Double-Stranded Strong-Stop DNA and the Second Template Switch in Human Immunodeficiency Virus (HIV) DNA Synthesis

Li, Peng; Stephenson, Alice J.; Kuiper, Lara J.; Burrell, Christopher J.

doi:10.1006/viro.1993.1237

Cited by 16 publications

(13 citation statements)

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“…However, unlike the DNA species detected by the previous three pairs of primers, which are present twice in the full-length HIV proviral genome and once in the double-stranded strong-stop DNA population (steps G and H in Fig. 1, also see Li et al, 1993), the species of DNA as detected by U3.1/PBS1 primers, appeared only once in the full-length HIV DNA and once in the doublestranded strong-stop DNA, hence the actual success rate for the initiation of plus-strand DNA synthesis is expected to be higher. The copy number of the near-fully extended minus-strand DNA as detected by the gagl/ gag2 primer, was estimated at ~ 500 000.…”

Section: R' Us'mentioning

confidence: 73%

“…This process can be divided into several discrete steps: (i) synthesis of a short stretch of DNA ('the minus-strand strong-stop DNA') from the primer binding site (PBS) near the 5' end of one genomic viral RNA molecule, using a tRNA as primer; (ii) transfer of the minus-strand strong-stop DNA from the 5' end of the viral RNA to the 3' end of the same, or a second molecule of viral RNA; (iii) continued synthesis of the minus-strand DNA, accompanied by degradation of the RNA template by RNase H activity of the viral reverse transcriptase [this process leaves a polypurine tract (PPT) at specific sites in the 3' end of the viral RNA to serve as the primer for the synthesis of the plus-strand viral DNA using the minus-strand DNA as template]; * Author for correspondence. Fax + 61 8 228 7538. e-mail jmcinnes @ microb.adelaide.edu.au (iv) this newly synthesized short stretch of plus-strand DNA ('plus-strand strong-stop DNA') is then transferred from the 5' end to the 3' end of newly made, partially completed minus-strand DNA; (v) after the second template transfer, the remainder of the plusstrand DNA as well as the 3' end of the minus-strand DNA is synthesized, to yield a linear double-stranded DNA with all sequences in the viral RNA plus duplicated U3 and U5 sequences present in the long terminal repeats (for reviews see Varmus & Brown, 1989;Coffin, 1990;Li et al, 1993;Jones et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

“…In the present study, we report a quantitative PCR analysis of the kinetics of HIV reverse transcription in this model. In this study, PCR was employed essentially for its ability to quantify DNA structures heterogeneous in length but representing completion of each of the ordered sequence of specific steps during reverse transcription (Varmus & Brown, 1989;Zack et al, 1990;Li et al, 1993). Different efficiencies arising from using different primer pairs and other PCR conditions were normalized by conversion of each PCR product signal to copy numbers by comparison with known standards.…”

Section: Introductionmentioning

confidence: 99%

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Stepwise analysis of reverse transcription in a cell-to-cell human immunodeficiency virus infection model: kinetics and implications

Karageorgos

Burrell

1995

Journal of General Virology

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We have investigated the kinetics of human immunodeficiency virus (HIV) reverse transcription in infected T cells, using a synchronized, one-step, cell-to-cell infection model and quantitative PCR assays for the different DNA intermediate structures that are found sequentially during reverse transcription. Different efficiencies that might arise from the use of different primers and other PCR conditions were normalized by conversion of each PCR product signal to copy numbers by comparing with standards. After an initial lag period, the minus-strand strong-stop viral DNA was detected first. This was followed by the post-transfer newly extended minus-strand viral DNA and then by the plusstrand strong-stop DNA and fully extended minusstrand DNA. Kinetic data indicated that, once reverse transcription was initiated, the HIV reverse transcriptase synthesized minus-strand DNA at a rate of 150-180 bases/min, and that the first template transfer and the initiation of the plus-strand DNA synthesis imposed specific time delays. In contrast, minus-strand viral DNA synthesized after the second template transfer appeared at a time point very close to the time of the appearance of the last piece of DNA synthesized just before the second template switch, suggesting that the second switch occurred very rapidly. Taken together, our results define more accurately than was previously possible the rates of several of the steps in HIV reverse transcription in infected T cell lines and indicate different mechanisms for the two distinct template switches during retrovirus reverse transcription.

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Section: R' Us'mentioning

confidence: 73%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Stepwise analysis of reverse transcription in a cell-to-cell human immunodeficiency virus infection model: kinetics and implications

Karageorgos

Burrell

1995

Journal of General Virology

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“…This intramolecular process leads to the generation of a circular molecule (Withcomb et al, 1990;Figure 1, step 9). Plus-strand strong-stop DNAs which represent the partially completed, newly made DNA prior to the second template switch, have been shown to accumulate during reverse transcription (Varmus and Shank, 1976;Coffin and Haseltine, 1977;Li R et al, 1993). The plusstrand is then fully replicated from the upstream PPT primer through a strand displacement DMA synthesis mechanism (Huber eta/., 1989;Hottiger et al, 1994).…”

Section: Plus-strand Dna Synthesismentioning

confidence: 99%

Review

1996

Biological Chemistry Hoppe-Seyler

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Proteolytic enzymes have many physiological functions in plants, bacteria, viruses, protozoa and mammals. They play a role in processes such as food digestion, complement activation or blood coagulation. The action of proteolytic enzymes is biologically controlled by proteinase inhibitors and increasing attention is being paid to the physiological significance of these natural inhibitors in pathological processes. The reason for this growing interest is that uncontrolled proteolysis can lead to irreversible damage e.g. in chronic inflammation or tumor metastasis. This review focusses on the possible role of the cystatins, natural and specific inhibitors of the cysteine proteinases, in pathological processes. Key words: Cystatins / Cysteine proteinases / Inflammation / Inhibitors / Pathological processes. Cysteine ProteinasesCysteine Proteinases, synonymous with thiol proteinases, are small proteins with molecular masses varying from 23 to 24 kDa and with two catalytic residues, Cys25 and His159, which are involved in the hydrolytic reaction. Cysteine proteinases catalyze the hydrolysis of various polypeptide substrates and are most active under reducing and mildly acidic conditions (pH 5 -6.5). They have been detected in and isolated from a large number of biological sources including plants, bacteria, animals and humans. Bacterial cysteine proteinases are produced by Clostridium histolyticum, named clostripain, by hemolytic group A streptococci and by the pathogenic anaerobic Porphyromonas gingivalis, a bacterium associated with periodontitis. Bacterial cysteine proteinases probably play a role in the penetration of normal tissues by the bacteria and in the food digestion. Viral cysteine proteinases from picornaviruses, e.g. the poliovirus and rhinovirus type I, are involved in proteolytic cleavage of precursor proteins for virus replication and for the production of new virus particles. A virus-coded cysteine proteinase is involved in this process (Kay and Dunn, 1990; Körant et a/., 1985;. HIV-I also needs proteolytic processing by cysteine proteinases to regulate the expression of viral proteins (Guy ef a/., 1991). Protozoal cysteine proteinases are produced by a number of protozoan parasites to facilitate host invasion, metabolize host proteins, to degrade the host immune molecules or to use them for intracellular replication. Cysteine proteinases are produced by e.g. Trypanosoma cruzi, the causative agent of American trypanosomiasis or by Leishmania species, causing one of the six major parasitic diseases. Furthermore, human malarial parasites produce a broad scala of proteinases including cysteine proteinase which are involved in erythrocyte invasion and rupture (McKerrow, 1993). Mammalian cysteine proteinases are present in the lysosomes of cells. Lysosomes contain different cysteine proteinases, the most important being the cathepsins B, H, L and S Kirschke, 1981, Barrett et a/., 1988). These cathepsins have common ancestors and are related to papain. Cathepsin B has been detected in every tissue or cel...

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“…In this DNA, a single-stranded region was localized near the middle of the molecule (Kupiec eta]., 1988), and nucleotide sequence analysis showed a PPT duplication in the same region (Kupiec eta]., 1988,1991). The gap was then shown to be located on the plus DNA strand and to be 120 nucleotides long, Li et al (1993) for the second jump.…”

Section: The ' Gap 'mentioning

confidence: 99%