UMLS:C0021051 - FACTA Search

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Query: UMLS:C0021051 (immunodeficiency)
71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The zinc-bound form of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, p7, aggregates into particles visible by electron microscopy. The HIV primer tRNA(Lys,3) forms similar high molecular weight complexes with p7 that are also detected by gel mobility shift assays. RNA oligonucleotides of the three stem-loop structures in tRNA(Lys,3) were assayed for the competitive inhibition of p7-tRNA(Lys,3) binding by the intensities of free tRNA(Lys,3) bands on native gels. This reveals that the p7 binds specifically to the central domain of tRNA(Lys,3) where the D and T psi C loops come together, but not the anticodon stem-loop.
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PMID:Chemically synthesised human immunodeficiency virus P7 nucleocapsid protein can self-assemble into particles and binds to a specific site on the tRNA(Lys,3) primer. 869 11

Retroviral reverse transcription starts near the 5' end of unspliced viral RNA at a sequence called the primer binding site (PBS), where the tRNA primer anneals to the RNA template for initiation of DNA synthesis. We have investigated the roles of NCp7 in annealing of primer tRNA(Lys3) to the PBS and in reverse transcriptase (RT) activity, using a cell-free reverse transcription reaction mixture consisting of various 5' viral RNA templates, natural primer tRNA(Lys3) or synthetic primer, human immunodeficiency virus type I (HIV-1) nucleocapsid protein (NCp7), and HIV-1 RT. In the presence of tRNA(Lys3), NCp7 was found to stimulate synthesis of minus-strand strong-stop DNA [(-)ssDNA], consistent with previous reports. However, specific DNA synthesis was observed only at a NCp7/RNA ratio similar to that predicted to be present in virions. Moreover, at these concentrations, NCp7 inhibited the synthesis of nonspecific reverse-transcribed DNA products, which are initiated because of self-priming by RNA templates. In contrast to results obtained with tRNA(Lys3) as primer, NCp7 inhibited the synthesis of (-)ssDNA products primed by an 18-nucleotide (nt) ribonucleotide (rPR), complementary to the PBS, even though rPR can initiate synthesis of such material in the absence of preannealing with NCp7. Primer placement band shift assays showed that NCp7 was necessary for efficient formation of the tRNA-RNA complex. In contrast, NCp7 was found to prevent formation of the rPR-RNA complex. Since NCp7 appears to exert opposite effects (annealing versus dissociation) on tRNA(Lys3) and rPR substrates, the non-PBS binding regions of the tRNA(Lys3) molecule may play a role in the annealing of tRNA to the template. We also investigated the roles of an A-rich loop upstream of the PBS, a 7-nt region immediately downstream of the PBS, and a 54-nt deletion further downstream of the PBS in interactions with tRNA(Lys3). We found that deletions in the 54-nt region that may prevent formation of the U5-leader stem prevented tRNA(Lys3) placement and priming, while deletions in the A-rich loop or the 7-nt sequence had relatively minor effects in this regard.
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PMID:Human immunodeficiency virus Type 1 nucleocapsid protein (NCp7) directs specific initiation of minus-strand DNA synthesis primed by human tRNA(Lys3) in vitro: studies of viral RNA molecules mutated in regions that flank the primer binding site. 876 6

Coaxially stacked RNA helices are a determined of RNA tertiary structure, but their presence is rarely detected using conventional chemical modification methods. In this report we describe a porphyrin ion photoreaction that enables one to monitor RNA stacking interactions and the folding of coaxially stacked RNA helices. The porphyrin cations meso-tetrakis(4-N-methylpyridyl)porphine, meso-tetrakis-(para-N-trimethylanilinium)porphine, and meso-tetrakis(2-N-methylpyridyl)porphine were used to characterize tRNA(Phe) and the human immunodeficiency virus type-I Rev response element RNA. Nucleosides at the bases of contiguous RNA helices in each RNA are efficiently modified by the porphyrin cations following irradiation of porphyrin-RNA mixtures. These photomodifications are markedly reduced for RNA equilibrated in ionic buffers that lead to enhanced stabilization of coaxially stacked helices. The porphyrin cation photoreaction specifically modifies G18, G20, and G34 in the tRNA folding produced by Mg(II). These nucleobases are exposed to solvent in the native tRNA structure and thus available to stack with solvent-borne porphyrin molecules. The describe porphyrin cation photochemical method provides a novel approach to study the solvent accessibility of nucleobases in RNA structure and to monitor the folding of coaxially stacked helices in RNA.
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PMID:Efficient modification of RNA by porphyrin cation photochemistry: monitoring the folding of coaxially stacked RNA helices in tRNA(Phe) and the human immunodeficiency virus type 1 rev response element RNA. 881 Sep 11

Initiation of minus (-) strand DNA synthesis was examined on templates containing R, U5, and primer-binding site regions of the human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) genomic RNA. DNA synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic tRNA(3Lys), and (iii) natural tRNA(3Lys), by the reverse transcriptases of HIV-1, FIV, EIAV, simian immunodeficiency virus, HIV type 2 (HIV-2), Moloney murine leukemia virus, and avian myeloblastosis virus. All enzymes used an oligonucleotide on wild-type HIV-1 RNA, whereas only a limited number initiated (-) strand DNA synthesis from either tRNA(3Lys). In contrast, all enzymes supported efficient tRNA(3Lys)-primed (-) strand DNA synthesis on the genomes of FIV and EIAV. This may be in part attributable to the observation that the U5-inverted repeat stem-loop of the EIAV and FIV genomes lacks an A-rich loop shown with HIV-1 to interact with the U-rich tRNA anticodon loop. Deletion of this loop in HIV-1 RNA, or disrupting a critical loop-loop complex by tRNA(3Lys) extended by 9 nt, restored synthesis of HIV-1 (-) strand DNA from primer tRNA(3Lys) by all enzymes. Thus, divergent evolution of lentiviruses may have resulted in different mechanisms to use the same host tRNA for initiation of reverse transcription.
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PMID:Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases. 881 51

The interactions between the Reverse Transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) and the natural tRNA(Lys3) primer for initiation of viral DNA synthesis were examined. We constructed a set of HIV-1 RNA templates in which the wild-type primer binding site (PBS(Lys3)) is replaced by sequences complementary to tRNA(lle), tRNA(Lys1,2), tRNA(Phe), tRNA(Pro) or tRNA(Trp) and tested the ability of RT enzymes of different retroviral species to initiate cDNA synthesis from self versus non-self tRNA primers. We demonstrate that initiation of HIV-1 reverse transcription is a specific process that is most efficient with the self tRNA(Lys3) primer. Interestingly, the property of HIV-1 RT to discriminate against non-self tRNA primers is lost upon extension of the tRNA by only two deoxyribonucleotides. Furthermore, selective tRNA priming by HIV-1 RT was not observed with viral RNA-tRNA(Lys3) duplexes isolated from HIV-1 virion particles, suggesting that the majority of tRNA(Lys3) primers annealed to viral RNA in particles is extended by a variable number of deoxyribonucleotides. This result indicates that reverse transcription is initiated relatively early in nascently assembled virions.
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PMID:HIV-1 reverse transcriptase discriminates against non-self tRNA primers. 895 74

The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome requires cellular tRNA(Lys,3) as a primer and occurs at a site in the viral RNA genome, designated as the primer binding site (PBS), which is complementary to the 3'-terminal 18 nucleotides of tRNA(Lys,3). We previously described an HIV-1 virus [designated as HXB2(His-AC)], which contained a sequence within the U5 region complementary to the anticodon region of tRNA(His) in addition to a PBS complementary to the 3'-terminal 18 nucleotides of the tRNA(His). That virus maintained a PBS complementary to tRNA(His) after extended in vitro culture (Wakefield et al., J. Virol. 70, 966-975, 1996). In the present study, we report that subcloning a 200-base-pair DNA fragment encompassing the U5 and PBS regions from an integrated provirus of HXB2(His-AC) back into the wild-type genome (pHXB2) resulted in an infectious virus, designated as HXB2(His-AC-gac), which again stably maintained a PBS complementary to tRNA(His). DNA sequence analysis of the 200-base-pair region revealed only three nucleotide changes from HXB2(His-AC): a T-to-G change at nucleotide 174, a G-to-A change at nucleotide 181, and a T-to-C change at nucleotide 200. The new mutant virus replicated in CD4+ Sup T1 cells similarly to the wild-type virus. Comparison of the nucleotide sequence of nucleocapsid gene of the wild-type and HXB2 (His-AC-gac) virus revealed no differences. Although we found numerous mutations in the reverse transcriptase gene in proviral clones derived from HXB2 (His-AC-gac), no common mutations were found among the 13 clones examined. Comparison of the virion-associated tRNAs of HXB2(His-AC-gac) with those of the wild type revealed that both viruses incorporated a similar subset of cellular tRNAs, with tRNA(Lys,3) being the predominant tRNA found within virions. There was no selective enrichment for tRNA(His) within virions of HXB2(His-AC-gac) virus which selectively use tRNA(His) to initiate reverse transcription. The results of these studies suggest that the U5 and PBS regions in the viral RNA genome are important determinants for HXB2(His-AC) viruses in the selective use of tRNA(His) to initiate reverse transcription.
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PMID:Nucleotide sequences within the U5 region of the viral RNA genome are the major determinants for an human immunodeficiency virus type 1 to maintain a primer binding site complementary to tRNA(His). 895 50

Initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) occurs by extension from the 3' end of a cellular tRNA complexed to the primer binding site (PBS) located near the 5' end of the viral RNA genome. Although the PBSs for all naturally occurring HIV-1 viruses are complementary to the 3'-terminal 18 nucleotides of tRNA(Lys)3, we identified an HIV-1 virus which contained a PBS complementary to the 3' nucleotides of tRNA(Met); the PBS of this virus was not stable upon extended culture and reverted back to the wild type (S.-M. Kang, J. K. Wakefield, and C. D. Morrow, Virology 222:401-414, 1996). To further characterize the virus with a PBS complementary to tRNA(Met), a DNA fragment encompassing the PBS and U5 region from this proviral genome was substituted for the same region in the infectious HIV-1 proviral clone [named pHXB2(AC-Met)]. Three additional proviral genomes were also created: pHXB2(Met), which is isogenic with pHXB2 except for the PBS complementary to tRNA(Met); pHXB2(Met-AC-Met), which contains the PBS sequence complementary to the 3'-terminal nucleotides and the sequence upstream of this PBS in U5 complementary to the anticodon region of tRNA(Met); and pHXB2(Met-C-Met), which contains two G-to-C changes predicted to disrupt complementarity within the tRNA(Met) anticodon region. Viruses derived from the transfection of these proviral genomes were infectious, although the appearance of the viruses was delayed compared to that of the wild-type virus. PCR amplification and DNA sequence analysis of the PBS regions from proviral genomes revealed that the PBSs from viruses derived from pHXB2(Met) and pHXB2(AC-Met) reverted back to the wild type by days 16 and 44 postcoculture, respectively. Two new, novel mutant viruses were identified among viruses derived from pHXB2(Met-C-Met) at day 35 postcoculture: one contained a PBS complementary to tRNA(Lys)1,2, while the second maintained a PBS complementary to tRNA(Met) but contained a 26-nucleotide deletion in U5 upstream of the anticodon-complementary region. By day 125 postcoculture, the PBS in the virus from this culture had reverted back to the wild type, complementary to tRNA(Lys)3. In contrast, the viruses derived from pHXB2(Met-AC-Met) stably maintained a PBS complementary to tRNA(Met) during the 125-day culture period examined. The results of these studies support the idea that HIV-1 can maintain a PBS complementary to alternative tRNAs provided that the appropriate complementarity exists between the U5-PBS region of the viral RNA genome and the tRNA molecule used to initiate reverse transcription.
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PMID:Identification of a sequence within U5 required for human immunodeficiency virus type 1 to stably maintain a primer binding site complementary to tRNA(Met). 898 40

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNALys,3 positioned at an 18-nucleotide sequence in the RNA genome referred to as the primer-binding site (PBS). We have found that mutations within the PBS and a region upstream in U5, designated the A loop, influenced the selection of the tRNA primer used to initiate reverse transcription. Surprisingly, a proviral genome that contained a PBS and A loop complementary to tRNAPro resulted in the generation of viruses that contained two PBSs within the same genome: one of the PBSs in the virus was complementary to tRNALys,3 while the second PBS was complementary to tRNAIle, tRNAPro, or tRNALys,3. There were 14 nucleotides separating the two PBSs in the viral genome. In the current study, DNA encompassing U5 and the dual PBS complementary to the different tRNAs were amplified by PCR and exchanged for the corresponding region in an infectious HIV-1 clone, HXB2. Transfection of the different proviruses into cells resulted in the production of viruses that were infectious as determined by coculture with SupT1 cells. PCR was used to amplify the PBS regions from the different proviral DNAs followed by DNA sequencing of individual PCR clones. Proviruses containing the dual PBS complementary to tRNALys,3 and tRNAIle stably maintained the dual PBS complementary to both of these tRNAs following in vitro culture, although we noted consistent G-to-T and AA-to-GG substitutions in the 14-nucleotide region between the PBSs. The viruses derived from genomes that contained the dual PBS complementary to tRNALys,3 and tRNAPro also maintained both PBSs following in vitro culture; a single mutation was noted after in vitro culture in the 14-nucleotide region between the PBSs, which changed a consensus integration site (CA dinucleotide) prior to the PBS complementary to tRNAPro. In contrast, the proviral genomes containing the dual PBS complementary to tRNALys,3 were not stable and reverted back to a single PBS complementary to tRNALys,3. The results of our studies suggest that only the 5'-proximal PBS has been used to initiate reverse transcription. On the basis of our results, a mechanism is proposed for the generation of a dual PBS, which provides new insights into HIV-1 reverse transcription.
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PMID:Stability of HIV type 1 proviral genomes that contain two distinct primer-binding sites. 911 13

RNA-DNA hybrid model substrates which mimic an intermediate of Moloney murine leukemia virus (M-MuLV) reverse transcription at the stage where the tRNAPro is removed were constructed. This substrate was used to assay the ability of M-MuLV reverse transcriptase (RT) to cleave the RNA portion of the substrate. The cleavage specificities of the cognate M-MuLV RT and the heterologous enzyme from the human immunodeficiency virus type-1 (HIV-1) were compared. M-MuLV and HIV-1 RT recognize and cleave the RNA at distinct positions. The site of the initial RNase H cleavage in vitro was determined using 3' end nearest neighbor analysis of the initial cleavage product. M-MuLV RT/RNase H removed the model tRNAPro between the terminal ribo-A and ribo-C, resulting in a terminal ribo-A attached to the viral DNA, whereas HIV-1 RT/RNase H was shown to cleave at the RNA-DNA junction. Analysis of the DNA over time indicated that the ribo-A is subsequently removed by M-MuLV RT. In vivo analysis from double-LTR circle junctions illustrated that 16 of the 23 clones isolated possessed the predicted junction if complete removal of the tRNA primer were to occur. The predicted junction for complete removal of the tRNA primer was CATT-AATG. One aberrant circle junction was isolated which could result from the use of an alternative primer. In contrast with HIV, no M-MuLV circle junctions were isolated which indicated processing of a single-LTR terminus by integrase. Analysis from in vivo and in vitro studies indicate that the M-MuLV tRNAPro primer is completely removed after plus-strand strong-stop synthesis.
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PMID:RNase H cleavage of tRNAPro mediated by M-MuLV and HIV-1 reverse transcriptases. 912 56

Retroviruses use a cellular tRNA molecule as primer for reverse transcription. The complementarity between the 3' end of this tRNA and a sequence near the 5' end of the viral RNA, the primer-binding site (PBS), allows the primer to anneal onto the viral RNA. During reverse transcription 18 nucleotides of the tRNA primer are copied into the viral cDNA, thereby regenerating the PBS sequence of the progeny. Thus, the PBS sequence reveals which primer was used. Human immunodeficiency viruses are known to replicate efficiently with tRNA(Lys3) as primer. Examination of the PBS sequence in natural and laboratory isolates indicates that a variant tRNA(Lys) is occasionally used as primer. This variant, for which the murine genomic sequence was described previously, was termed tRNA(Lys5) and differs from tRNA(Lys3) at five nucleotide positions. These results suggest that HIV uses both tRNA(Lys3) and tRNA(Lys5) molecules as primer, causing a switch of the PBS sequence.
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PMID:Sequence variation of the human immunodeficiency virus primer-binding site suggests the use of an alternative tRNA(Lys) molecule in reverse transcription. 912 56

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