Primer For Reverse Transcription Research Articles

Effect of altering the tRNA(Lys)(3) concentration in human immunodeficiency virus type 1 upon its annealing to viral RNA, GagPol incorporation, and viral infectivity.

Human immunodeficiency virus type 1 (HIV-1) uses tRNA(Lys)(3) as a primer for reverse transcription and, during viral assembly, this tRNA is selectively packaged into the virus along with the other major tRNA(Lys), tRNA(Lys)(3). Increasing the cytoplasmic concentration of tRNA(Lys)(3) through transfection of cells with a plasmid containing both HIV-1 proviral DNA and a tRNA(Lys)(3) gene results in a greater incorporation of tRNA(Lys)(3) into virions, which is accompanied by increased annealing of tRNA(Lys)(3) to the viral genome and increased infectivity of the viral population. Increased viral tRNA(Lys)(3) is accompanied by decreased viral tRNA(Lys)(3), with the total tRNA(Lys)/virion and the GagPol/Gag ratios remaining unchanged. Viral tRNA(Lys) can be doubled, with increases in both tRNA(Lys)(3) and tRNA(Lys)(1,2) concentrations, by overexpressing lysyl tRNA synthetase. This also results in increased tRNA(Lys)(3) annealing to the viral RNA and increased viral infectivity but, again, no change in the GagPol/Gag ratio was observed. This result indicates that GagPol, whose interaction is required during packaging, is not a limiting factor during tRNA(Lys) incorporation into HIV-1, whereas LysRS is.

Priming reverse transcription with oligo(dT) does not yield representative samples of Mycobacterium tuberculosis cDNA.

Several recent publications have suggested that oligo(dT) can prime reverse transcription of several mycobacterial mRNAs. To determine if this is the case for most Mycobacterium tuberculosis mRNA species, reverse transcription reactions of M. tuberculosis RNA were primed with oligo(dT) or with other primers that did not target polyadenylylated sequences, and the resultant cDNA product was evaluated. Priming with oligo(dT) yielded more cDNA than priming with an arbitrary primer for only 1 of 12 unrelated M. tuberculosis genes, as measured by competitive PCR. Priming with oligo(dT) yielded cDNA for only 30% of the genes primed for by 37 M. tuberculosis genome-directed oligonucleotides, as assessed by hybridization of cDNA with an M. tuberculosis microarray. These data demonstrate that priming of reverse transcription of mycobacterial mRNA with oligo(dT) does not yield representative samples of cDNA.

DNA repair mediated by endonuclease-independent LINE-1 retrotransposition.

Long interspersed elements (LINE-1s) are abundant retrotransposons in mammalian genomes that probably retrotranspose by target site-primed reverse transcription (TPRT). During TPRT, the LINE-1 endonuclease cleaves genomic DNA, freeing a 3' hydroxyl that serves as a primer for reverse transcription of LINE-1 RNA by LINE-1 reverse transcriptase. The nascent LINE-1 cDNA joins to genomic DNA, generating LINE-1 structural hallmarks such as frequent 5' truncations, a 3' poly(A)+ tail and variable-length target site duplications (TSDs). Here we describe a pathway for LINE-1 retrotransposition in Chinese hamster ovary (CHO) cells that acts independently of endonuclease but is dependent upon reverse transcriptase. We show that endonuclease-independent LINE-1 retrotransposition occurs at near-wildtype levels in two mutant cell lines that are deficient in nonhomologous end-joining (NHEJ). Analysis of the pre- and post-integration sites revealed that endonuclease-independent retrotransposition results in unusual structures because the LINE-1s integrate at atypical target sequences, are truncated predominantly at their 3' ends and lack TSDs. Moreover, two of nine endonuclease-independent retrotranspositions contained cDNA fragments at their 3' ends that are probably derived from the reverse transcription of endogenous mRNA. Thus, our results suggest that LINE-1s can integrate into DNA lesions, resulting in retrotransposon-mediated DNA repair in mammalian cells.

Correlation Between tRNALys3 Aminoacylation and Its Incorporation into HIV-1

During human immunodeficiency virus type 1 (HIV-1) assembly, tRNA(Lys) is selectively packaged into the virus, where tRNA(Lys3) serves as the primer for reverse transcription. Lysyl-tRNA synthetase is also selectively incorporated into HIV-1 and is therefore a strong candidate for being the signal by which viral proteins interact with tRNA(Lys) isoacceptors. Previously, mutations in the tRNA(Lys3) anticodon have been shown to strongly inhibit the charging of tRNA(Lys3) by lysyl-tRNA synthetase in vitro, and we show here that in vivo aminoacylation is also inhibited by anticodon changes. The order of decreasing in vivo aminoacylation for tRNA(Lys3) anticodon mutants is: wild-type SUU (where S = mcm(5)S(2)U) 100%) --> SGU (49%) --> CGU (40%) --> SGA (0%) and CGA (0%). We found that the ability of these tRNA(Lys3) anticodon variants to be aminoacylated in vivo is directly correlated with their ability to be packaged into HIV-1. These data showed that the anticodon is a major determinant for tRNA(Lys3) packaging and support the conclusion that its productive interaction with lysyl-tRNA synthetase is important for tRNA(Lys3) incorporation into HIV-1.

In Vitro Evidence That the Untranslated Leader of the HIV-1 Genome Is an RNA Checkpoint That Regulates Multiple Functions through Conformational Changes

The HIV-1 RNA genome forms dimers through base pairing of a palindromic 6-mer sequence that is exposed in the loop of the dimer initiation signal (DIS) hairpin structure (loop-loop kissing). The HIV-1 leader RNA can adopt a secondary structure conformation that is not able to dimerize because the DIS hairpin is not folded. Instead, this DIS motif is base-paired in a long distance interaction (LDI) that extends the stem of the primer-binding site domain. In this study, we show that targeting of the LDI by either antisense oligonucleotides or specific mutations can induce the conformational switch to a branched multiple hairpin (BMH) structure, and this LDI-to-BMH switch coincides with increased RNA dimerization. Another interesting finding is that the extended LDI stem can resist a certain level of destabilization, indicating that a buffer is created to prevent a premature conformational switch and early dimerization. Because the tRNA(Lys3) primer for reverse transcription anneals to multiple sequence elements of the HIV-1 leader RNA, including sequences in the LDI stem, we tested whether tRNA-annealing can destabilize the LDI stem such that RNA dimerization is triggered. Using a combination of stem-destabilizing approaches, we indeed measured a small but significant effect of tRNA-annealing on the ability of the RNA template to form dimers. This observation suggests that HIV-1 RNA can act as a checkpoint to control and coordinate different leader functions through conformational switches. This in vitro result should be verified in subsequent in vivo studies with HIV-infected cells.

Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription.

We have analyzed a systematic flaw in the current system of gene identification: the oligo(dT) primer widely used for cDNA synthesis generates a high frequency of truncated cDNAs through internal poly(A) priming. Such truncated cDNAs may contribute to 12% of the expressed sequence tags in the current dbEST database. By using a synthetic transcript and real mRNA templates as models, we characterized the patterns of internal poly(A) priming by oligo(dT) primer. We further demonstrated that the internal poly(A) priming can be effectively diminished by replacing the oligo(dT) primer with a set of anchored oligo(dT) primers for reverse transcription. Our study indicates that cDNAs designed for genomewide gene identification should be synthesized by use of the anchored oligo(dT) primers, rather than the oligo(dT) primers, to diminish the generation of truncated cDNAs caused by internal poly(A) priming.

Switching the in vitro tRNA usage of HIV-1 by simultaneous adaptation of the PBS and PAS.

Reverse transcription of the HIV-1 RNA genome is primed by the cellular tRNA(lys3) molecule that anneals to a complementary sequence in the viral genome, the primer-binding site (PBS). Additional interactions between the tRNA primer and the viral RNA were proposed to play a role in reverse transcription. We recently identified an 8-nt element in the U5 region upstream of the PBS that is critical for initiation and processive elongation of reverse transcription. This motif was termed the primer activation signal (PAS), and is proposed to interact with the "antiPAS sequence" in the TphiC arm of tRNA(lys3). In this study, we demonstrate that the efficiency of initiation of reverse transcription can be modulated by PAS mutations that strengthen or weaken the interaction with antiPAS. These results provide further evidence for a direct base-pairing interaction between the PAS in the viral RNA and the antiPAS in the tRNA(lys3) molecule. A broad phylogenetic survey indicated that a PAS element is present in all retroviral RNA genomes, suggesting that the process of reverse transcription is regulated by a common mechanism in all retroviridae. It has proven very difficult to change the identity of the tRNA primer for HIV-1 reverse transcription by changing the PBS sequence. Using in vitro reverse transcription assays, we demonstrate that the identity of the priming tRNA species can be switched by simultaneous alteration of the PBS and PAS motifs to accommodate a new tRNA primer. These results indicate that the PAS-antiPAS interaction is important for both primer selection and efficient reverse transcription.

Non-cognate template usage and alternative priming by a group II intron-encoded reverse transcriptase

Group II introns are retroelements that site-specifically insert into DNA through homing. They are also implicated in related phenomena such as ectopic site insertions and precise intron deletions, but little is known about how group II intron reverse transcriptases (RTs) interact with non-cognate substrates. Here we show that wild-type aI2 RT readily reverse transcribes non-cognate RNAs in mitochondrial RNP particles when the aI2 intron structure is misfolded. In two closely related priming mutants, 1°2 ΔD5 and 1 +2 ΔD5, which contain wild-type RT but a disrupted intron structure, the RT has substantially lost specificity for aI2 RNA and copies multiple RNAs present in the RNP particles, using an alternative priming mechanism. The RT in 1°2 ΔD5 RNP particles can also copy exogenous RNAs but unlike the endogenous templates, a complementary primer is required, suggesting that the alternative priming event is specific to RT-RNA interactions formed in vivo. Alternatively primed cDNAs from strains 1°2 ΔD5, 1 +2 ΔD5 and 1°2 P714T (containing the mutation P714T in the RT) do not use homing site DNA as a primer, but appear to utilize a non-complementary DNA primer of approximately ten nucleotides. The alternative priming mechanism and reverse transcription of non-cognate templates has implications for in vivo reverse transcription of non-intronic RNAs, which is expected to occur during intron deletions and other retroprocessing events.

Twin Priming: A Proposed Mechanism for the Creation of Inversions in L1 Retrotransposition

L1 retrotransposons are pervasive in the human genome. Approximately 25% of recent L1 insertions in the genome are inverted and truncated at the 5' end of the element, but the mechanism of L1 inversion has been a complete mystery. We analyzed recent L1 inversions from the genomic database and discovered several findings that suggested a mechanism for the creation of L1 inversions, which we call twin priming. Twin priming is a consequence of target primed reverse transcription (TPRT), a coupled reverse transcription/integration reaction that L1 elements are thought to use during their retrotransposition. In TPRT, the L1 endonuclease cleaves DNA at its target site to produce a double-strand break with two single-strand overhangs. During twin priming, one of the overhangs anneals to the poly(A) tail of the L1 RNA, and the other overhang anneals internally on the RNA. The overhangs then serve as primers for reverse transcription. The data further indicate that a process identical to microhomology-driven single-strand annealing resolves L1 inversion intermediates.

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.

CDNA - protein fusions: covalent protein - gene conjugates for the in vitro selection of peptides and proteins.

We report a method for the synthesis of covalent cDNA-protein fusions for protein display applications. A branched mRNA template was developed which carries a peptidyl acceptor and a reverse transcription primer at the 3'-end. Translation in vitro followed by reverse transcription produced a protein covalently bonded to its encoding cDNA. Both single- and double-stranded cDNA-protein fusions were prepared. cDNA-protein fusions are stable in alkali and resistant to ribonucleases. Their simple preparation and their resistance towards degradation should make cDNA-protein fusions a useful tool for the in vitro selection and evolution of high affinity ligands from large libraries of polypeptides.

CDNA–Protein Fusions: Covalent Protein–Gene Conjugates for the In Vitro Selection of Peptides and Proteins

We report a method for the synthesis of covalent cDNA-protein fusions for protein display applications. A branched mRNA template was developed which carries a peptidyl acceptor and a reverse transcription primer at the 3'-end. Translation in vitro followed by reverse transcription produced a protein covalently bonded to its encoding cDNA. Both single- and double-stranded cDNA-protein fusions were prepared. cDNA-protein fusions are stable in alkali and resistant to ribonucleases. Their simple preparation and their resistance towards degradation should make cDNA-protein fusions a useful tool for the in vitro selection and evolution of high affinity ligands from large libraries of polypeptides.

Incorporation of lysyl-tRNA synthetase into human immunodeficiency virus type 1.

During human immunodeficiency virus type 1 (HIV-1) assembly, tRNA(Lys) isoacceptors are selectively incorporated into virions and tRNA(Lys)3 is used as the primer for reverse transcription. We show herein that the tRNA(Lys)-binding protein, lysyl-tRNA synthetase (LysRS), is also selectively packaged into HIV-1. The viral precursor protein Pr55gag alone will package LysRS into Pr55gag particles, independently of tRNA(Lys). With the additional presence of the viral precursor protein Pr160gag-pol, tRNA(Lys) and LysRS are both packaged into the particle. While the predominant cytoplasmic LysRS has an apparent M(r) of 70,000, viral LysRS associated with tRNA(Lys) packaging is shorter, with an apparent M(r) of 63,000. The truncation occurs independently of viral protease and might be required to facilitate interactions involved in the selective packaging and genomic placement of primer tRNA.

Improvement of RNA fingerprinting efficiency for the analysis of differential gene expression in human cardiac macro- and microvascular endothelial cells

RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) is a powerful tool to screen differential gene expression. However, PCR-based screening techniques show a high incidence of false positive results (40–90%). In order to increase the efficiency and feasibility of RAP-PCR, the original protocol was modified and applied to analyse differential gene expression in human coronary macro- (HCEC) and microvascular (HCMEC) endothelial cells. The major modifications introduced were: (i) the use of two primers for PCR amplification, instead of reverse-transcription primer alone; (ii) the use of three cycles at low stringency followed by further amplification at high stringency; (iii) optimization of amplification cycle number, template amount, and concentration of primers, dNTP, Mg2+; (iv) detection of fingerprints by silver staining; and (v) direct sequencing using RAP-PCR primers. Analysis of untreated and TNF α -stimulated (100 U ml−1for 1, 4, and 24 h) HCEC and HCMEC displayed 11 differentially expressed products by 18 primer combinations. Confirmation of results by RT-PCR showed that the rate of false positives attributable to our screening method was less than 20%. Among detected RAP-PCR products, the expression of Mn-superoxide dismutase, A20 zinc finger protein, and three novel genes (A/a, 4/d, 7/c) was more strongly modulated by TNF in HCEC than HCMEC. A further novel gene (B/e) was strongly expressed in HCMEC while only barely detectable in HCEC. In conclusion, modification of RAP-PCR strongly reduced the incidence of false positives, eliminated a radioactive requirement, and allowed sequencing without prior cloning, supplying an improved technology able to identify new differentially expressed genes between macro- and microvascular endothelial cells.

Identification of Critical Elements in the tRNA Acceptor Stem and TΨC Loop Necessary for Human Immunodeficiency Virus Type 1 Infectivity

A mutant human immunodeficiency virus type 1 (HIV-1) with a primer binding site (PBS) complementary to yeast tRNA(Phe) (psHIV-Phe), which relies on exogenous yeast tRNA(Phe) as reverse transcription primer, was used to investigate elements in the tRNA acceptor stem and T(Psi)C stem-loop required for the tRNA primer selection and use in HIV-1 replication. tRNA(Phe) mutants with two- or four-nucleotide deletions in the 3' end retained the capacity to complement replication of psHIV-Phe. tRNA(Phe) mutants with an extended 5' end had reduced capacity for complementation, which could be restored by extension of the 3' end of these tRNA(Phe) mutants with sequences complementary to the HIV-1 U5 region. Further analysis of mutations in the acceptor stem of tRNA(Phe) suggested that an intact acceptor stem RNA structure is important for complementation. Analysis of single-nucleotide changes in the T(Psi)C stem-loop of tRNA(Phe) revealed an unexpected, essential role of this region for rescue of psHIV-Phe.

Essential regions of the tRNA primer required for HIV-1 infectivity.

Human immunodeficiency virus (HIV), like all retroviruses, requires a cellular tRNA as a primer for initiation of reverse transcription. In a previous study, we demonstrated that an HIV-1 with a primer binding site complementary to yeast tRNA(Phe) (psHIV-Phe) was not infectious unless yeast tRNA(Phe) was supplied in trans. This unique in vivo complementation system has now been used to define the elements of the tRNA required for HIV-1 replication. Mutant tRNA(Phe) with deletions in TPsiC stem-loop, anticodon stem-loop or D stem-loop of the tRNA were generated and assessed for the capacity to rescue psHIV-Phe. Mutant tRNA(Phe) with disrupted TPsiC stem-loop did not rescue psHIV-Phe. In contrast, a mutant tRNA(Phe) without the D stem-loop was fully functional for the rescue. The tRNA anticodon stem-loop region was found to be important for efficient complementation. The results of our studies demonstrate for the first time the importance of specific structural and sequence elements of the tRNA primer for HIV-1 reverse transcription and define new targets for interruption of HIV-1 replication.

The crystal structure of HIV reverse-transcription primer tRNA(Lys,3) shows a canonical anticodon loop.

We have solved to 3.3 A resolution the crystal structure of the HIV reverse-transcription primer tRNA(Lys,3). The overall structure is exactly comparable to the well-known L-shape structure first revealed by yeast tRNA(Phe). In particular, it unambiguously shows a canonical anticodon loop. This contradicts previous results in short RNA fragment studies and leads us to conclude that neither frameshifting specificities of tRNA(Lys) nor tRNA(Lys,3) primer selection by HIV are due to a specific three-dimensional anticodon structure. Comparison of our structure with the results of an NMR study on a hairpin representing a nonmodified anticodon stem-loop makes plausible the conclusion that chemical modifications of the wobble base U34 to 5-methoxycarbonyl-methyl-2-thiouridine and of A37 to 2-methylthio-N-6-threonylcarbamoyl-adenosine would be responsible for a canonical 7-nt anticodon-loop structure, whereas the unmodified form would result in a noncanonical UUU short triloop. The hexagonal crystal packing is remarkable and shows tight dimers of tRNAs forming a right-handed double superhelix. Within the dimers, the tRNAs are associated head-to-tail such that the CCA end of one tRNA interacts with the anticodon of the symmetry-related tRNA. This provides us with a partial view of a codon-anticodon interaction and gives insights into the positioning of residue 37, and of its posttranscriptional modifications, relative to the first base of the codon.

Evidence for the packaging of multiple copies of Tf1 mRNA into particles and the trans priming of reverse transcription.

Long terminal repeat (LTR)-containing retrotransposons and retroviruses are close relatives that possess similar mechanisms of reverse transcription. The particles of retroviruses package two copies of viral mRNA that both function as templates for the reverse transcription of the element. We studied the LTR-retrotransposon Tf1 of Schizosaccharomyces pombe to test whether multiple copies of transposon mRNA participate in the production of cDNA. Using the unique self-priming property of Tf1, we obtained evidence that multiple copies of Tf1 mRNA were packaged into virus-like particles. By coexpressing two distinct versions of Tf1, we found that the bulk of reverse transcription that was initiated on one mRNA template was subsequently transferred to others. In addition, the first 11 nucleotides of one mRNA were able to prime, in trans, the reverse transcription of another mRNA.

Quantitative detection of dengue 2 virus using fluorogenic RT-PCR based on 3′-noncoding sequence

A fluorescent DNA probe (DV2.P1) specific to the conserved distal 3′-noncoding region (nucleotides 10 653–10 678) of dengue 2 virus and a pair of flanking primers (DV2.L1 and DV2.U2) were designed to formulate a dengue 2-specific fluorogenic polymerase chain reaction (PCR). In addition, DV2.L1 was also used as a reverse transcription (RT) primer to generate superior cDNA from dengue viral RNA. Optimal assay conditions with zero background were established to detect low levels of dengue 2 virus from clinical specimens. The range of dengue virus detection in spiked human sera was determined to be from 10 to10 6 infectious virions per milliliter (plaque forming units determined using Vero cell line). Dengue 2 virus isolates from different geographic regions can be detected universally and identified by the fluorogenic RT-PCR assay. Moreover, the assay is specific for dengue 2 virus and does not recognize other related flaviviruses, including dengue serotypes 1, 3 and 4, Japanese encephalitis, St. Louis encephalitis, yellow fever, and Kunjin viruses. The assay also detected efficiently immunocomplexed dengue viruses. In practice, the fluorogenic RT-PCR assay detected readily viremia in sera collected from individuals ill with dengue fever. The rise and fall of dengue 2 virus concentrations in rhesus monkeys, reflecting viral proliferation and clearance, was also clearly illustrated by the assay.

Quantitative Estimation of Cross-Hybridization in DNA Microarrays Based on a Linear Model

Microarray expression data contains possible errors that could be classified into two types, one representing systematic errors and the other representing random errors. Systematic errors may occur due to different factors such as shapes of spots, purity of probes, sequences of reverse transcription (RT) primers, higher order structures of target nucleotides and probe-target cross-hybridization [1, 2]. In this study, we propose a linear model of systematic errors caused by probe-target cross-hybridization. We estimate the influences of such systematic errors so as to reconstruct more realistic gene regulatory networks and co-expression gene groups by the profiles [3].