scholarly journals Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

2002 ◽  
Vol 278 (10) ◽  
pp. 8006-8017 ◽  
Author(s):  
Yan Chen ◽  
Mini Balakrishnan ◽  
Bernard P. Roques ◽  
Philip J. Fay ◽  
Robert A. Bambara
Keyword(s):  
Reverse Transcription ◽  
Minus Strand ◽  
Hiv 1 ◽  
Strong Stop

scholarly journals Determination of the Ex Vivo Rates of Human Immunodeficiency Virus Type 1 Reverse Transcription by Using Novel Strand-Specific Amplification Analysis

2007 ◽  
Vol 81 (9) ◽  
pp. 4798-4807 ◽  
Author(s):  
David C. Thomas ◽  
Yegor A. Voronin ◽  
Galina N. Nikolenko ◽  
Jianbo Chen ◽  
Wei-Shau Hu ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Reverse Transcription ◽  
Ex Vivo ◽  
Dna Transfer ◽  
Minus Strand ◽  
Immunodeficiency Virus ◽  
Specific Amplification ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACT Replication of human immunodeficiency virus type 1 (HIV-1), like all organisms, involves synthesis of a minus-strand and a plus-strand of nucleic acid. Currently available PCR methods cannot distinguish between the two strands of nucleic acids. To carry out detailed analysis of HIV-1 reverse transcription from infected cells, we have developed a novel strand-specific amplification (SSA) assay using single-stranded padlock probes that are specifically hybridized to a target strand, ligated, and quantified for sensitive analysis of the kinetics of HIV-1 reverse transcription in cells. Using SSA, we have determined for the first time the ex vivo rates of HIV-1 minus-strand DNA synthesis in 293T and human primary CD4+ T cells (∼68 to 70 nucleotides/min). We also determined the rates of minus-strand DNA transfer (∼4 min), plus-strand DNA transfer (∼26 min), and initiation of plus-strand DNA synthesis (∼9 min) in 293T cells. Additionally, our results indicate that plus-strand DNA synthesis is initiated at multiple sites and that several reverse transcriptase inhibitors influence the kinetics of minus-strand DNA synthesis differently, providing insights into their mechanism of inhibition. The SSA technology provides a novel approach to analyzing DNA replication processes and should facilitate the development of new antiretroviral drugs that target specific steps in HIV-1 reverse transcription.

scholarly journals Single-Molecule FRET Studies of Important Intermediates in the Nucleocapsid-Protein-Chaperoned Minus-Strand Transfer Step in HIV-1 Reverse Transcription

2005 ◽  
Vol 89 (5) ◽  
pp. 3470-3479 ◽  
Author(s):  
Hsiao-Wei Liu ◽  
Gonzalo Cosa ◽  
Christy F. Landes ◽  
Yining Zeng ◽  
Brandie J. Kovaleski ◽  
...  
Keyword(s):  
Single Molecule ◽  
Reverse Transcription ◽  
Nucleocapsid Protein ◽  
Strand Transfer ◽  
Minus Strand ◽  
Single Molecule Fret ◽  
Transfer Step ◽  
Hiv 1

scholarly journals Sequences in the 5′ and 3′ R Elements of Human Immunodeficiency Virus Type 1 Critical for Efficient Reverse Transcription

2000 ◽  
Vol 74 (18) ◽  
pp. 8324-8334 ◽  
Author(s):  
Yuki Ohi ◽  
Jared L. Clever
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Strand Transfer ◽  
Minus Strand ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The genome of human immunodeficiency virus type 1 (HIV-1) contains two direct repeats (R) of 97 nucleotides at each end. These elements are of critical importance during the first-strand transfer of reverse transcription, during which the minus-strand strong-stop DNA (−sssDNA) is transferred from the 5′ end to the 3′ end of the genomic RNA. This transfer is critical for the synthesis of the full-length minus-strand cDNA. These repeats also contain a variety of other functional domains involved in many aspects of the viral life cycle. In this study, we have introduced a series of mutations into the 5′, the 3′, or both R sequences designed to avoid these other functional domains. Using a single-round infectivity assay, we determined the ability of these mutants to undergo the various steps of reverse transcription utilizing a semiquantitative PCR analysis. We find that mutations within the first 10 nucleotides of either the 5′ or the 3′ R sequence resulted in virions that were markedly defective for reverse transcription in infected cells. These mutations potentially introduce mismatches between the full-length −sssDNA and 3′ acceptor R. Even mutations that would create relatively small mismatches, as little as 3 bp, resulted in inefficient reverse transcription. In contrast, virions containing identically mutated R elements were not defective for reverse transcription or infectivity. Using an endogenous reverse transcription assay with disrupted virus, we show that virions harboring the 5′ or the 3′ R mutations were not intrinsically defective for DNA synthesis. Similarly sized mismatches slightly further downstream in either the 5′, the 3′, or both R sequences were not detrimental to continued reverse transcription in infected cells. These data are consistent with the idea that certain mismatches within 10 nucleotides downstream of the U3-R junction in HIV-1 cause defects in the stability of the cDNA before or during the first-strand transfer of reverse transcription leading to the rapid disappearance of the −sssDNA in infected cells. These data also suggest that the great majority of first-strand transfers in HIV-1 occur after the copying of virtually the entire 5′ R.

scholarly journals A Succession of Mechanisms Stimulate Efficient Reconstituted HIV-1 Minus Strand Strong Stop DNA Transfer†

Biochemistry ◽  
2009 ◽  
Vol 48 (8) ◽  
pp. 1810-1819 ◽  
Author(s):  
Min Song ◽  
Mini Balakrishnan ◽  
Robert J. Gorelick ◽  
Robert A. Bambara
Keyword(s):  
Dna Transfer ◽  
Minus Strand ◽  
Hiv 1 ◽  
Strong Stop

scholarly journals In Vitro Analysis of Human Immunodeficiency Virus Type 1 Minus-Strand Strong-Stop DNA Synthesis and Genomic RNA Processing

2001 ◽  
Vol 75 (2) ◽  
pp. 672-686 ◽  
Author(s):  
Mark D. Driscoll ◽  
Marie-Pierre Golinelli ◽  
Stephen H. Hughes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Synthesis ◽  
Viral Genome ◽  
Rnase H ◽  
Minus Strand ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Strong Stop

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), nucleocapsid protein (NC), genomic RNA, and the growing DNA strand all influence the copying of the HIV-1 RNA genome into DNA. A detailed understanding of these activities is required to understand the process of reverse transcription. HIV-1 viral DNA is initiated from a tRNA3 Lys primer bound to the viral genome at the primer binding site. The U3 and R regions of the RNA genome are the first sequences to be copied. The TAR hairpin, a structure found within the R region of the viral genome, is the site of increased RT pausing, RNase H activity, and RT dissociation. Template RNA was digested approximately 17 bases behind the site where polymerase paused at the base of TAR. In most template RNAs, this was the only cleavage made by the RT responsible for initiating polymerization. If the RT that initiated DNA synthesis dissociated from the base of the TAR hairpin and an RT rebound at the end of the primer, there was competition between the polymerase and RNase H activities. After the complete heteroduplex was formed, there were additional RNase H cleavages that did not involve polymerization. Levels of NC that prevented TAR DNA self-priming did not protect genomic RNA from RNase H digestion. RNase H digestion of the 100-bp heteroduplex produced a 14-base RNA from the 5′ end of the RNA that remained annealed to the 3′ end of the minus-strand strong-stop DNA only if NC was present in the reaction.

scholarly journals Steps of the Acceptor Invasion Mechanism for HIV-1 Minus Strand Strong Stop Transfer

2003 ◽  
Vol 278 (40) ◽  
pp. 38368-38375 ◽  
Author(s):  
Yan Chen ◽  
Mini Balakrishnan ◽  
Bernard P. Roques ◽  
Robert A. Bambara
Keyword(s):  
Minus Strand ◽  
Invasion Mechanism ◽  
Hiv 1 ◽  
Strong Stop

scholarly journals Proximity and Branch Migration Mechanisms in HIV-1 Minus Strand Strong Stop DNA Transfer

2007 ◽  
Vol 283 (6) ◽  
pp. 3141-3150 ◽  
Author(s):  
Min Song ◽  
Vandana P. Basu ◽  
Mark N. Hanson ◽  
Bernard P. Roques ◽  
Robert A. Bambara
Keyword(s):  
Dna Transfer ◽  
Minus Strand ◽  
Branch Migration ◽  
Hiv 1 ◽  
Strong Stop

scholarly journals Interactions between Ty1 Retrotransposon RNA and the T and D Regions of the tRNAiMet Primer Are Required for Initiation of Reverse Transcription In Vivo

1998 ◽  
Vol 18 (2) ◽  
pp. 799-806 ◽  
Author(s):  
S. Friant ◽  
T. Heyman ◽  
A. S. Byström ◽  
M. Wilhelm ◽  
F. X. Wilhelm
Keyword(s):  
Reverse Transcription ◽  
Primer Binding Site ◽  
Minus Strand ◽  
Genomic Rna ◽  
Complementary Sequences ◽  
Compensatory Mutations ◽  
Ty1 Retrotransposon ◽  
Ty1 Retrotransposition ◽  
Strong Stop

ABSTRACT Reverse transcription of the Saccharomyces cerevisiaeTy1 retrotransposon is primed by tRNAi Met base paired to the primer binding site (PBS) near the 5′ end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNAi Met. A structural probing study of the interactions between the Ty1 RNA template and the tRNAi Met primer showed that besides interactions between the PBS and the 3′ end of tRNAi Met, three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNAi Met. To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNAi Metwere tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNAi Met contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNAi Met and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNAi Met.

scholarly journals Structural Insights into the HIV-1 Minus-strand Strong-stop DNA

2015 ◽  
Vol 291 (7) ◽  
pp. 3468-3482 ◽  
Author(s):  
Yingying Chen ◽  
Ouerdia Maskri ◽  
Françoise Chaminade ◽  
Brigitte René ◽  
Jessica Benkaroun ◽  
...  
Keyword(s):  
Minus Strand ◽  
Structural Insights ◽  
Hiv 1 ◽  
Strong Stop
Sign in / Sign up

Export Citation Format

Share Document