with most RT and IN combinations tested at around equimolar RT/IN ratios. The disintegration activity of IN was also inhibited by RTs. Neither DNA synthesis nor the ribonuclease H (RNase H) domain of RT were involved in IN inhibition, since specific DNA polymerase inhibitors did not affect the level of IN inhibition, and the p51 isoform of HIV-1 RT (which lacks the RNase H domain) is as effective in inhibiting IN as the heterodimeric p66/p51 isoform. On the other hand, the catalytic activities of HIV RTs were not affected by the INs, showing that RTs can inhibit IN activities, whereas INs do not inhibit RTs. We postulate that sequences and/or three-dimensional protein structures common to RTs interact with INs and inhibit their activities. We show evidence for this hypothesis and discuss the possible sites of IN involved in this interaction.

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