Specific Recognition and Cleavage of the Plus-Strand Primer by Reverse Transcriptase

ABSTRACT Reverse transcriptases (RTs) of retroviruses and long terminal repeat (LTR)-retrotransposons possess DNA polymerase and RNase H activities. During reverse transcription these activities are necessary for the programmed sequence of events that include template switching and primer processing. Integrase then inserts the completed cDNA into the genome of the host cell. The RT of the LTR-retrotransposon Tf1 was subjected to random mutagenesis, and the resulting transposons were screened with genetic assays to test which mutations reduced reverse transcription and which inhibited integration. We identified a cluster of mutations in the RNase H domain of RT that were surprising because they blocked integration without reducing cDNA levels. The results of immunoblots demonstrated that these mutations did not reduce levels of RT or integrase. DNA blots showed that the mutations did not lower the amounts of full-length cDNA. The sequences of the 3′ ends of the cDNA revealed that mutations within the cluster in RNase H specifically reduced the removal of the polypurine tract (PPT) primer from the ends of the cDNA. These results indicate that primer removal is not a necessary component of reverse transcription. The residues mutated in Tf1 RNase H are conserved in human immunodeficiency virus type 1 and make direct contact with DNA opposite the PPT. Thus, our results identify a conserved element in RT that contacts the PPT and is specifically required for PPT removal.

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Template Dimerization Promotes an Acceptor Invasion-Induced Transfer Mechanism during Human Immunodeficiency Virus Type 1 Minus-Strand Synthesis

Journal of Virology ◽

10.1128/jvi.77.8.4710-4721.2003 ◽

2003 ◽

Vol 77 (8) ◽

pp. 4710-4721 ◽

Cited By ~ 44

Author(s):

Mini Balakrishnan ◽

Bernard P. Roques ◽

Philip J. Fay ◽

Robert A. Bambara

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Rnase H ◽

Template Switching ◽

Acceptor Interaction ◽

Immunodeficiency Virus ◽

Donor Acceptor ◽

Hiv 1

ABSTRACT The biochemical mechanism of template switching by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the role of template dimerization were examined. Homologous donor-acceptor template pairs derived from the HIV-1 untranslated leader region and containing the wild-type and mutant dimerization initiation sequences (DIS) were used to examine the efficiency and distribution of transfers. Inhibiting donor-acceptor interaction was sufficient to reduce transfers in DIS-containing template pairs, indicating that template dimerization, and not the mere presence of the DIS, promotes efficient transfers. Additionally, we show evidence that the overall transfer process spans an extended region of the template and proceeds through a two-step mechanism. Transfer is initiated through an RNase H-facilitated acceptor invasion step, while synthesis continues on the donor template. The invasion then propagates towards the primer terminus by branch migration. Transfer is completed with the translocation of the primer terminus at a site distant from the invasion point. In our system, most invasions initiated before synthesis reached the DIS. However, transfer of the primer terminus predominantly occurred after synthesis through the DIS. The two steps were separated by 60 to 80 nucleotides. Sequence markers revealed the position of primer terminus switch, whereas DNA oligomers designed to block acceptor-cDNA interactions defined sites of invasion. Within the region of homology, certain positions on the template were inherently more favorable for invasion than others. In templates with DIS, the proximity of the acceptor facilitates invasion, thereby enhancing transfer efficiency. Nucleocapsid protein enhanced the overall efficiency of transfers but did not alter the mechanism.

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Dimerization and Template Switching in the 5′ Untranslated Region between Various Subtypes of Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.77.5.3020-3030.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3020-3030 ◽

Cited By ~ 26

Author(s):

Ebbe Sloth Andersen ◽

Rienk E. Jeeninga ◽

Christian Kroun Damgaard ◽

Ben Berkhout ◽

Jørgen Kjems

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Untranslated Region ◽

Template Switching ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) particle contains two identical RNA strands, each corresponding to the entire genome. The 5′ untranslated region (UTR) of each RNA strand contains extensive secondary and tertiary structures that are instrumental in different steps of the viral replication cycle. We have characterized the 5′ UTRs of nine different HIV-1 isolates representing subtypes A through G and, by comparing their homodimerization and heterodimerization potentials, found that complementarity between the palindromic sequences in the dimerization initiation site (DIS) hairpins is necessary and sufficient for in vitro dimerization of two subtype RNAs. The 5′ UTR sequences were used to design donor and acceptor templates for a coupled in vitro dimerization-reverse transcription assay. We showed that template switching during reverse transcription is increased with a matching DIS palindrome and further stimulated proportional to the level of homology between the templates. The presence of the HIV-1 nucleocapsid protein NCp7 increased the template-switching efficiency for matching DIS palindromes twofold, whereas the recombination efficiency was increased sevenfold with a nonmatching palindrome. Since NCp7 did not effect the dimerization of nonmatching palindromes, we concluded that the protein most likely stimulates the strand transfer reaction. An analysis of the distribution of template-switching events revealed that it occurs throughout the 5′ UTR. Together, these results demonstrate that the template switching of HIV-1 reverse transcriptase occurs frequently in vitro and that this process is facilitated mainly by template proximity and the level of homology.

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Replication of Phenotypically Mixed Human Immunodeficiency Virus Type 1 Virions Containing Catalytically Active and Catalytically Inactive Reverse Transcriptase

Journal of Virology ◽

10.1128/jvi.75.14.6537-6546.2001 ◽

2001 ◽

Vol 75 (14) ◽

pp. 6537-6546 ◽

Cited By ~ 96

Author(s):

John G. Julias ◽

Andrea L. Ferris ◽

Paul L. Boyer ◽

Stephen H. Hughes

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Virus Replication ◽

Human Immunodeficiency Virus Type ◽

Reverse Transcription ◽

Rnase H ◽

Wild Type ◽

Immunodeficiency Virus ◽

Catalytically Active

ABSTRACT The amount of excess polymerase and RNase H activity in human immunodeficiency virus type 1 virions was measured by using vectors that undergo a single round of replication. Vectors containing wild-type reverse transcriptase (RT), vectors encoding the D110E mutation to inactivate polymerase, and vectors encoding mutations D443A and E478Q to inactivate RNase H were constructed. 293 cells were cotransfected with different proportions of plasmids encoding these vectors to generate phenotypically mixed virions. The resulting viruses were used to infect human osteosarcoma cells, and the relative infectivity of the viruses was determined by measuring transduction of the murine cell surface marker CD24, which is encoded by the vectors. The results indicated that there is an excess of both polymerase and RNase H activities in virions. Viral replication was reduced to 42% of wild-type levels in virions with where half of the RT molecules were predicted to be catalytically active but dropped to 3% of wild-type levels when 25% of the RT molecules were active. However, reducing RNase H activity had a lesser effect on viral replication. As expected, based on previous work with murine leukemia virus, there was relatively inefficient virus replication when the RNase H and polymerase activities were encoded on separate vectors (D110E plus E478Q and D110E plus D443A). To determine how virus replication failed when polymerase and RNase H activities were reduced, reverse transcription intermediates were measured in vector-infected cells by using quantitative real-time PCR. The results indicated that using the D11OE mutation to reduce the amount of active polymerase reduced the number of reverse transcripts that were initiated and also reduced the amounts of products from the late stages of reverse transcription. If the E478Q mutation was used to reduce RNase H activity, the number of reverse transcripts that were initiated was reduced; there was also a strong effect on minus-strand transfer.

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Antiretroviral Drug Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Increase Template-Switching Frequency

Journal of Virology ◽

10.1128/jvi.78.16.8761-8770.2004 ◽

2004 ◽

Vol 78 (16) ◽

pp. 8761-8770 ◽

Cited By ~ 60

Author(s):

Galina N. Nikolenko ◽

Evguenia S. Svarovskaia ◽

Krista A. Delviks ◽

Vinay K. Pathak

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Rnase H ◽

Switching Frequency ◽

Template Switching ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Template-switching events during reverse transcription are necessary for completion of retroviral replication and recombination. Structural determinants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that influence its template-switching frequency are not known. To identify determinants of HIV-1 RT that affect the frequency of template switching, we developed an in vivo assay in which RT template-switching events during viral replication resulted in functional reconstitution of the green fluorescent protein gene. A survey of single amino acid substitutions near the polymerase active site or deoxynucleoside triphosphate-binding site of HIV-1 RT indicated that several substitutions increased the rate of RT template switching. Several mutations associated with resistance to antiviral nucleoside analogs (K65R, L74V, E89G, Q151N, and M184I) dramatically increased RT template-switching frequencies by two- to sixfold in a single replication cycle. In contrast, substitutions in the RNase H domain (H539N, D549N) decreased the frequency of RT template switching by twofold. Depletion of intracellular nucleotide pools by hydroxyurea treatment of cells used as targets for infection resulted in a 1.8-fold increase in the frequency of RT template switching. These results indicate that the dynamic steady state between polymerase and RNase H activities is an important determinant of HIV-1 RT template switching and establish that HIV-1 recombination occurs by the previously described dynamic copy choice mechanism. These results also indicate that mutations conferring resistance to antiviral drugs can increase the frequency of RT template switching and may influence the rate of retroviral recombination and viral evolution.

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RNase H Requirements for the Second Strand Transfer Reaction of Human Immunodeficiency Virus Type 1 Reverse Transcription

Journal of Virology ◽

10.1128/jvi.73.8.6573-6581.1999 ◽

1999 ◽

Vol 73 (8) ◽

pp. 6573-6581 ◽

Cited By ~ 24

Author(s):

Christine M. Smith ◽

Jeffrey S. Smith ◽

Monica J. Roth

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcription ◽

Transfer Reaction ◽

Rnase H ◽

Strand Transfer ◽

Transfer Event ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Retroviral reverse transcriptase (RT) enzymes are responsible for transcribing viral RNA into double-stranded DNA. An in vitro assay to analyze the second strand transfer event during human immunodeficiency virus type 1 (HIV-1) reverse transcription has been developed. Model substrates were constructed which mimic the viral intermediate found during plus-strand strong-stop synthesis. Utilizing wild-type HIV-1 RT and a mutant E478Q RT, the requirement for RNase H activity in this strand transfer event was analyzed. In the presence of Mg2+, HIV-1 RT was able to fully support the second strand transfer reaction in vitro. However, in the presence of Mg2+, the E478Q RT mutant had no detectable RNase H activity and was unable to support strand transfer. In the presence of Mn2+, the E478Q RT yields the initial endoribonucleolytic cleavage at the penultimate C residue of the tRNA primer yet does not support strand transfer. This suggests that subsequent degradation of the RNA primer by the RNase H domain was required for strand transfer. In reactions in which the E478Q RT was complemented with exogenous RNase H enzymes, strand transfer was supported. Additionally, we have shown that HIV-1 RT is capable of supporting strand transfer with substrates that mimic tRNAHis as well as the authentic tRNA3 Lys.

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Pseudodiploid Genome Organization Aids Full-Length Human Immunodeficiency Virus Type 1 DNA Synthesis

Journal of Virology ◽

10.1128/jvi.02100-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2376-2384 ◽

Cited By ~ 11

Author(s):

Steven R. King ◽

Nisha K. Duggal ◽

Clement B. Ndongmo ◽

Crystal Pacut ◽

Alice Telesnitsky

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Full Length ◽

Template Switching ◽

Genomic Rnas ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Template switching between copackaged human immunodeficiency virus type 1 (HIV-1) genomic RNAs is genetically silent when identical RNAs are copackaged but yields recombinants when virions contain two distinct RNAs. Sequencing has revealed that errors at retroviral recombination junctions are infrequent, suggesting that template switching is not intrinsically mutagenic. Here, we tested the hypothesis that template switching may instead contribute to replication fidelity. This hypothesis predicts that reverse transcription of a single-copy gene will be more error prone than replication in the presence of a second copy. To test this, HIV-1-based vectors containing both lacZ and the puromycin resistance marker were expressed either alone or with an excess of an “empty” vector lacking lacZ and puro. This resulted in virions with either RNA homodimers or haploid genomes with only a single lacZ-puro RNA. In untreated cells, lacZ inactivation rates suggested that haploid vector reverse transcription was slightly more error prone than that of homodimerized pseudodiploid vectors. Haploid reverse transcription was at least threefold more error prone than pseudodiploid-templated synthesis when slowed by hydroxyurea treatment or stopped prematurely with zidovudine. Individual products of one- and two-copy genes revealed both nucleotide substitutions and deletions, with deletions more frequent than point mutations among haploid genome products. Similar spectra of defective products were observed at early reverse transcription time points and among products of haploid virions. These results indicate that faithful, full-length reverse transcription products were underrepresented in the absence of a reserve of genetic information and suggest that template switching contributes to HIV-1 genomic integrity.

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