Interaction of tRNA(Lys-3) with multiple forms of human immunodeficiency virus reverse transcriptase.

Richter-Cook, Nancy J.; Howard, Kathryn J.; Cirino, Nick M.; Wöhrl, Birgitta M.; Grice, S F Le

doi:10.1016/s0021-9258(19)49626-9

Cited by 34 publications

(5 citation statements)

References 31 publications

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“…The best fit of the data to a quadratic equation (see Materials and Methods) describing the binding the tRNA Phe to a single site on the enzyme yielded a K d value of 9.3 ( 2.7 nM that agrees well with that determined from the RT tryptophan fluorescence titration (K d ) 9.8 ( 1.9 nM). Further evaluation of the titration data shows that the stoichiometry of the binding is 1:1 (RT:tRNA Phe ), in contrast to a 2:1 ratio estimated previously by gel-shift analyses (Richter-Cook et al, 1992). Titration of the Y base fluorescence of tRNA Phe with RT also revealed a lower affinity under higher salt conditions (K d value of 59.8 ( 15.7 nM in buffer containing 50 mM KCl, data not shown) as was detected by the titrations of RT tryptophan fluorescence.…”

Section: Resultscontrasting

confidence: 75%

“…In its in Vitro, and presumably in ViVo, fully processed form, RT exists as a heterodimeric enzyme, composed of an asymmetric arrangement of a 66 kDa (p66) subunit, containing the polymerase and RNaseH active sites, and a 51 kDa (p51) subunit, derived from proteolytic truncation of the larger subunit (LeGrice, 1993). The in Vitro interaction between tRNA 3 Lys and RT has been detected in several labs by gel-shift assay (Barat et al, 1989(Barat et al, , 1993Richter-Cook et al, 1992;Oude-Essink et al, 1995), footprinting (Sarih-Cottin et al, 1992;Wo ¨hrl et al, 1993), and crosslinking studies (Barat et al, 1989;Mishima & Steitz, 1995). Taken together, these studies suggested that both the p51 and p66 subunits of RT interact with tRNA and that several portions of the tRNA molecule, including the anticodon loop and the D and TψC loops, as well as the acceptor stem of the tRNA (see Figure 1) interact with RT.…”

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confidence: 99%

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Evaluation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Primer tRNA Binding by Fluorescence Spectroscopy: Specificity and Comparison to Primer/Template Binding

et al. 1996

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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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Section: Resultscontrasting

confidence: 75%

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confidence: 99%

Evaluation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Primer tRNA Binding by Fluorescence Spectroscopy: Specificity and Comparison to Primer/Template Binding

et al. 1996

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“…Lys (1,2,17,21,22,25,27,30,33,35,37,38). Others have previously studied the role of RT in the placement of tRNA 3 Lys by means of a large deletion (ϳ2 kb) that included both RT and integrase (22).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, mutated forms of NCp were not able to efficiently place primer tRNA Trp onto the avian leukosis virus RNA genome (23). The viral RT enzyme may also be involved in this process, since RTs possess higher affinities for primer tRNA species than do other forms of tRNA (1,2,17,21,22,25,27,30,33,35,37,38) and are responsible for the selective packaging of primer tRNA into the virion (12,18,19). However, RT itself cannot efficiently place primer tRNA onto viral RNA and may therefore be only indirectly involved (16).…”

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confidence: 97%

The roles of the human immunodeficiency virus type 1 Pol protein and the primer binding site in the placement of primer tRNA(3Lys) onto viral genomic RNA

Liang

Morin

et al. 1997

J Virol

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Factors that modulate the placement of primer tRNA(3Lys) onto the viral RNA genome in human immunodeficiency virus type 1 (HIV-1) were investigated through analysis of reverse-transcribed products that are extended from the tRNA(3Lys) primer. Mutations were introduced into the HIV-1 pol gene to result in the appearance of a stop codon in the open reading frame of the reverse transcriptase (RT) gene. These constructs, BH10-RT1 and BH10-RT2, yielded viruses with truncated Pol proteins. Alternatively, we altered the sequences involved in frameshifting by generating the construct BH10-FS. With each of these mutated viruses, we found that the primer tRNA(3Lys) that was placed onto viral genomic RNA was present in an unextended state. In contrast, as expected, tRNA(3Lys) in the case of wild-type BH10 virus had been extended by 2 bases. Furthermore, the amount of tRNA(3Lys) that was placed onto viral RNA in mutated viruses was significantly less than that placed in the wild-type virus. We also generated a mutant within the polymerase-active site of RT (D185H) (Asp-->His) that eliminated RT polymerase activity. We found that the placement of primer tRNA(3Lys) onto viral genomic RNA was independent of enzyme function; however, the tRNA(3Lys) that was placed was present in an unextended state due to the loss of RT activity. In contrast, the elimination of protease activity through a D25A (Asp-->Ala) point mutation in the protease-active site (construct BH10-PR) did cause a drop in the efficiency of tRNA(3Lys) placement. In this situation, a proportion of the placed tRNA(3Lys) was found to be extended by 2 bases, although not to the extent found with wild-type virus (BH10), due to a decrease in RT activity associated with unprocessed Gag-Pol protein that could not be cleaved because of the loss of protease activity. We also investigated the role of the primer binding site (PBS) in the placement of tRNA(3Lys) through a series of 2-, 4-, and 8-nucleotide (nt) deletions at the 3' end of the PBS, i.e., BH10-PBS2, BH10-PBS4, and BH10-PBS8, respectively. In mutated viruses BH10-PBS2 and BH10-PBS4, the 2-base-extended form of tRNA(3Lys) was still detected. However, less primer tRNA(3Lys) was placed onto viral genomic RNA as more nucleotides were deleted until the percentage of placement seen with wild-type BH10 virus dropped to only 4% in the virus with 8 nt deleted (BH10-PBS8). Consistently, these mutated viruses possessed decreased initial replication capacity compared with that of the wild-type virus, with the extent of incapacity corresponding to the size of the deletion. However, after several days, an increase in replication potential was accompanied by a reversion to a wild-type PBS.

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“…Le Grice (21). A 256 bp region of the clone containing the T7 promoter upstream of the full-length tRNA gene was amplified by polymerase chain reaction as described by Richter et al (22). The PCR product was used as the template for in Vitro transcription of tRNA 3 Lys using T7 RNA polymerase (23).…”

Section: Methodsmentioning

confidence: 99%

Polyamide Nucleic Acid Targeted to the Primer Binding Site of the HIV-1 RNA Genome Blocks in Vitro HIV-1 Reverse Transcription

et al. 1998

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We report here that polyamide nucleic acid (PNA) as well as a polyamide nucleic acid-DNA chimera complementary to the primer binding site of the HIV-1 genome can completely block priming by tRNA3Lys and consequently the in vitro initiation of reverse transcription by HIV-1 RT. Conventional heating and cooling is not required for annealing PNA analogs to the complementary nucleotide sequence as effective blockage of reverse transcription results from their invasion in the duplex region of preprimed U5-PBS HIV-1 RNA template-primer and was seen even at ambient temperature. Further, the extension of the initiated nascent (-) strand DNA can also be blocked by inclusion of another PNA, targeted to upstream sequences in the U5 region of the viral RNA. Interestingly, a PNA chimera having only two DNA nucleotides annealed with the U5-PBS RNA is recognized as a bonafide primer by HIV-1 RT, as the 3'OH end of the chimeric molecule is extended by the enzyme in the presence of dNTPs. A significant observation was that RNA/PNA or RNA/(PNA-DNA) hybrids were entirely resistant to the RNase H activity of HIV-1 RT. Furthermore, PNA invasion into the RNA/DNA hybrid completely prevented the cleavage of the RNA strand, suggesting that the RNase H activity of HIV-1 RT which was required in reverse transcription may also be inhibited by the PNA oligomer. These observations suggest that oligomeric PNAs targeted to various critical regions of the viral genome are likely to have strong therapeutic potential for interrupting multiple steps involved in the replication of HIV-1 and warrant serious investigation especially in the area of an effective delivery system.

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Interaction of tRNA(Lys-3) with multiple forms of human immunodeficiency virus reverse transcriptase.

Cited by 34 publications

References 31 publications

Evaluation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Primer tRNA Binding by Fluorescence Spectroscopy: Specificity and Comparison to Primer/Template Binding

Evaluation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Primer tRNA Binding by Fluorescence Spectroscopy: Specificity and Comparison to Primer/Template Binding

The roles of the human immunodeficiency virus type 1 Pol protein and the primer binding site in the placement of primer tRNA(3Lys) onto viral genomic RNA

Polyamide Nucleic Acid Targeted to the Primer Binding Site of the HIV-1 RNA Genome Blocks in Vitro HIV-1 Reverse Transcription

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