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. 2002 Jul 9;99(14):9515-20.
doi: 10.1073/pnas.142123199. Epub 2002 Jul 1.

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

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Free PMC article

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

John G Julias et al. Proc Natl Acad Sci U S A. .
Free PMC article

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-long-terminal 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.

Figures

Figure 1
Figure 1
Reverse transcription of viral RNA. (i) Minus-strand DNA synthesis is initiated from a tRNALys-3 primer (gray arrow) annealed to the pbs. (ii) The U5 and R regions of the viral RNA (thin line) are copied into DNA (thick line), and the RNase H activity of RT degrades the viral RNA (the dotted line represents degraded RNA). (iii) Minus-strand DNA can be transferred to the 3′ end of the viral RNA because of the complementarity of the viral RNA R region and the minus-strand DNA. (iv) Minus-strand DNA elongation copies the RNA genome, and the RNase H activity of RT degrades the viral RNA. The ppt is resistant to RNase H cleavage and serves as the primer for plus-strand DNA synthesis. Two specific cleavages by RNase H generate the ppt primer (arrowheads). (v) Plus-strand DNA synthesis is initiated from the ppt and copies the U3, R, and U5 regions of the minus-strand DNA. RNase H removes the tRNA primer 1 base from the RNA/DNA junction and the ppt at the RNA/DNA junction (arrowheads). (vi) Plus-strand transfer occurs using the complementarity of the pbs and the ends of the viral DNA. Extension of the plus- and minus-strands complete the synthesis of the viral DNA. The ribonucleotide (rA) at the 5′ end of the minus strand is derived from the tRNA. HIV-1 RT removes the tRNA one base from the RNA-DNA junction (–18); other retroviral RTs remove the entire tRNA (2). (vii) The structure of full-length viral DNA.
Figure 2
Figure 2
Stereo diagram showing the structure of the RNase H primer grip. The RNA template strand is shown in turquoise, the DNA primer strand, in purple. The scissile phosphate is designated with a red arrow pointing to the phosphate. The RNase H domain is shown in orange; the connection subdomain is in yellow. Amino acids contacting the nucleic acid are labeled. Amino acids that were not mutated are labeled in black. Amino acids that had minimal (<2-fold) effects on titer when converted to alanine are labeled in green. Amino acids where alanine substitutions had a moderate (>5-fold) or strong effect on titer are labeled in blue. Contacts between individual amino acids and the RNA template are shown in black, contacts with the DNA primer are shown in red. Most of the contacts are shown as dotted lines; contacts with Q475 (which contacts both strands) are shown as solid lines. The nucleic acid strands are numbered relative to the site of cleavage on the RNA strand.
Figure 3
Figure 3
(A) The HIV-1 vector pNLNgoMIVRE.HSA expresses the murine cell surface marker CD24 (heat-stable antigen, HSA) from the nef ORF. The env and the vpr genes have been inactivated. The drawing is not to scale. (B) The effect of mutations in the RNase H domain on virus titer. The relative infectivity of the mutants, normalized to the p24 concentration, is shown on the y axis and the mutants are shown on the x axis.
Figure 4
Figure 4
Viral DNA synthesis by RNase H mutants. The amount of viral DNA synthesized by the RNase H primer grip mutants relative to wild-type is shown on the y axis. The virus used to infect cells is indicated on the x axis the different steps of reverse transcription that were monitored are shown by the differently shaded bars, as designated in the figure key. The amount of virus used for the infections was measured by using a p24 ELISA. The copy numbers of different products measured for the different steps of reverse transcription for wild-type virus were 6 × 105 copies of RU5 specific DNA, 5 × 105 copies of U3 specific DNA, 4 × 105 copies of gag-specific DNA, and 2 × 105 copies of DNA specific for plus-strand transfer.
Figure 5
Figure 5
Frequency of different classes of 2-LTR circle junctions. 2-LTR circle junctions were analyzed from cells infected with the wild-type vector (WT), vector with reduced RNase H activity (WT + E478Q), and vectors containing the RNase H primer grip mutations Q475A, Y501A, and N474A + Q475A. At the top is a schematic diagram of the unintegrated viral DNA. The pbs is shown in navy blue, the viral leader sequence 3′ of the pbs is shown in light blue, the region 5′ of the ppt is shown in gray, the U-tract is red, and the ppt is in green. The drawing is not to scale. (i) The consensus 2-LTR circle junction arising from the ligation of the ends of unintegrated viral DNA. The underlined T is derived from the ribonucleotide A at the 3′ end of the tRNALys-3 primer used for minus-strand DNA synthesis. (ii) “Simple” ppt insertions (green box) that contain part or all of the ppt but no upstream flanking sequences. This class of defective circle junctions may also contain deletions in the U5 region. (iii) Insertion of the ppt (green box) along with short flanking sequences (adjacent red box) immediately upstream of the ppt. These flanking sequences are 1–5 bp in length and are probably caused by improper cleavages by RNase H (see Fig. 6 and Results and Discussion). (iv) Insertion of the ppt with long flanking sequences (gray box) upstream of the ppt. This class of inserts may also include deletions in the U5 region. (v) Insertions of part of the tRNA Lys-3 sequences (blue box) at the 2-LTR circle junction. Only a portion of the tRNA is reverse transcribed and inserted; this portion corresponds to the tRNA sequences that anneal to the pbs. This class of defective circle junctions may also include deletions in the U3 region. (vi) Insertion of tRNA (navy blue box) and viral leader sequence from downstream of the pbs (light blue box). During reverse transcription, plus-strand DNA synthesis is initiated from the ppt primer and copies both the minus-strand DNA and the portion of the tRNA primer that hybridizes to the pbs (Fig. 1). Normally, this plus strand is transferred to the minus strand after the tRNA is removed (see Fig. 1). However, if RNase H fails to remove the tRNA primer before the plus strand is extended a second time to complete the synthesis of viral DNA, the tRNA can be copied a second time. If this happens, the DNA could undergo another strand-transfer reaction, and RT could then copy the adjacent sequences on the leader, which could be captured at the circle junction. This class of defective circle junctions may also include deletions in the U3 region. (vii) Small deletions (1–5 bp) at the circle junction in the U3, U5, or both U3 and U5 (white to black gradation). (viii) Large deletions in the U5, the U3 or both U5 and U3 (white to black box overlapping the circle junction). (ix) Insertions (yellow box) at the 2-LTR circle junction that do not come from either the ppt or the tRNA primer. (x) Deletions with insertions that do not derive from the ppt or the tRNA primer.
Figure 6
Figure 6
Miscleavages by RNase H lead to insertions of short flanking sequences 5′ of the ppt and to small deletions in the U3 region. (Top) The RNA⋅DNA duplex containing the ppt (green lettering). Wild-type RT cleaves at the U-tract/ppt junction (black arrow) and at the ppt/U3 junction (gray arrow) to generate the normal ppt primer used for plus-strand DNA synthesis. Miscleavage (orange arrow) in the U-tract (orange lettering) can lead to the insertion of flanking sequences if the ppt is retained. Miscleavage in U3 (blue arrow) generates an incorrect ppt primer, which, if it is removed by RNase H, creates a 5-bp deletion in U3.

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