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 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 Stimulation of HIV-1 Minus Strand Strong Stop DNA Transfer by Genomic Sequences 3′ of the Primer Binding Site

2006 ◽  
Vol 281 (34) ◽  
pp. 24227-24235 ◽  
Author(s):  
Min Song ◽  
Mini Balakrishnan ◽  
Yan Chen ◽  
Bernard P. Roques ◽  
Robert A. Bambara
Keyword(s):  
Binding Site ◽  
Dna Transfer ◽  
Genomic Sequences ◽  
Primer Binding Site ◽  
Minus Strand ◽  
Hiv 1 ◽  
Strong Stop ◽  
Stimulation Of

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 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 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 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

scholarly journals Actinomycin D Inhibits Human Immunodeficiency Virus Type 1 Minus-Strand Transfer in In Vitro and Endogenous Reverse Transcriptase Assays

1998 ◽  
Vol 72 (8) ◽  
pp. 6716-6724 ◽  
Author(s):  
Jianhui Guo ◽  
Tiyun Wu ◽  
Julian Bess ◽  
Louis E. Henderson ◽  
Judith G. Levin
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nucleic Acid ◽  
Actinomycin D ◽  
Strand Transfer ◽  
Minus Strand ◽  
Nucleic Acid Chaperone ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT In this report we demonstrate that human immunodeficiency virus type 1 (HIV-1) minus-strand transfer, assayed in vitro and in endogenous reactions, is greatly inhibited by actinomycin D. Previously we showed that HIV-1 nucleocapsid (NC) protein (a nucleic acid chaperone catalyzing nucleic acid rearrangements which lead to more thermodynamically stable conformations) dramatically stimulates HIV-1 minus-strand transfer by preventing TAR-dependent self-priming from minus-strand strong-stop DNA [(−) SSDNA]. Despite this potent activity, the addition of NC to in vitro reactions with actinomycin D results in only a modest increase in the 50% inhibitory concentration (IC50) for the drug. PCR analysis of HIV-1 endogenous reactions indicates that minus-strand transfer is inhibited by the drug with an IC50 similar to that observed when NC is present in the in vitro system. Taken together, these results demonstrate that NC cannot overcome the inhibitory effect of actinomycin D on minus-strand transfer. Other experiments reveal that at actinomycin D concentrations which severely curtail minus-strand transfer, neither the synthesis of (−) SSDNA nor RNase H degradation of donor RNA is affected; however, the annealing of (−) SSDNA to acceptor RNA is significantly reduced. Thus, inhibition of the annealing reaction is responsible for actinomycin D-mediated inhibition of strand transfer. Since NC (but not reverse transcriptase) is required for efficient annealing, we conclude that actinomycin D inhibits minus-strand transfer by blocking the nucleic acid chaperone activity of NC. Our findings also suggest that actinomycin D, already approved for treatment of certain tumors, might be useful in combination therapy for AIDS.

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