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 initiation of reverse transcription of a retroviral RNA genome occurs by a tRNA primer bound near the 5' end of the genomic RNA at a position called the primer-binding site (PBS). To understand the molecular basis for this RNA-RNA interaction, the secondary structure of the leader RNA of the human immunodeficiency virus type 2 (HIV-2) RNA was analyzed. In vitro synthesized HIV-2 RNA was probed with various structure-specific enzymes and chemicals. A computer program was then used to predict the secondary structure consistent with these data. In addition, the nucleotide sequences of different HIV-2 isolates were used to screen for the occurrence of covariation among putative base pairs. The primary sequences have diverged rapidly in some HIV-2 isolates, however, some strikingly conserved secondary structure elements were identified. Most nucleotides in the leader region are involved in base pairing. An exception is the PBS sequence, of which 15 out of 18 nucleotides are exposed in an internal loop. These findings suggest that the overall structure of the HIV-2 genome has evolved to facilitate an optimal interaction with its tRNA primer.
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PMID:Secondary structure of the HIV-2 leader RNA comprising the tRNA-primer binding site. 846 1

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

The nucleocapsid (NC) protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is important for encapsidation of the virus genome, RNA dimerization, and primer tRNA annealing in vitro. Here we present evidence from gel mobility-shift experiments indicating that NCp7 binds specifically to an RNA sequence. Two complexes were identified in native gels. The more slowly migrating complex contained two RNA molecules and one peptide, while the more rapidly migrating one is composed of one RNA and one peptide. Further, mutational analysis of the RNA shows that the predicted stem and loop structure of stem-loop 1 plays a critical role. Our results show that NCp7 binds to a unique RNA structure within the psi region; in addition, this structure is necessary for RNA dimerization. We propose that NCp7 binds to the RNA via a direct interaction of one zinc-binding motif to stem-loop 1 followed by binding of the other zinc-binding motif to stem-loop 1, stem-loop 2, or the linker region of the second RNA molecule, forming a bridge between the two RNAs.
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PMID:Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein. 850 69

The comparison of Km and Vmax values for various primers in the reaction of polymerization catalyzed by the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase was carried out. The primers were: (a) complementary to the template, (b) partially complementary with mismatched nucleotides at different positions from the 3' end or (c) non-complementary. Non-complementary primers were not elongated by HIV-1 reverse transcriptase. However, if they contained only one residue complementary to the template or an abasic unit at the 3' end, they could serve as primers. The most effective discrimination between matched and mismatched primers, due to a decrease in the affinity and Vmax, was found in the case of oligonucleotides containing non-complementary bases at the second or third position from the 3' end of the primer. The efficiency of discrimination by HIV-1 reverse transcriptase between matched and mismatched base-paired primers was about 1-1.5 orders of magnitude lower than that of procaryotic, eucaryotic and archaebacterial DNA polymerases and avian myeloblastosis virus reverse transcriptase. Oligonucleotides such as (dT)4(dCdG)k(dT)4 showed higher affinity for the enzyme than (dT)4 or (dT)8 primers. These data suggest that HIV-1 reverse transcriptase, in contrast to procaryotic, eucaryotic and archaebacterial DNA polymerases, forms additional contacts with the 5'-end region of the non-complementary primer. In addition, using tRNA(3Lys), the natural primer of HIV-1, it was shown that the p66 subunit of reverse transcriptase can be crosslinked, in the presence of a platinum derivative, to the 5' end of tRNA. Thus, besides the normal binding site for the 3' end of tRNA, which is crucial for the initiation of cDNA synthesis, the 5' end of the tRNA also interacts with a specific site on the enzyme.
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PMID:High-affinity interaction of human immunodeficiency virus type-1 reverse transcriptase with partially complementary primers. 852 51

Translational incorporation of the unusual amino acid selenocysteine in eukaryotes requires a coding region UGA codon (which otherwise serves as a termination signal), a selenocysteine insertion sequence (SECIS) in the 3'-untranslated region of the mRNA, and selenocysteyl-tRNA. The mechanisms involved in SECIS recognition by the eukaryotic translational machinery remain unknown. We report the detection of RNA-binding proteins that specifically recognize the SECIS from human cellular glutathione peroxidase (GPX1) transcripts. RNA gel shift assays showed three retarded bands after incubation with COS-1 whole cell lysate or S-100 cytosol fraction or with extracts from hepatoma cell lines HepG2 and Hep3B. The specificity of the binding was demonstrated by competition by cold unlabeled SECIS RNA and by lack of competition by other RNA species with similar stem-loop secondary structures, such as the human immunodeficiency virus (HIV) transactivation-response region of HIV mRNA element, and mutated SECIS constructs. UV cross-linking and SDS-polyacrylamide gel electrophoresis revealed at least two proteins, with estimated molecular masses of 55,000 and 65,000 Da, that bind to the SECIS. Examination of a series of insertion and deletion SECIS mutants indicated recognition of the SECIS primarily through the basal stem region, although the upper stem, loop, and two of three short conserved sequences also appear to contribute to the affinity of the binding.
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PMID:RNA-binding proteins that specifically recognize the selenocysteine insertion sequence of human cellular glutathione peroxidase mRNA. 853 Apr 73

The initiation of human immunodeficiency virus type 1 reverse transcription occurs by extension of a tRNA(Lys3) primer bound near the 5' end of the viral RNA genome which is designated the primer binding site (PBS). Sequences within the viral genome upstream of the PBS which are complementary to the anticodon loop (USUU) and the T psi C loop and arm (AGGGTm psi) of tRNA(Lys3) are postulated to play a role in maintaining the selective use of tRNA(Lys3) in reverse transcription. To investigate this possibility, proviral genomes which contain a PBS complementary to the 3'-terminal 18 nucleotides of tRNA(His) [pHXB2(His)] as well as sequences upstream of this PBS which are complementary to either the anticodon loop [CCACAA; pHXB2(His-AC)] or T psi C loop [GACCGAGG; pHXB2(His-T psi C)] of tRNA(His) were constructed. Infectious virus was recovered upon transfection into COS-1 cells of pHXB2(His), pHXB2(His-AC), or pHXB2(His-T psi C). The appearance of infectious virus after cocultivation with SupT1 cells was delayed for the proviruses containing a PBS complementary to tRNA(His) compared with that obtained by transfection of the wild-type provirus [pHXB2(WT)]. However, by several passages in SupT1 cells, the mutant viruses demonstrated replication kinetics similar to those of the wild-type virus. A DNA sequence analysis of the PBS region from integrated proviruses revealed that by day 15 of culture, the PBS of viruses derived from pHXB2(His) and pHXB2(His-T psi C) reverted back to the wild-type PBS complementary to tRNA(Lys3). In contrast, viruses derived from pHXB2(His-AC) maintained a PBS complementary to tRNA(His) for over 4 months in culture encompassing 12 serial passages. This study, then, is the first report of a stable human immunodeficiency virus type 1 which utilizes an alternative tRNA primer and suggests that interactions between the primer tRNA anticodon loop and viral sequences upstream of the PBS contribute to the specificity of the tRNA primer used in reverse transcription.
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PMID:Construction of a type 1 human immunodeficiency virus that maintains a primer binding site complementary to tRNA(His). 855 37

A host cell-derived tRNA3Lys molecule is utilized by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to prime DNA synthesis from the viral RNA genome. We performed fluorescence titration experiments to characterize the interaction between RT and its natural primer, tRNA3Lys, and to address RT's putative role in the required and specific packaging of tRNA3Lys into the budding virus. Titration of RT with tRNA3Lys resulted in a 30% maximal quenching of RT tryptophan fluorescence, from which a dissociation constant (Kd) of 57.6 +/- 7.5 nM was derived. Titration of RT with Escherichia coli tRNA2Glu, E. coli tRNA2Tyr, E. coli tRNALys, yeast tRNAPhe, or in vitro-synthesized human tRNA3Lys (no base modifications) resulted in similar fluorescence changes and Kd values as obtained for the natural tRNA3Lys. The specific interaction between RT and tRNA3Lys during viral assembly suggested by previous in vivo studies is therefore not present in the fully processed, in vitro form of RT. Other factors during viral assembly must therefore cooperate in the packaging of tRNA3Lys. The nonspecific and ionic strength dependent RT-tRNA interaction detected in the present studies suggests that the overall shape and charges of tRNA constitute recognition features for RT binding. The fluorescence of the wyebutine base contained on the anticodon loop of yeast tRNAPhe was found to increase upon RT binding, supporting speculation that RT interacts with the anticodon loop of tRNA. The individual tRNAs also displaced a fluorescent DNA primer/template (p/t) substrate from RT, indicating overlapping tRNA and p/t binding sites. Cubic fit evaluation of the displacement titrations allowed further assessment of the affinities of the two competing ligands. The presence of both overlapping and separate p/t and tRNA binding regions on RT was tested by examination of the affinity of a possible RT bisubstrate type inhibitor, containing motifs proposed to be essential for both tRNA and p/t binding. Reverse transcriptase was found to bind to the mutant tRNA 10-fold more tightly than to the unaltered tRNA (Kd = 4.5 +/- 1.0 and 44.6 +/- 6.6 nM, respectively). Further analyses revealed that the tighter affinity is probably due to a preferred p/t binding mode and not to one expected if separate tRNA and p/t binding regions are accessed simultaneously by the same molecule.
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PMID:Evaluation of human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: specificity and comparison to primer/template binding. 860 12

The mechanism for the initiation of reverse transcription in human immunodeficiency virus type 1 (HIV-1) was studied utilizing a unique reverse transcriptase (RT) mutant altered in its noncatalytic p51 subunit. This mutant (p66/p51Delta13) retains full DNA- and RNA-dependent DNA polymerase activity but has reduced affinity for tRNA3Lys, the cognate HIV primer. When the ability to support(-)-strand DNA synthesis on a viral RNA template was evaluated, this mutant initiated from an 18-nucleotide (nt) oligoribo- or oligodeoxyribonucleotide primer complementary to the primer binding site (pbs). However, it failed to do so from natural and synthetic versions of tRNA3Lys. tRNA-primed(-)-strand synthesis could, however, be rescued by substituting the 76-nt tRNA3Lys with 81- and 107-nt tRNA-DNA chimeras, i.e. tRNA3Lys extended by 5 and 31 deoxyribonucleotides complementary to the viral genome upstream of the pbs. These findings imply that through interactions involving its p51 subunit, RT may be required to disrupt additional tRNA-viral RNA duplexes outside the pbs to proceed into productive(-)-strand DNA synthesis. Alternatively, specific interactions between tRNA3Lys and HIV-1 RT may be necessary for efficient initiation of(-)-strand DNA synthesis.
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PMID:Restoration of tRNA3Lys-primed(-)-strand DNA synthesis to an HIV-1 reverse transcriptase mutant with extended tRNAs. Implications for retroviral replication. 862 54

Different strategies proposed in the literature to attempt gene therapy of AIDS are based mainly on the intracellular production of RNA and protein therapeutics. This report describes the construction and the anti-human immunodeficiency virus type 1 (HIV-1) activity of a new type of antisense tRNA directed against a nucleotide region in the first coding exon of HIV-1 tat (nucleotides 5924 to 5943; Los Alamos data bank) which is conserved among many HIV-1 clones. The anti-tat antisense sequence was inserted into a tRNA(Pro) backbone by replacement of the anticodon loop, without altering the tRNA canonic tetraloop structure. The antisense tRNA was able to interact effectively with its target in vitro. Jurkat cells that constitutively expressed the anti-tat tRNA following retroviral vector transduction exhibited significant resistance to HIV-1 de novo infection. Resistance seemed to correlate with the level of antisense expression. This is the first time that such a tRNA antisense strategy has been shown to be effective as a genetic treatment of HIV-1 infection in tissue culture. The construct design proposed in this report has some intrinsic advantages: the transcript is driven by a polymerase III promoter, the short length of the RNA minimizes effects of intramolecular base pairing that may impair target recognition, and the antisense RNA has the stability and intracellular fate of a native tRNA molecule.
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PMID:A new antisense tRNA construct for the genetic treatment of human immunodeficiency virus type 1 infection. 864 37

tRNA(3Lys) is a primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1), and the anticodon of tRNA(3Lys) has been implicated in playing a role in both its placement onto the HIV-1 genome and its interaction with HIV-1 reverse transcriptase (RT). In this work, the anticodon in a tRNA(3Lys) gene was changed from UUU to CUA (tRNA(3Lys)Su+) or, in addition, G-73 was altered to A (tRNA(3Lys)Su+G73A). COS-7 cells were transfected with either wild-type or mutant tRNA(3Lys) genes, and both the wild-type and mutant tRNA(3Lys) produced were purified by using immobilized tRNA-specific hybridization probes. Each mutant tRNA(3Lys) was tested for its ability to prime reverse transcription in vitro, either alone or in competition with wild-type tRNA(3Lys). Short RT extensions of wild-type and mutant tRNALys could be distinguished from each other by their different mobilities in one-dimensional single-stranded conformation polymorphism polyacrylamide gel electrophoresis. These reverse transcription products show that heat-annealed tRNA(3Lys)Su+ has the same ability as heat-annealed wild-type tRNA(3Lys) to prime RT and competes equally well with wild-type tRNA(3Lys) for priming RT. tRNA(3Lys)Su+G73A has 60% of the wild-type ability to prime RT but competes poorly with wild-type tRNA(3Lys) for priming RT. However, the priming abilities of wild-type and mutant tRNA(3) are quite different when in vivo-placed tRNA is examined. HIV-1 produced in COS cells transfected with a plasmid containing both the HIV-1 proviral DNA and DNA coding for tRNA(3Lys)Su+ contains both endogenous, cellular wild-type tRNA(3Lys) and mutant tRNA(3Lys). When total viral RNA is used as the source of primer tRNA placed onto the genomic RNA in vivo, only wild-type tRNA(3Lys) is used as a primer. If the total viral RNA is first heated and exposed to hybridizing conditions, then both the wild-type and mutant tRNA(3Lys) act as primers for RT. These results indicate that the tRNA(3Lys)Su+ packaged into the virions is unable to act as a primer for RT, and a model is proposed to explain the disparate results between heat-annealed and in vivo-placed primer tRNA.
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PMID:Effects of modifying the tRNA(3Lys) anticodon on the initiation of human immunodeficiency virus type 1 reverse transcription. 867 96

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