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

Abstract

Retroviral reverse transcriptases contain a DNA polymerase activity that can copy an RNA or DNA template and an RNase H activity that degrades the viral RNA genome during reverse transcription. RNase H makes both specific and nonspecific cleavages; specific cleavages are used to generate and remove the polypurine tract primer used for plus-strand DNA synthesis and to remove the tRNA primer used for minus-strand DNA synthesis. We generated mutations in an HIV-1-based vector to change amino acids in the RNase H domain that contact either the RNA and DNA strands. Some of these mutations affected the initiation of DNA synthesis, demonstrating an interdependence of the polymerase and RNase H activities of HIV-1 reverse transcription during viral DNA synthesis. The ends of the linear DNA form of the HIV-1 genome are defined by the specific RNase H cleavages that remove the plus-and minus-strand primers; these ends can be joined to form two-longterminal repeat circles. Analysis of two-long-terminal repeat circle junctions showed that mutations in the RNase H domain affect the specificity of RNase H cleavage.H IV-1 reverse transcriptase (RT) is the virally encoded enzyme that converts the single-stranded RNA genome found in virions into double-stranded DNA. The conversion of the RNA genome into DNA is accomplished through the collaboration of the two enzymatic activities of RT: a DNA polymerase that can use either RNA or DNA as a template and an RNase H that cleaves RNA if (and only if) it is present in an RNA⅐DNA duplex (reviewed in refs. 1-3). Both polymerase and RNase H activities are required for the conversion of the RNA genome into double-stranded DNA; mutations that inactivate either the polymerase or RNase H block viral replication (4-7).In vitro assays showed that polymerase and RNase H activities of HIV-1 RT are interdependent (8-13). Both the polymerase and RNase H domains of HIV-1 RT simultaneously contact the nucleic acid substrate and contribute to the binding (and proper positioning) of the nucleic acid. In in vitro assays, mutations in the RNase H domain can affect polymerase activity (and vice versa). We found that reducing RNase H activity by cotransfecting a mixture of wild-type HIV-1 vector DNA and DNA encoding HIV-1 vectors with mutations in the RNase H active site reduced the efficiency of initiation of DNA synthesis (14). On the basis of these observations, we wanted to determine whether mutating amino acids that make specific contacts with the nucleic acid substrate in the vicinity of the RNase H active site would also affect the initiation of DNA synthesis and͞or the specificity of RNase H cleavage.Reverse transcription requires that RNase H make both specific and nonspecific cleavages. The process of reverse transcription is shown schematically in Fig. 1 (reviewed in refs. 1 and 3). The RNase H cleavages that remove the tRNA primer, and the cleavages that generate and remove the polypurine tract (ppt) primer define the ends of the unintegrated linear viral DNA that is the substrate for the int...

Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir

“…From these experiments, we conclude that, in MLV replication: (i) Raltegravir inhibits the DNA strand transfer (27) but not the 3Ј processing; (ii) Because it increases rather than decreases 2-LTR circle concentration, reverse transcriptase activity and DNA repair are not inhibited either; (iii) Trex1 degrades the 3Ј ends of retroviral DNA and also is not inhibited by raltegravir; and (iv) Because insertions at the circle junctions are generated by defective RNase H activity (35), but raltegravir treatment does not seem to increase their number (see Fig. 1B), RNase H probably is also unaffected.…”

FIV: from lentivirus to lentivector

“…After HIV-1 vectors were described, replication-defective systems were engineered from molecular clones of non-primate and other primate lentiviruses [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. In addition to their use in pre-clinical gene therapy research, lentiviral vectors have been important tools for basic science [20][21][22][23][24][25][26][27][28]. This review concentrates on virological fundamentals of FIV-based lentiviral vector development, and discusses some recent lentivirological controversies and vector applications.…”

Dynamic binding orientations direct activity of HIV reverse transcriptase

“…These residues are conserved between RNases H found in viruses, bacteria and humans 22,31,32 . Replacement of these residues with alanine in HIV-1 RT decreases the DNA synthesis rate and inhibits virus infectivity 33 . Overall, the backbone composition of nucleotides near the 59 end of the primer had a greater influence on enzyme binding orientation than those near the 39 end (Fig.…”