Plus-strand Strong-stop DNA Research Articles

The bipartite structure of the tRNA m1A58 methyltransferase from S. cerevisiae is conserved in humans

Among all types of RNA, tRNA is unique given that it possesses the largest assortment and abundance of modified nucleosides. The methylation at N(1) of adenosine 58 is a conserved modification, occurring in bacterial, archaeal, and eukaryotic tRNAs. In the yeast Saccharomyces cerevisiae, the tRNA 1-methyladenosine 58 (m(1)A58) methyltransferase (Mtase) is a two-subunit enzyme encoded by the essential genes TRM6 (GCD10) and TRM61 (GCD14). While the significance of many tRNA modifications is poorly understood, methylation of A58 is known to be critical for maintaining the stability of initiator tRNA(Met) in yeast. Furthermore, all retroviruses utilize m(1)A58-containing tRNAs to prime reverse transcription, and it has been shown that the presence of m(1)A58 in human tRNA(3) (Lys) is needed for accurate termination of plus-strand strong-stop DNA synthesis during HIV-1 replication. In this study we have identified the human homologs of the yeast m(1)A Mtase through amino acid sequence identity and complementation of trm6 and trm61 mutant phenotypes. When coexpressed in yeast, human Trm6p and Trm61p restored the formation of m(1)A in tRNA, modifying both yeast initiator tRNA(Met) and human tRNA(3) (Lys). Stable hTrm6p/hTrm61p complexes purified from yeast maintained tRNA m(1)A Mtase activity in vitro. The human m(1)A Mtase complex also exhibited substrate specificity--modifying wild-type yeast tRNA(i) (Met) but not an A58U mutant. Therefore, the human tRNA m(1)A Mtase shares both functional and structural homology with the yeast tRNA m(1)A Mtase, possessing similar enzymatic activity as well as a conserved binary composition.

Inhibition of HIV Type 1 Replication Using Lentiviral-Mediated Delivery of Mutant tRNALys3A58U

In previous studies, we showed that residue A58 of cellular tRNALys3 is necessary for appropriate termination of viral plus-strand strong-stop DNA (+SS DNA), and therefore plays a critical role in the life cycle of HIV-1. We also performed proof-of-principle studies that established that a mutant form of this tRNA primer (tRNA(Lys3)A58U, which lacks the M1A58 residue necessary for +SS DNA termination) could inhibit HIV-1 replication. In the present work, we examined whether a third generation lentiviral vector (SIN) could be used to deliver tRNA(Lys3)A58U to CEM cells. Using both viral kinetic studies and limiting dilution assays (LDA), we observed significant impairment of HIV-1 replication, up to 3 logs in the LDA, in CEM sublines expressing mutant tRNA(Lys3)A58U. No inhibition occurred in cells that either expressed wild-type tRNA(Lys3) or were transduced with empty SIN vector. Further, we observed impairment of viral replication using primary isolates of both HIV-1 and HIV-2 in sublines containing tRNA(Lys3)A58U. We also detected "breakthrough" HIV-1 replication in some tRNA(Lys3)A58U-expressing cultures. Interestingly, analyzed breakthrough viruses appeared to be both genetically and phenotypically wild type. One possible explanation for virological breakthrough is that it reflects the gradual accumulation of HIV-1 within the infected cell culture, to a level that ultimately exceeds the containment "threshold" conferred by tRNA(Lys3)A58U. The fact that HIV-1 does not appear to acquire heritable resistance to tRNA(Lys3)A58U-mediated blockade differentiates this antiviral modality from other therapeutic interventions. It also suggests that tRNA-mediated inhibition of viral replication might be a valuable adjunct to other antiviral approaches.

Mutation of the methylated tRNA(Lys)(3) residue A58 disrupts reverse transcription and inhibits replication of human immunodeficiency virus type 1.

Cellular tRNA(Lys)(3) serves as the primer for reverse transcription of human immunodeficiency virus type 1 (HIV-1). tRNA(Lys)(3) interacts directly with HIV-1 reverse transcriptase (RT), is packaged into viral particles, and anneals to the primer-binding site (PBS) of the HIV-1 genome in order to initiate reverse transcription. Residue A58 of tRNA(Lys)(3), which lies outside the PBS-complementary region, is posttranscriptionally methylated to form 1-methyladenosine 58 (M(1)A58). This methylation is thought to serve as a pause signal for plus-strand strong-stop DNA synthesis during reverse transcription. However, formal proof that the methylation is necessary for the pausing of RT has not been obtained in vivo. In the present study, we investigated the role of tRNA(Lys)(3) residue A58 in the replication cycle of HIV-1 in living cells. We have developed a mutant tRNA(Lys)(3) derivative, tRNA(Lys)(3)A58U, in which A58 was replaced by U. This mutant tRNA was expressed in CEM cells. We demonstrate that the presence of M(1)A58 is necessary for the appropriate termination of plus-strand strong-stop DNA synthesis and that the absence of M(1)A58 allows RT to read the tRNA sequences beyond residue 58. In addition, we show that replacement of M(1)A58 with U inhibits the replication of HIV-1 in vivo. These results highlight the importance of tRNA primer residue A58 in the reverse transcription process. Inhibition of reverse transcription with mutant tRNA primers constitutes a novel approach for therapeutic intervention against HIV-1.

Zinc finger structures in the human immunodeficiency virus type 1 nucleocapsid protein facilitate efficient minus- and plus-strand transfer.

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) has two zinc fingers, each containing the invariant metal ion binding residues CCHC. Recent reports indicate that mutations in the CCHC motifs are deleterious for reverse transcription in vivo. To identify reverse transcriptase (RT) reactions affected by such changes, we have probed zinc finger functions in NC-dependent RT-catalyzed HIV-1 minus- and plus-strand transfer model systems. Our approach was to examine the activities of wild-type NC and a mutant in which all six cysteine residues were replaced by serine (SSHS NC); this mutation severely disrupts zinc coordination. We find that the zinc fingers contribute to the role of NC in complete tRNA primer removal from minus-strand DNA during plus-strand transfer. Annealing of the primer binding site sequences in plus-strand strong-stop DNA [(+) SSDNA] to its complement in minus-strand acceptor DNA is not dependent on NC zinc fingers. In contrast, the rate of annealing of the complementary R regions in (-) SSDNA and 3' viral RNA during minus-strand transfer is approximately eightfold lower when SSHS NC is used in place of wild-type NC. Moreover, unlike wild-type NC, SSHS NC has only a small stimulatory effect on minus-strand transfer and is essentially unable to block TAR-induced self-priming from (-) SSDNA. Our results strongly suggest that NC zinc finger structures are needed to unfold highly structured RNA and DNA strand transfer intermediates. Thus, it appears that in these cases, zinc finger interactions are important components of NC nucleic acid chaperone activity.

A mechanism for plus-strand transfer enhancement by the HIV-1 nucleocapsid protein during reverse transcription.

The HIV-1 nucleocapsid protein (NC) functions as a nucleic acid chaperone during the plus-strand transfer step in reverse transcription by facilitating annealing of the primer binding site (PBS) sequence in the short plus-strand strong-stop DNA fragment [(+) SSDNA] to a complementary site located near the 3' end of the minus-strand DNA [(-) PBS DNA]. To investigate the mechanism by which NC performs this function, we have prepared an 18-nucleotide (-) PBS DNA for nuclear magnetic resonance (NMR) based structural and NC binding studies. The (-) PBS DNA forms a stable hairpin (T(m) approximately 42 +/- 5 degrees C) that contains a five-residue loop and a bulged thymine in a guanosine-cytosine-rich stem. Addition of substoichiometric amounts of NC results in significant broadening and reductions in NMR signal intensities of the Watson-Crick base-paired imino protons and a reduction by 20 degrees C in the upper temperature at which the imino proton signals are detectable, consistent with destabilization of the structure. The results suggest that inefficient annealing in the absence of NC may be due to the intrinsic stability of an internal (-) PBS DNA hairpin and that NC facilitates strand transfer by destabilizing the hairpin and exposing stem nucleotides for base pairing with the PBS sequence in (+) SSDNA.

Molecular requirements for human immunodeficiency virus type 1 plus-strand transfer: analysis in reconstituted and endogenous reverse transcription systems.

We have developed a reconstituted system which models the events associated with human immunodeficiency virus type 1 (HIV-1) plus-strand transfer. These events include synthesis of plus-strand strong-stop DNA [(+) SSDNA] from a minus-strand DNA donor template covalently attached to human tRNA3Lys, tRNA primer removal, and annealing of (+) SSDNA to the minus-strand DNA acceptor template. Termination of (+) SSDNA synthesis at the methyl A (nucleotide 58) near the 3' end of tRNA3Lys reconstitutes the 18-nucleotide primer binding site (PBS). Analysis of (+) SSDNA synthesis in vitro and in HIV-1 endogenous reactions indicated another major termination site: the pseudouridine at nucleotide 55. In certain HIV-1 strains, complementarity between nucleotides 56 to 58 and the first three bases downstream of the PBS could allow all of the (+) SSDNA products to be productively transferred. Undermodification of the tRNA may be responsible for termination beyond the methyl A. In studies of tRNA removal, we find that initial cleavage of the 3' rA by RNase H is not sufficient to achieve successful strand transfer. The RNA-DNA hybrid formed by the penultimate 17 bases of tRNA still annealed to (+) SSDNA must also be destabilized. This can occur by removal of additional 3'-terminal bases by RNase H (added either in cis or trans). Alternatively, the nucleic acid chaperone activity of nucleocapsid protein (NC) can catalyze this destabilization. NC stimulates annealing of the complementary PBS sequences in (+) SSDNA and the acceptor DNA template. Reverse transcriptase also promotes annealing but to a lesser extent than NC.

Reverse transcription of the yeast ty1 retrotransposon: the mode of first strand transfer is either intermolecular or intramolecular

Replication of the yeast Ty1 retrotransposon occurs by a mechanism similar to that of retroviruses. According to the current model of retroviral reverse transcription, two strand transfers (the so-called minus-strand and plus-strand strong-stop DNA transfers) are required to produce full-length preintegrative DNA. Because two genomic RNA molecules are packaged inside the viral particles, the strand transfers can be either intra- or intermolecular. To study the mode of transfer of minus-strand strong-stop DNA during reverse transcription of the yeast Ty1 retrotransposon, we have analyzed the cDNA products that accumulate in the cytoplasmic virus-like particles of yeast cells harboring two marked Ty1 elements. Our results indicate that Ty1 minus-strand transfer occurs in a random manner with approximately similar frequencies of intra- and intermolecular transfer. It has been observed recently that intra- and intermolecular minus-strand transfer occur at similar frequencies during replication of a complex retrovirus such as HIV-1. These results together with the observation that genetic recombination occurs with a high frequency during minus-strand synthesis suggest that both packaged RNA molecules are needed for the synthesis of one minus-strand DNA.

Plus-strand strong-stop DNA transfer in yeast Ty retrotransposons.

The yeast Ty1 LTR retrotransposon replicates by reverse transcription and integration; the process shows many similarities to the retroviral life cycle. However, we show that plus strand strong-stop DNA transfer in yeast Ty1 elements differs from the analogous retroviral process. By analysis of the native structure of the Ty1 primer binding site and by a series of manipulations of this region and assessment of the effects on retrotransposition, we show that primer binding site inheritance is not from the tRNA primer, which is inconsistent with classical retroviral models. This unusual inheritance pattern holds even when the Ty1 primer binding site is lengthened in order to be more retrovirus-like. Finally, the distantly related Ty3 element has an inheritance pattern like Ty1, indicating evolutionary conservation of the alternative pathway used by Ty1. Based on these results we arrive at a plus strand primer recycling model that explains Ty1 plus strand strong-stop DNA transfer and inheritance patterns in the primer binding site.

Heterogeneous terminal structure of Ty1 and Ty3 reverse transcripts.

A specific terminal structure of preintegrative DNA is required for transposition of retroviruses and LTR-retrotransposons. We have used an anchored PCR technique to map the 3'ends of DNA intermediates synthesized inside yeast Ty1 and Ty3 retrotransposon virus-like particles. We find that, unlike retroviruses, Ty1 replicated DNA does not have two extra base pairs at its 3'ends. In contrast some Ty3 preintegrative DNA molecules have two extra nucleotides at the 3'end of upstream and downstream long terminal repeats. Moreover we find that some molecules of replicated Ty3 DNA have more than two extra nucleotides at the 3'end of the upstream LTR. This observation could be accounted for by imprecise RNAse H cutting of the PPT sequence. The site of Ty1 and Ty3 plus-strand strong-stop DNA termination was also examined. Our results confirm that the prominent Ty1 and Ty3 plus-strand strong-stop molecules harbor 12 tRNA templated bases but also show that some Ty1 and Ty3 plus-strand strong-stop DNA molecules harbor less tRNA templated bases. We propose that these less than full length plus-strand molecules could be active intermediates in Ty retrotransposon replication.

Enhancement or inhibition of HIV-1 replication by intracellular expression of sense or antisense RNA targeted at different intermediates of reverse transcription.

To construct retroviral vectors expressing sense or antisense RNA targeted at HIV reverse transcription intermediates, and to test the anti-HIV properties of these constructs in transduced T cells. Five double-copy retroviral vectors were constructed, in which the expression of the sense or antisense RNA corresponding to HIV minus- or plus-strand strong-stop DNA was driven by the human tRNA(met) promoter. The templates for the sense or antisense RNA were polymerase chain reaction-cloned from HIV pNL43 into a murine leukaemia virus-based vector and corresponding defective virions were packaged in PA317 cells. Human Jurkat T cells transduced with these vectors were challenged with HIV and monitored for viral RNA, viral DNA and p24 production for 23 weeks. Intracellular expression of HIV sense RU5 sequences (RNA complementary to minus-strand strong-stop DNA) enhanced HIV replication in T cells. Expression of HIV sense or antisense U3RU5 sequences (identical or complementary to plus-strand strong-stop DNA) conferred long-term inhibition of HIV replication, despite continuous presence of viral challenge in the transduced cell cultures. Plus-strand strong-stop DNA as an intermediate in the early process of viral reverse transcription can be explored as an additional target for anti-HIV gene therapy.

Ty3 integrase mutants defective in reverse transcription or 3'-end processing of extrachromosomal Ty3 DNA

Ty3, a retroviruslike element in Saccharomyces cerevisiae, encodes an integrase (IN) which is essential for position-specific transposition. The Ty3 integrase contains the highly conserved His-Xaa(3-7)-His-Xaa(23-32)-Cys-Xaa(2)-Cys and Asp, Asp-Xaa(35)-Glu [D,D(35)E] motifs found in retroviral integrases. Mutations were introduced into the coding region for the Ty3 integrase to determine the effects in vivo of changes in conserved residues of the putative catalytic triad D,D(35)E and the nonconserved carboxyl-terminal region. Ty3 viruslike particles were found to be associated with significant amounts of linear DNA of the approximate size expected for a full-length reverse transcription product and with plus-strand strong-stop DNA. The full-length, preintegrative DNA has at each 3' end 2 bp that are removed prior to or during integration. Such 3'-end processing has not been observed for other retroviruslike elements. A mutation at either D-225 or E-261 of the Ty3 integrase blocked transposition and prevented processing of the 3' ends of Ty3 DNA in vivo, suggesting that the D,D(35)E region is part of the catalytic domain of Ty3 IN. Carboxyl-terminal deletions of integrase caused a dramatic reduction in the amount of Ty3 DNA in vivo and a decrease in reverse transcriptase activity in vitro but did not affect the apparent size or amount of the 55-kDa reverse transcriptase in viruslike particles. The 115-kDa viruslike particle protein, previously shown to react with antibodies to Ty3 integrase, was shown to be a reverse transcriptase-IN fusion protein. These results are consistent with a role for the integrase domain either in proper folding of reverse transcriptase or as part of a heterodimeric reverse transcriptase molecule.

Plus-strand DNA Synthesis of the Yeast Retrotransposon Ty1 is Initiated at Two Sites, PPT1 Next to the 3′ LTR and PPT2 Within the polGene. PPT1 is Sufficient for Ty1 Transposition

Long terminal repeat elements and retroviruses require primers for initiation of minus and plus-strand DNA synthesis by reverse transcriptase. Here we demonstrate genetically that plus-strand DNA synthesis of the yeast Ty1 element is initiated at two sites located at the 5′ boundary of the 3′ long terminal repeat (PPT1) and near the middle of the polgene in the integrase coding sequence (PPT2). A consequence of the presence of two PPTs is that Ty1 plus-strand DNA exists as segments at some time during replication. Three fragments have been identified: the plus-strand strong-stop DNA initiated at PPT1, a downstream fragment initiated at PPT2 and an upstream fragment spanning the 5′-terminal part of Tyr1 and a portion of the TyB gene. Characterization of the 3′ ends of the plus-strand DNA fragments reveals (1) that the upstream fragment is elongated beyond PPT2 creating a plus-strand overlap and (2) that the majority of plus-strand strong-stop DNA fragments bear a copy of the minus-strand primer binding site in agreement with the accepted model of retroviral genomic RNA reverse transcription. The two polypurine tracts, PPT1 and PPT2, have an identical sequence GGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.

Stepwise analysis of reverse transcription in a cell-to-cell human immunodeficiency virus infection model: kinetics and implications.

We have investigated the kinetics of human immunodeficiency virus (HIV) reverse transcription in infected T cells, using a synchronized, one-step, cell-to-cell infection model and quantitative PCR assays for the different DNA intermediate structures that are found sequentially during reverse transcription. Different efficiencies that might arise from the use of different primers and other PCR conditions were normalized by conversion of each PCR product signal to copy numbers by comparing with standards. After an initial lag period, the minus-strand strong-stop viral DNA was detected first. This was followed by the post-transfer newly extended minus-strand viral DNA and then by the plus-strand strong-stop DNA and fully extended minus-strand DNA. Kinetic data indicated that, once reverse transcription was initiated, the HIV reverse transcriptase synthesized minus-strand DNA at a rate of 150-180 bases/min, and that the first template transfer and the initiation of the plus-strand DNA synthesis imposed specific time delays. In contrast, minus-strand viral DNA synthesized after the second template transfer appeared at a time point very close to the time of the appearance of the last piece of DNA synthesized just before the second template switch, suggesting that the second switch occurred very rapidly. Taken together, our results define more accurately than was previously possible the rates of several of the steps in HIV reverse transcription in infected T cell lines and indicate different mechanisms for the two distinct template switches during retrovirus reverse transcription.

Double-Stranded Strong-Stop DNA and the Second Template Switch in Human Immunodeficiency Virus (HIV) DNA Synthesis

Synthesis of unintegrated retroviral DNA via reverse transcription is thought to involve two separate template switches. Minus-strand strong-stop DNA, a major product of in vitro reactions using detergent-treated virions, is synthesized prior to the first template switch. Plus-strand strong-stop DNA, which is found base-paired to near full-length minus-strand DNA in infected cells, is believed to be synthesized before the second template switch. Using a synchronized, one-step HIV infection model, we report here the detection in acutely infected cells of a novel double-stranded strong-stop HIV DNA with a discrete length of ∼650 base pairs, commencing at or near the left hand end of the right-hand U3 region of the HIV long terminal repeat. The plus-strand of this double-stranded strong-stop DNA possesses the primer binding site sequence and appears to be synthesized prior to the completion of the synthesis of its complementary minus-strand. In contrast, the minus-strand of the double-stranded strong-stop DNA lacks the primer binding site sequence after RNaseH digestion. We propose that a transient free plus-strand strong-stop DNA is released from its template by displacement synthesis and subsequently used as template for the synthesis of its complementary minus-strand. The proposed transient free plus-strand strong-stop DNA may also mediate the second template switch.

Characterization of large deletions occurring during a single round of retrovirus vector replication: novel deletion mechanism involving errors in strand transfer

Retroviruses mutate at a high rate during replication. We used a spleen necrosis virus-based vector system and helper cell line to characterize mutations occurring during a single round of retrovirus replication. The vector used, JD216HyNeo, codes for two drug resistance genes, hygromycin resistance (hygro) and neomycin resistance (neo). The downstream neo gene is expressed only when a mutation alleviates a block to splicing which is located in the upstream hygro gene. The mutations allowing splicing were large deletions, ranging in size from about 500 to about 2,000 bp. Most of the mutant proviruses lacked the encapsidation sequence, as shown by our inability to rescue the mutant proviruses with wild-type reticuloendotheliosis virus strain A and confirmed by Southern blotting and direct DNA sequence analysis. We therefore concluded that most of the deletions arose during reverse transcription in the target cell, rather than during transcription in the host cell. The sequence data also indicated that the deletions occurred by at least three different mechanisms: (i) misalignment of the growing point; (ii) incorrect synthesis and termination in the primer-binding sequence during synthesis of the plus-strand strong-stop DNA; and (iii) incorrect synthesis and termination before the primer-binding sequence during synthesis of the plus-strand strong-stop DNA. The second mechanism also led to the incorporation of cellular sequences into the proviral genome, pointing to a potential novel mechanism by which retroviruses can acquire cellular genes.

Abortive reverse transcription by mutants of Moloney murine leukemia virus deficient in the reverse transcriptase-associated RNase H function

The reverse transcriptase enzymes of retroviruses are multifunctional proteins containing both DNA polymerase activity and a nuclease activity, termed RNase H, specific for RNA in RNA-DNA hybrid form. To determine the role of RNase H activity in retroviral replication, we constructed a series of mutant genomes of Moloney murine leukemia virus that encoded reverse transcriptase enzymes that were specifically altered to retain polymerase function but lack RNase H activity. The mutant genomes were all replication defective. Analysis of in vitro reverse transcription reactions carried out by mutant virions showed that minus-strand strong-stop DNA was formed but did not efficiently translocate to the 3' end of the genome; rather, the DNA was stably retained in RNA-DNA hybrid form. Plus-strand strong-stop DNA was not detected. These results suggest that RNase H normally promotes strong-stop translocation, perhaps by exposing single-stranded DNA sequences for base pairing. Four new DNA species were also detected among the reaction products. Analysis of these DNAs suggested that they were minus-strand DNAs formed from VL30 RNAs encoded by the mouse genome. We suggest that reverse transcriptase can initiate DNA synthesis at any one of four alternate tRNA primer-binding sites near the 5' ends of VL30 RNAs.

Discontinuities in the DNA synthesized by an avian retrovirus.

The unintegrated linear DNA synthesized in cells infected by Rous sarcoma virus is a predominantly double-stranded structure in which most of the minus-strand DNA, complementary to the viral RNA genome, is genome sized, whereas the plus-strand DNA is present as subgenomic fragments. We previously reported the application of benzoylated naphthoylated DEAE-cellulose chromatography to demonstrate that of the linear viral DNA species synthesized in quail embryo fibroblasts infected with Rous sarcoma virus greater than 99.5% contain single-stranded regions and these regions are predominantly composed of plus-strand DNA sequences (T. W. Hsu and J. M. Taylor, J. Virol. 44:47-53, 1982). We now present the following additional findings. (i) There were on the average 3.5 single-stranded regions per linear viral DNA, and these single-stranded regions could occur at many locations. (ii) With a probe to the long terminal repeat, we detected, in addition to a heterogeneous size distribution of subgenomic plus-strand DNA species, at least three prominent discrete size classes. Each of these discrete species had its own specific initiation site, but all had the same termination site. Such species were analogous to those reported by Kung et al. (J. Virol. 37: 127-138, 1981). (iii) These discrete size classes of plus-strand DNA were present not only on the major size class of linear DNA but also on a heterogeneous of slower-sedimenting species, which we have called immature linears. Our interpretation is that we have thus detected several additional sites for the initiation of plus-strand DNA. (iv) The 340-base plus-strand strong-stop DNA was only found associated with the immature linears. (v) From a size and hybridization comparison of these discrete size classes of plus-strand DNA with minus-strand DNA species, as synthesized in the endogenous reaction of melittin-disrupted virions, it was found that the putative additional initiation sites for plus-strand DNA synthesis corresponded to many of the pause sites in the synthesis of minus-strand DNA.