Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Base-pair formation between two hairpin loops--a "kissing" complex--is an RNA-folding motif that links two elements of RNA secondary structure. It is also a unique protein recognition site involved in regulation of ColE1 plasmid DNA replication. The trans-activation response element (TAR), a hairpin and bulge at the 5' end of the untranslated leader region of the human immunodeficiency virus 1 mRNA, enhances the transcription of the virus and is necessary for viral replication. Gel electrophoresis and absorbance melting curves indicate that a synthesized RNA hairpin (Tar*-16) with a loop sequence complementary to the TAR loop sequence (CUGGGA) associates specifically with a 16-nucleotide TAR hairpin (Tar-16) to form a stable complex. RNase T1 probing indicates that the three guanines in the Tar-16 loop become inaccessible in the complex. NMR imino proton spectra reveal that 5 base pairs are formed between the two hairpin loops (Tar-16 and Tar*-16); only the adenine at the 3' terminus of the TAR loop does not form a base pair with the 5'-terminal uracil of the complementary loop. A 14-nucleotide hairpin [CCUA(UCCCAG)UAGG] with a loop sequence complementary to the TAR loop is conserved within the gag gene of human immunodeficiency virus 1. A synthesized RNA hairpin corresponding to this conserved sequence also binds to the Tar-16 hairpin with high affinity. It is possible that the same RNA loop-loop interaction occurs during the viral life cycle.
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PMID:Characterization of a "kissing" hairpin complex derived from the human immunodeficiency virus genome. 807 46

The structure of the TAR RNA element transcribed at the 5' end of the R region of the human immunodeficiency virus is compared to the structure of its duplicate sequence in the 3' R region of the viral genome. Based on the 5'-TAR secondary structure already described, we assessed by RNase T1 primer extension assay the degree of similarity between the 5'-TAR and the 3'-TAR RNA secondary structures. We also analysed the influence of modifications in the flanking sequences. We show that the secondary structures of the 5'-TAR and the 3'-TAR are very similar and are not influenced by the flanking sequences.
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PMID:Similarity of the 5' and 3'-TAR secondary structures in HIV-1. 837 97

We have identified the tRNAs which are incorporated into both wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1IIIB) produced in COS-7 cells transfected with HIV-1 proviral DNA and mutant, noninfectious HIV-1Lai particles produced in a genetically engineered Vero cell line. The mutant proviral DNA contains nucleotides 678 to 8944; i.e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, both mutant and wild-type HIV-1 contain four major-abundance tRNA species, which include tRNA(1,2Lys), tRNA(3Lys) (the putative primer for HIV-1 reverse transcriptase) and tRNA(Ile). Identification was accomplished by comparing the electrophoretic mobilities and RNase T1 digests with those of tRNA(3Lys) and tRNA(1,2Lys) purified from human placenta and comparing the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNA(Lys) incorporation into HIV-1. However, only the wild-type virus contains tRNA(3Lys) tightly associated with the viral RNA genome. The identification of the tightly associated tRNA as tRNA(3Lys) is based upon an electrophoretic mobility identical to that of tRNA(3Lys) and the ability of this RNA to hybridize with a tRNA(3Lys)-specific DNA probe. In addition to the four wild-type tRNA species, the mutant HIV-1-like particle contains two tRNA(His) species and three tRNA-sized species that we have been unable to identify. Their absence in wild-type virus makes it unlikely that they are required for viral infectivity.
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PMID:Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1. 849 49

A targeted RNase would be ideal for gene therapy of several acquired and inherited disorders. Such an RNase may be engineered to contain a ribonucleolytic domain and a specific target RNA binding domain. To demonstrate the feasibility of this approach, an RNase targeted against human immunodeficiency virus (HIV) RNA--Tev-RNase T1--was designed and tested for its use in HIV-1 gene therapy. A human CD4+ T lymphoid (MT4) cell line and human peripheral blood lymphocytes (PBLs) were transduced with retroviral vectors lacking or expressing the tevT1 gene. Expression of enzymatically functional Tev-RNase T1 protein and its lack of toxicity was demonstrated in stable MT4 transductants. Compared with control cells lacking this protein, both transduced MT4 cells and PBLs expressing Tev-RNase T1 delayed HIV-1 replication. Tev-RNase T1 was shown to act after integration, since HIV-1 proviral DNA could be detected, but the amount of HIV-1 RNA produced in MT4 cells and PBLs was significantly decreased. This study demonstrates the feasibility of a targeted RNase strategy for therapeutic use.
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PMID:Targeted RNases: a feasibility study for use in HIV gene therapy. 1050 17

The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F.
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PMID:Cooperative and specific binding of Vif to the 5' region of HIV-1 genomic RNA. 1623 19

The mechanism of human immunodeficiency virus 1 (HIV-1) minus strand transfer was examined using a genomic RNA sequence-based donor-acceptor template system. The donor RNA, D199, was a 199-nucleotide sequence from the 5'-end of the genome to the primer binding site (PBS) and shared 97 nucleotides of homology with the acceptor RNA. To investigate the influence of RNA structure on transfer, a second donor RNA, D520, was generated by extending the 3'-end of D199 to include an additional 321 nucleotides of the genome. The position of priming, length of homology with the acceptor, and length of cDNA synthesized were identical with the two donors. Interestingly, at 200% NC coating, donor D520 yielded a transfer efficiency of about 75% compared with about 35% with D199. A large proportion of the D520 promoted transfers occurred after the donor RNA was copied to the end. Analysis of donor RNA cleavage, the acceptor invasion site and R homology requirements indicated that transfers with D520 involved a similar but more efficient acceptor invasion mechanism compared with D199. RNA structure probing by RNase T1 and the RT pause profile during synthesis indicated conformational differences between D199 and D520 in the starting structure, and in dynamic structures formed during synthesis within the R region. Overall observations suggest that regions 3' of the primer binding site influence the conformation of the R region of D520 to facilitate steps that promote strand transfer.
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PMID:Stimulation of HIV-1 minus strand strong stop DNA transfer by genomic sequences 3' of the primer binding site. 1678 13