Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An oligonucleotide-peptide conjugate, having dual binding capability for a designated RNA, was designed. The peptide portion of the conjugate interacts with a folded domain in the RNA, whereas the oligonucleotide portion hybridizes with a nearby single-stranded region in the RNA. The dual specificity was proven in a model HIV-1 TAR RNA system using an RNase H cleavage assay to assess antisense binding to this RNA. The peptide portion of the conjugate was shown to confer increased specificity on the oligonucleotide.
 ...
PMID:Dual-specificity interaction of HIV-1 TAR RNA with Tat peptide-oligonucleotide conjugates. 763 1

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.
 ...
PMID:Actinomycin D inhibits human immunodeficiency virus type 1 minus-strand transfer in in vitro and endogenous reverse transcriptase assays. 965 19

Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (-sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with -sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic -sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3' end of the -sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.
 ...
PMID:Human immunodeficiency virus type 1 nucleocapsid protein can prevent self-priming of minus-strand strong stop DNA by promoting the annealing of short oligonucleotides to hairpin sequences. 1098 20

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 tRNA(3)(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.
 ...
PMID:In vitro analysis of human immunodeficiency virus type 1 minus-strand strong-stop DNA synthesis and genomic RNA processing. 1113 81

Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 5'R and the 3'R. There is accumulating evidence that retroviral R regions contain features other than sequence complementarity that stimulate this critical nucleic acid hybridization step. The R region of the human immunodeficiency virus type 1 (HIV-1) is relatively extended (97 nt) and encodes two well-conserved stem-loop structures, the TAR and poly(A) hairpins. The role of these motifs was studied in an in vitro strand-transfer assay with two separate templates, the 5'R donor and the 3'R acceptor, and mutants thereof. The results indicate that the upper part of the TAR hairpin structure in the 5'R donor is critical for efficient strand transfer. This seems to pose a paradox, as the 5'R template is degraded by RNase H before strand transfer occurs. We propose that it is not the RNA hairpin motif in the 5'R donor, but rather the antisense motif in the ssDNA copy, which can also fold a hairpin structure, that is critical for strand transfer. Mutation of the loop sequence in the TAR hairpin of the donor RNA, which is copied in the loop of the cDNA hairpin, reduces the transfer efficiency more than fivefold. It is proposed that the natural strand-transfer reaction is enhanced by interaction of the anti-TAR ssDNA hairpin with the TAR hairpin in the 3'R acceptor. Base pairing can occur between the complementary loops ("loop-loop kissing"), and strand transfer is completed by the subsequent formation of an extended RNA-cDNA duplex.
 ...
PMID:Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription. 1149 29

Minus strand strong stop transfer is obligatory for completion of HIV-1 minus strand synthesis. We previously showed evidence for an acceptor invasion-initiated mechanism for minus strand transfer. In the present study, we examined the major acceptor invasion initiation site using a minus strand transfer system in vitro, containing the 97-nucleotide full-length R region. A series of DNA oligonucleotides complementary to different regions of the cDNA was designed to interfere with transfer. Oligomers covering the region around the base of the TAR hairpin were most effective in inhibiting transfer, suggesting that the hairpin base is a preferred site for acceptor invasion. The strong pausing of reverse transcriptase at the base of the TAR and the concomitant RNase H cleavages 10-19 nucleotides behind the pause site correlated with the location of the invasion site. Oligomers closer to the 5'-end of R also inhibited transfer, though less effectively, presumably by blocking strand exchange and branch migration. We propose that pausing of reverse transcriptase at the base of TAR increases RNase H cleavages, creating gaps for acceptor invasion and transfer initiation. Strand exchange then propagates by branch migration, displacing the fragmented donor RNA, including the fragment at the 5' terminus. The primer terminus switches to the acceptor, completing the transfer. Nucleocapsid (NC) protein stimulated transfer efficiency by 5-7-fold. NC enhanced RNase H cleavages close to the TAR base, creating more effective invasion sites for efficient transfer. Most likely, NC also stimulates transfer by promoting strand exchange invasion and branch migration.
 ...
PMID:Steps of the acceptor invasion mechanism for HIV-1 minus strand strong stop transfer. 1287 97

We previously proposed that HIV-1 minus strand transfer occurs by an acceptor invasion-initiated multi-step mechanism. During synthesis of minus strong stop DNA, reverse transcriptase (RT) transiently pauses at the base of TAR before continuing synthesis. Pausing promotes RT-RNase H cleavage of the donor RNA, exposing regions of the cDNA. The acceptor RNA then invades at these locations to interact with the minus strong stop DNA. Whereas primer extension continues on the donor RNA, the cDNA-acceptor hybrid expands by branch migration until transfer of the primer terminus is completed. We present results here showing that the interaction of the acceptor RNA and the cDNA can be determined by examining the time-dependent cleavage of the acceptor RNA by RNase H. Our approach utilizes a combination of RT-RNase H and Escherichia coli RNase H to allow assessment of acceptor-cDNA interactions at high sensitivity. Results show an initial interaction of the acceptor RNA with cDNA at the base of TAR. We observe a time-dependent shift in RNase H susceptibility along the length of the acceptor toward the 5' end, suggesting hybrid propagation from the initial invasion point. Control experiments validate that the RNase H cleavage profile represents the formation and expansion of the acceptor-DNA interaction and that the process is promoted by the nucleocapsid. Observations with this new approach lend additional support to the proposed multistep transfer mechanism.
 ...
PMID:Acceptor RNA cleavage profile supports an invasion mechanism for HIV-1 minus strand transfer. 1565 44