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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The three-dimensional solution structure of the hybrid-chimeric duplex r(gcca)d(CTGC).d(GCAGTGGC) has been determined by two-dimensional NMR, restrained molecular dynamics (rMD), and NOE back-calculation methods. This chimera, consisting of a chimeric RNA-DNA strand and its complementary DNA strand, is formed after priming (-)-strand DNA synthesis by tRNA(Lys3) and subsequent (+)-strand DNA synthesis by reverse transcriptase and is an obligatory intermediate in the formation of double-stranded DNA prior to HIV-1 retrovirus integration. The duplex consists of two different types of double helix: a hybrid form (H-form) and a B-form structure connected by a junction. It is chemically similar to several other Okazaki fragments whose structures have been previously determined in our laboratory. However, some structural parameters are not the same and were found to be sequence dependent. In particular, the sugar conformations at the DNA base pair proximal to the hybrid segment vary from O4'-endo to C2'-endo depending on the base composition. The position of the transition from the relatively wide groove of H-form to the narrow groove of B-form is also sequence dependent, occurring either exactly at the RNA-DNA junction or within the purely DNA segment of the chimera-as is the case in the structure of the present HIV-1 (-)-strand primer. This structural change produces a kink at the DNA-DNA step adjacent to the RNA-DNA junction in the HIV-1 (-)-strand primer. The sequence dependence of structures of RNA-DNA chimeric duplexes may be responsible for the variable cleavage pattern of different Okazaki fragments by reverse transcriptase RNase H.
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PMID:Structural variation among retroviral primer-DNA junctions: solution structure of the HIV-1 (-)-strand Okazaki fragment r(gcca)d(CTGC).d(GCAGTGGC). 878 May 9

The transcription initiation primer for HIV-1 is a specific cellular tRNA species, tRNA(Lys3). We used several methods to assess the binding of tRNA by recombinant HIV-1 p51/p66 reverse transcriptase (RT), gel retardation analysis, intrinsic RT protein fluorescence quenching, and nitrocellulose filter binding assays. The binding of tRNA to RT was saturable, implying a distinct site or sites on the enzyme for tRNA interaction. However, this binding was non-selective, with all tRNA isoacceptors and total unfractionated tRNA binding with similar affinity as primer tRNA(Lys3). In contrast, no significant binding of tRNA by RT was noted. Our results show that HIV-1 RT has no specificity for the binding of primer tRNA(Lys3), and imply that factors other than RT sequences may be important for the selective incorporation of primer tRNA into the virion particle.
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PMID:HIV-1 reverse transcriptase shows no specificity for the binding of primer tRNA(Lys3). 878 Jun 99

RNAs play a crucial and central role in a large variety of biological functions obviously linked to the wide variety of structures that they can adopt. Understanding the function of RNAs thus requires the knowledge of their two- and three-dimensional structures. We describe in detail the way to access the secondary structure of RNAs, by combining sequence comparison, secondary structure prediction by computer and, mainly, experimental data obtained by probing with chemicals and ribonucleases. These approaches were used to investigate secondary structure of the region containing the primer binding site of HIV-1 genomic RNA either free or involved in the binary complex with the replication primer tRNA(3Lys).
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PMID:Determining the conformation of RNAs in solution. Application to a retroviral system: structure of the HIV-1 primer binding site region and effect of tRNA(3Lys) binding. 878 94

The initiation of HIV-1 reverse transcription is primed by a cellular tRNA(Lys),3 molecule which is bound to a complementary sequence near the 5' end of the viral RNA genome designated as the primer-binding site (PBS). Recent studies have suggested that sequences upstream of the PBS within U5 consisting of a stretch of adenine nucleotides (referred to as the A-loop) might be important in the selection and positioning of tRNALys,3 primer used to initiate reverse transcription. To further explore the role that the A-loop plays in reverse transcription, we have constructed proviral genomes in which the PBS was changed so as to be complementary to the 3'-terminal 18 nucleotides of tRNA(Ile), tRNA(Pro), or tRNA(Trp) [pHXB(Ile), pHXB(Pro), or pHXB(Trp), respectively]; a second set of proviral genomes was constructed which contained additional mutations so that the A-loop regions were complementary to the anticodon region of tRNA(Ile) [pHXB(Ile-AC)], tRNA(Pro) [pHXB(Pro-AC)], or tRNA(Trp) [pHXB(Trp-AC)]. Transfection of the proviruses into COS-1 cells followed by coculture with SupT1 cells resulted in production of infectious virus. PCR was used to amplify the PBS regions which were subcloned into M13mp18 followed by DNA sequence analysis. After short-term culture, the PBSs of proviruses derived from pHXB(Ile), pHXB(Pro), and pHXB(Trp) reverted to be complementary to tRNA(Lys),3. The PBSs of the viruses derived from pHXB(Ile-AC) also reverted to be complementary to tRNA(Lys),3; the A-loop region was still complementary to tRNA(Ile). In contrast, viruses derived from transfection of pHXB(Pro-AC) initially maintained a PBS complementary to tRNA(Pro). Upon extended culture, we identified proviruses which contained PBSs complementary to two additional tRNAs: tRNA(Ile) and tRNA(Lys),3. Furthermore, we found proviruses which contain two PBSs within the same genome: one complementary to tRNA(Lys),3 and a second complementary to tRNA(Pro) or tRNA(Ile). Viruses derived from transfection of pHXB(Trp-AC) were the most delayed in appearance following transfection. Analysis of the PBS revealed that early after transfection, the majority of the PBSs were complementary to tRNA(Trp). After further in vitro culture, proviruses were identified with a PBS complementary to a new tRNA, tRNA(Met). Finally, upon extended culture, the viruses derived from the transfection of pHXB(Ile-AC), pHXB(Pro-AC), and pHXB(Trp-AC) contained mutations upstream from the PBS in U5 that created a stretch of 3 adenine nucleotides. The results of these studies then highlight the flexibility that exists with respect to the selection of the tRNA primer used to initiate HIV-1 reverse transcription.
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PMID:Mutations in both the U5 region and the primer-binding site influence the selection of the tRNA used for the initiation of HIV-1 reverse transcription. 880 24

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 recognition of primer tRNA by retroviral reverse transcriptase is a crucial step in the replication of retroviruses. In the complex formed by HIV-1 reverse transcriptase and its natural primer tRNALys3, the heterodimeric enzyme, p66/p51, binds two molecules of tRNALys3 with different affinities. The same complex but in the presence of a non-complementary template, poly(A), gave higher Kd values. Preincubation of the reverse transcriptase with tRNA at concentrations comparable to the Kd2 value results in different levels of stimulation of the DNA polymerase activity: 300% in the absence and 70-80% in the presence of poly(A). The activation of the catalytically active p66 subunit is most probably mediated through tRNA interaction with the site of reverse transcriptase presenting the lower affinity. In this article, we describe the results obtained with new chemically reactive derivatives of tRNA bearing three or seven hydrophobic residues. Incubation of reverse transcriptase with tRNA derivatives, in the presence or absence of poly(A), leads to covalent binding of the reagents and inactivation of the enzymatic activity. However, during the initial step of the modification reaction, in the absence of poly(A), a slight stimulation of reverse transcriptase by tRNA derivatives took place, followed by a decrease in the enzymatic activity due to the covalent binding of tRNA derivatives to reverse transcriptase. In the presence of poly(A), enzyme inactivation occurs according to pseudo-first-order reaction kinetics. The affinities of tRNA derivatives for the p66/p51 heterodimer estimated from affinity modification data (Kd values) and from the inhibition of polymerization reaction (Ki values) were determined. Each analog of tRNA presented two Kd and two Ki values.
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PMID:Interaction of human immunodeficiency virus type 1 reverse transcriptase with primer tRNALys3 and affinity modification of the enzyme by tRNALys3 derivatives. 885 83

tRNALys3 is the primer for HIV-1 reverse transcriptase (RI) and is selectively incorporated into HIV-1 during viral assembly. While whole cell extracts of uninfected or infected cells contain only one detectable form of tRNALys3, multiple forms of tRNALys3 are detected in the virus released into the cell culture media. These tRNALys3 isoacceptors are found in HIV-1 produced from newly infected cord blood lymphocytes and from cells chronically infected with HIV-1, such as the lymphocytic cell line H9 and the monocytic cell lines U937 and PLB. They can be detected through the use of either RPC-5 column chromatography of tRNA aminoacylated with radioactive lysine or northern blot analysis using a tRNALys3-specific DNA hybridization probe. Both RPC-5 chromatography and northern blot analysis show the cytoplasmic form of tRNALys3 to be the major abundance form of tRNALys3 in the virus. Starting with the viral RNA isolated from HIV (PLB), the tRNALys3 species resolved by RPC-5 into peaks 2, 3, and 4 were deacylated and 3' end-labeled by heat-annealing the RNA in each peak to synthetic HIV genomic RNA, and extending the hybridized species one base using HIV-1 RT and radioactive dCTP. An electrophoretic comparison of the partial T1 digest pattern of purified human placental tRNALys3 with those of the RPC-5 resolved species showed that the labeled RNA species in each peak was tRNALys3. These radioactive tRNALys3 species retained their relative mobilities when rechromatographed on RPC-5. When total HIV (PLB) RNA was used as the source of primer/template, and similarly extended with RT in the presence of radioactive dCTP, the major priming tRNA resolved by RPC-5 had a chromatographic mobility identical to peak 3. This tRNA primer has a T1 digest pattern identifying it as tRNALys3. These results indicate that the major tRNALys3 species present in the virus is also the major tRNALys3 isoacceptor used as the primer for reverse transcription.
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PMID:Multiple forms of tRNA(Lys3) in HIV-1. 887 48

RNA decoys are oligonucleotides corresponding to the TAR and RRE sequences of HIV which inhibit the HIV-encoded regulatory proteins Tat and Rev, respectively. Adeno-associated viral vectors encoding RNA decoys stably transduced into the human T-cell line CEM-SS expressed transactivating region (TAR) and Rev-responsive element (RRE) RNA decoys from tRNA polIII promoters at high levels, without any apparent deleterious effects on cell growth or expression of CD4. DNA blot analysis indicated that RNA decoy-encoding vectors were not rearranged and were integrated into the genomic DNA of selected cell lines. Vector DNA with the appropriate TAR and RRE sequences was isolated from transduced cell lines after prolonged growth in culture, further confirming that the vector DNA was present in a stable form through multiple cell cycles. Cell lines expressing TAR and RRE decoys transiently inhibited HIV gene expression and replication by 70-99% as determined by measurement of intracellular and extracellular HIV p24 production. Adeno-associated vectors encoding RNA decoys may be useful for gene therapy of HIV infection.
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PMID:Transient protection of human T-cells from human immunodeficiency virus type 1 infection by transduction with adeno-associated viral vectors which express RNA decoys. 889 Nov 69

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


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