Requirements for DNA Strand Transfer During Reverse Transcription in Mutant HIV-1 Virions

Berkhout, Ben; Wamel, Jos van; Klaver, Bep

doi:10.1006/jmbi.1994.0475

Cited by 56 publications

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“…Thus, in our system, the extent of sequence complementarity between (Ϫ) SSDNA and the acceptor RNA need not be extremely long for successful minus-strand transfer. This result is in excellent agreement with previous studies showing that the full-length R region is not required for minusstrand transfer both in vivo and in vitro (26,44,51,(77)(78)(79). Similarly, the size of the homology region is not the most critical factor for efficient recombination via internal strand transfer (80).…”

Section: Discussionsupporting

confidence: 92%

Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein

Heilman-Miller

Levin

2004

Journal of Biological Chemistry

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During human immunodeficiency virus type 1 minusstrand transfer, the nucleocapsid protein (NC) facilitates annealing of the complementary repeat regions at the 3-ends of acceptor RNA and minus-strand strong-stop DNA ((؊) SSDNA). In addition, NC destabilizes the highly structured complementary trans-activation response element (TAR) stem-loop (TAR DNA) at the 3-end of (؊) SSDNA and inhibits TAR-induced self-priming, a deadend reaction that competes with minus-strand transfer. To investigate the relationship between nucleic acid secondary structure and NC function, a series of truncated (؊) SSDNA and acceptor RNA constructs were used to assay minus-strand transfer and self-priming in vitro. The results were correlated with extensive enzymatic probing and mFold analysis. As the length of (؊) SSDNA was decreased, self-priming increased and was highest when the DNA contained little more than TAR DNA, even if NC and acceptor were both present; in contrast, truncations within TAR DNA led to a striking reduction or elimination of self-priming. However, destabilization of TAR DNA was not sufficient for successful strand transfer: the stability of acceptor RNA was also crucial, and little or no strand transfer occurred if the RNA was highly stable. Significantly, NC may not be required for in vitro strand transfer if (؊) SSDNA and acceptor RNA are small, relatively unstructured molecules with low thermodynamic stabilities. Collectively, these findings demonstrate that for efficient NC-mediated minus-strand transfer, a delicate thermodynamic balance between the RNA and DNA reactants must be maintained.Reverse transcription consists of a complex series of reactions catalyzed by the virion-associated enzyme reverse transcriptase (RT) 1 that lead to conversion of the single-stranded RNA genome into an integration-competent linear doublestranded DNA (1). This process is facilitated by host and viral accessory proteins, one of which is the viral nucleocapsid protein (NC). Human immunodeficiency virus type 1 (HIV-1) NC is a small, highly basic, nucleic acid-binding protein with two zinc fingers, each containing the invariant CCHC metal ion-binding motif (2-5). NC functions as a nucleic acid chaperone in an ATP-independent manner (6) and catalyzes nucleic acid conformational rearrangements that lead to the formation of the most thermodynamically stable structure ). This activity is required for efficient reverse transcription and allows NC to promote intermolecular annealing of nucleic acids with significant stretches of base complementarity (7, 12-26), destabilization of secondary structures in RNA and DNA templates that are responsible for RT pausing (27-30), unwinding of primer tRNA (19, 31-34), primer placement (12,15,19,32,33,(35)(36)(37)(38), and the initiation step (39 -41).NC nucleic acid chaperone activity is also critical for the minus-strand (reviewed in Refs. 8, 10, and 11) and plus-strand (18,20,22,42) transfer events that are required to complete elongation of minus-and plus-strand DNAs and to generate the long te...

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Section: Discussionsupporting

confidence: 92%

Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein

Heilman-Miller

Levin

2004

Journal of Biological Chemistry

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“…pLAI-R37 has a deletion in the R-U5 region of the 3Ј LTR. The WT 3Ј LTR is restored during the reverse transcription process in the first round of virus replication (47).…”

Section: Methodsmentioning

confidence: 99%

A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

Bel

Das

Cornelissen

et al. 2014

Journal of Biological Chemistry

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Background: Retroviruses package a genomic RNA dimer. In vitro studies suggested kissing loop and extended dimer (ED) RNA-RNA interactions. Results: HIV-1 mutants with ED defects are replication-impaired, and revertant viruses with compensatory RNA mutations were selected. Conclusion: We describe a correlation between in vitro ED defects and poor virus replication. Significance: We present, to our knowledge, the first in vivo evidence for the importance of the ED.

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“…The R1 PCR fragment was sequenced, subsequently digested with HindIII and ClaI, and cloned into the Blue-5ЈLTR vector containing the XbaI-ClaI fragment of the pLAI proviral clone (36). The R1 mutated XbaI-ClaI fragment was finally cloned into pLAI-R37, a derivative of the full-length infectious clone pLAI (10). The mutant proviral construct was designated pLAI-R37-M-R1, in which M indicates the original PAS 1,2 Lys and PBS 1,2 Lys mutations (T128A, G129C, G190A, A192C, C193G, A194T, and A198G).…”

Section: Methodsmentioning

confidence: 99%

Forced Selection of a Human Immunodeficiency Virus Type 1 Variant That Uses a Non-Self tRNA Primer for Reverse Transcription: Involvement of Viral RNA Sequences and the Reverse Transcriptase Enzyme

Abbink

Beerens

Berkhout

2004

J Virol

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Human immunodeficiency virus type 1 uses the tRNA 3Lys molecule as a selective primer for reverse transcription. This primer specificity is imposed by sequence complementarity between the tRNA primer and two motifs in the viral RNA genome: the primer-binding site (PBS) and the primer activation signal (PAS). In addition, there may be specific interactions between the tRNA primer and viral proteins, such as the reverse transcriptase (RT) enzyme. We constructed viruses with mutations in the PAS and PBS that were designed to employ the nonself primer tRNA Pro or tRNA 1,2 Lys . These mutants exhibited a severe replication defect, indicating that additional adaptation of the mutant virus is required to accommodate the new tRNA primer. Multiple independent virus evolution experiments were performed to select for fast-replicating variants. Reversion to the wild-type PBS-lys3 sequence was the most frequent escape route. However, we identified one culture in which the virus gained replication capacity without reversion of the PBS. This revertant virus eventually optimized the PAS motif for interaction with the nonself primer. Interestingly, earlier evolution samples revealed a single amino acid change of an otherwise well-conserved residue in the RNase H domain of the RT enzyme, implicating this domain in selective primer usage. We demonstrate that both the PAS and RT mutations improve the replication capacity of the tRNA 1,2 Lys -using virus.

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Requirements for DNA Strand Transfer During Reverse Transcription in Mutant HIV-1 Virions

Cited by 56 publications

References 0 publications

Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein

Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein

A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

Forced Selection of a Human Immunodeficiency Virus Type 1 Variant That Uses a Non-Self tRNA Primer for Reverse Transcription: Involvement of Viral RNA Sequences and the Reverse Transcriptase Enzyme

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