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Casey D Morrow - One of the best experts on this subject based on the ideXlab platform.

  • analysis of the replication of hiv 1 forced to use trnamet i supports a link between Primer selection translation and encapsidation
    Retrovirology, 2007
    Co-Authors: Uros V Djekic, Casey D Morrow
    Abstract:

    Background Previous studies have suggested that the process of HIV-1 tRNA Primer selection and encapsidation of genomic RNA might be coupled with viral translation. In order to further investigate this relationship, proviruses were constructed in which the Primer-Binding Site (PBS) was altered to be complementary to elongator tRNAMet (tRNAMet(e)) (HXB2-Met(e)) or initiator tRNAMet (tRNAMet(i)) (HXB2-Met(i)). These tRNAMet not only differ with respect to the 3' terminal 18-nucleotides, but also with respect to interaction with host cell proteins during protein synthesis.

  • murine leukemia virus with a Primer Binding Site complementary to trnalys 3 adapts to select new trnas for replication following extended in vitro culture
    Virology, 2005
    Co-Authors: Matthew T Palmer, Sylvia A Mcpherson, Casey D Morrow
    Abstract:

    The preference of MuLV for the selection of tRNAPro as a replication Primer was investigated by altering the Primer-Binding Site (PBS) to be complementary to tRNALys,3. MuLV-based vectors with a PBS complementary to tRNALys,3 were found to be approximately 2-fold less infectious than vectors with the wild-type PBS complementary to tRNAPro. MuLV with a PBS complementary to tRNALys,3 was replication competent and maintained the PBS during early stages of in vitro culture. Upon extended culture, PBS were isolated which were complementary to tRNAArg. A second MuLV was generated in which the region upstream of the PBS which is predicted to form an RNA stem loop structure was altered so that the nucleotide sequence within the loop would be complementary to the anticodon of tRNALys,3. The virus with both the U5 and PBS complementary to tRNALys,3 was also replication competent. Upon extended in vitro culture though, this virus reverted to utilize tRNALys1,2. Analysis of the infectivity and replication of the wild-type and mutant viruses revealed that tRNAPro was the preferred tRNA for high-level replication. Viruses with a PBS complementary to tRNAArg or tRNALy1,2 replicated at levels approximately 30% and 10% as effective as the wild-type virus, while virus with a PBS complementary to tRNALys,3 had the slowest replication kinetics and least infectivity. Comparison of the virion tRNA content of the wild-type and mutant viruses revealed similar ratios with respect to levels of tRNAPro, tRNAArg and tRNALys. Modeling of the U5-PBS region revealed that the predicted RNA structure for the virus that selected tRNAArg was more similar to the wild type virus that uses tRNAPro than the virus which use tRNALys1,2 or tRNALys,3; the virus that uses tRNALys,3 had the most profound disruption in the predicted RNA structure. The results of these studies demonstrate that MuLV has evolved to preferentially select tRNAPro for high-level replication and are discussed with respect to common features of the Primer selection process between MuLV and other retroviruses.

  • inhibition of human immunodeficiency virus type 1 replication by sirna targeted to the highly conserved Primer Binding Site
    Virology, 2004
    Co-Authors: Wenlong Han, Megan Windrotolo, Richard Kirkman, Casey D Morrow
    Abstract:

    The initiation of HIV-1 reverse transcription occurs at an 18-nucleotide sequence in the viral genome designated as the Primer Binding Site (PBS), which is complementary to the 3' terminal nucleotides of tRNA(Lys,3). Since the PBS is highly conserved among all infectious HIV-1, it represents an attractive target for the development of new therapeutics to inhibit viral replication. In this study, we have evaluated three approaches using small interfering RNA (siRNAs) targeted to the PBS for the capacity to inhibit HIV-1 replication. In the first, transfection of a 21-nucleotide siRNA complementary to the PBS into cells inhibited production of HIV-1 following infection. Control siRNAs of the same length complementary to HIV-1 gag mRNA or to gfp mRNA decreased the production of virus or had no effect on virus replication, respectively. Analysis of the PBS of integrated proviruses derived from viruses that ultimately grew in cultures transfected with siRNA all contained wild-type PBS sequence, demonstrating that HIV-1 did not mutate to escape inhibition by siRNA. In the second approach, hairpin siRNA targeted to the wild-type PBS were expressed using an adeno-associated virus (AAV) vector. HIV-1 replication was inhibited in cells infected with AAV encoding the siRNA to the wild-type PBS, but not in cells infected with AAV encoding an siRNA of the same length targeted to an irrelevant PBS. Finally, studies from this laboratory have shown that alteration of the PBS to be complementary to tRNAHis results in the production of infectious virus that rapidly reverts to utilize tRNALys,3 following in vitro culture. A proviral genome containing a PBS complementary to tRNAHis that encodes an siRNA molecule complementary to the wild-type PBS under control of a U6 promoter within the nef gene was as infectious as the parent HIV-1 genome containing no insert in nef. The virus with the PBS only complementary to tRNAHis reverted to use tRNALys,3, coincident with rapid virus growth, while the virus encoding siRNA grew slower than the virus without siRNA and maintained the PBS complementary to tRNAHis longer in culture. At later times of infection, viruses with the PBS complementary to tRNAHis and the siRNA exhibited a rapid increase in p24 antigen in the culture. Analysis of the PBS revealed that it was now complementary to tRNALys,3. Analysis of the gene encoding the siRNA revealed that the reversion of the PBS coincided with the deletion of the gene encoding siRNA. The results of these studies show that siRNA targeted to the PBS of HIV-1 can inhibit virus replication, supporting the concept that HIV-1 has evolved a strong preference to select tRNALys,3 for high-level replication and establishing the PBS and Primer selection as a potential target for new therapeutics.

  • genetic analysis of a unique human immunodeficiency virus type 1 hiv 1 with a Primer Binding Site complementary to trnamet supports a role for u5 pbs stem loop rna structures in initiation of hiv 1 reverse transcription
    Journal of Virology, 1999
    Co-Authors: Sangmoo Kang, Casey D Morrow
    Abstract:

    Human immunodeficiency virus type 1 (HIV-1) exclusively uses tRNA 3 Lys to initiate reverse transcription. A novel HIV-1 mutant which stably utilizes tRNA Met rather than tRNA 3 Lys as a Primer was previously identified [HXB2(Met-AC] (S.-M. Kang, Z. Zhang, and C. D. Morrow, J. Virol. 71:207–217, 1997). Comparison of RNA secondary structures of the unique sequence (U5)-Primer Binding Site (PBS) viral RNA genome alone or complexed with tRNA Met of HXB2(Met-AC) revealed structural motifs in common with the U5-PBS of the wild-type virus. In the current study, mutations were constructed to alter the U5-PBS structure and disrupt the U5-PBS-tRNA Met interaction of the virus derived from HXB2(Met-AC). All of the mutant viruses were infectious following transfection and coculture with SupT1 cells. Analysis of the initiation of reverse transcription revealed that some of the mutants were impaired compared to HXB2(Met-AC). The genetic stability of the PBS from each virus was determined following in vitro culture. Two mutant proviral constructs, one predicted to completely disrupt the stem-loop structure in U5 and the other predicted to destabilize contact regions of U5 with tRNA Met , reverted back to contain a PBS complementary to tRNA 3 Lys . All other mutants maintained a PBS complementary to tRNA Met after in vitro culture, although all contained multiple nucleotide substitutions within the U5-PBS from the starting proviral clones. Most interestingly, a viral mutant containing a 32-nucleotide deletion between nucleotides 142 and 173, encompassing regions in U5 which interact with tRNA Met , maintained a PBS complementary to tRNA Met following in vitro culture. All of the proviral clones recovered from this mutant, however, contained an additional 19-nucleotide insertion in U5. RNA modeling of the U5-PBS from this mutant demonstrated that the additional mutations present in U5 following culture restored RNA structures similar to those modeled from HXB2(Met-AC). These results provide strong genetic evidence that multiple sequence and structural elements in U5 in addition to the PBS are involved in the interaction with the tRNA used for initiation of reverse transcription.

  • complementarity between 3 terminal nucleotides of trna and Primer Binding Site is a major determinant for selection of the trna Primer used for initiation of hiv 1 reverse transcription
    Virology, 1999
    Co-Authors: Casey D Morrow
    Abstract:

    Abstract The initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) exclusively utilizes tRNA Lys,3 as a Primer. Previous studies have shown that HIV-1 could use alternative tRNAs, such as tRNA Ile or tRNA His , to initiate reverse transcription only if the Primer Binding Site (PBS) was made complementary to the 3′ terminal 18 nucleotides of the cognate tRNA. However, upon in vitro culture, the viruses with a PBS complementary to the alternative tRNAs rapidly reverted to generate a PBS complementary to tRNA Lys,3 . To investigate the process of reversion, we have constructed defective proviral genomes that contain a PBS complementary to tRNA Ile or tRNA His . The genomes contain the gene for xanthine-guanosine phosphoribosyl transferase ( gpt ) in place of env. Cotransfection of these proviral genomes with a plasmid-encoding vesicular stomatitis virus G protein (VSV-G) results in viruses that undergo a single round of HIV-1 infection; successful infections are scored as cells resistant to the drug mycophenolic acid. Using this single-round infection system, we demonstrated that HIV-1 with a PBS complementary to tRNA Ile or tRNA His is three- to fivefold less efficient in replication as measured by production of drug-resistant cell colonies compared to the wild-type virus. These viruses predominantly used the cognate tRNA as Primer in their initial round of replication, although we did obtain a single cell colony in which the PBS was complementary to tRNA Lys,3 . Using an HIV-1 provirus with a PBS complementary to yeast tRNA Phe , we established a single-round infection system in which the infectivity of this mutant HIV-1 relies on transfected yeast tRNA Phe . The results of our studies suggest that the mechanism for selection of the tRNA Primer for initiation of reverse transcription relies primarily on the complementarity between the tRNA Primerthe PBS.

Finn Skou Pedersen - One of the best experts on this subject based on the ideXlab platform.

  • transfer of Primer Binding Site mutated simian immunodeficiency virus vectors by genetically engineered artificial and hybrid trna like Primers
    Journal of Virology, 2001
    Co-Authors: Anette Chemnitz Hansen, Anders H Lund, Mogens Duch, Thomas Grunwald, Alexander Schmitz, Klaus Uberla, Finn Skou Pedersen
    Abstract:

    Simian immunodeficiency viruses (SIV) harbor Primer Binding Sites (PBS) matching tRNA or tRNA. To study determinants of Primer usage in SIV, a SIVmac239-based vector was impaired by mutating the PBS to a sequence (PBS-X2) with no match to any tRNA. By cotransfection of a synthetic gene encoding a tRNAPro-like RNA with a match to PBS-X2, the activity of this vector could be restored to a transduction efficiency slightly lower than that of the wild-type vector. A vector with a PBS matching tRNAPro was functional at a level slightly below that of the wild-type vector, but higher transduction efficiency could be obtained by cotransfection of a gene for an engineered tRNAPro-tRNA hybrid with a match to PBS-Pro. The importance of tRNA backbone identity was further analyzed by complementing the PBS-X2 vector with a gene for a matching x2 Primer with a tRNA backbone, which led to three- to fourfold-higher titers than those observed for the x2 Primer with the tRNAPro backbone. In summary, our results demonstrate flexibility in PBS and Primer usage for SIVmac239, with PBS-Primer complementarity being the major determinant, in analogy with previous findings for murine leukemia viruses and human immunodeficiency virus type 1.

  • lack of shielding of Primer Binding Site silencer mediated repression of an internal promoter in a retrovirus vector by the putative insulators scs bead 1 and hs4
    Journal of Virology, 2000
    Co-Authors: Charlotte Modin, Finn Skou Pedersen, Mogens Duch
    Abstract:

    A major determinant for transcriptional incompetence of murine leukemia virus (MLV) and MLV-derived vectors in embryonal cells is located at the proline Primer Binding Site (PBS). The mechanism of silencing is unknown, yet the effect is capable of spreading to adjacent promoters. Based on a retroviral vector containing an internal promoter and the escape mutant B2 PBS with expressional capacity in embryonal cells, we have developed an assay to test the ability of putative insulators to shield the silencer at the PBS. Since the B2 PBS reverts to the wild-type PBS at high frequency, a shielding ability of a putative insulator can be assessed from the ratio of expressing B2 PBS to proline PBS proviruses in the target embryonal carcinoma cell population as measured by Primer extension. Our results show that none of the possible insulators, scs, BEAD-1, or HS4, is able to shield an internal promoter from the repressive effect of the silencer at the PBS region when inserted between the silencer and the promoter.

  • replication and pathogenicity of Primer Binding Site mutants of sl3 3 murine leukemia viruses
    Journal of Virology, 1999
    Co-Authors: Anders H Lund, Jorg Schmidt, Arne Luz, Annette Balle Sorensen, Mogens Duch, Finn Skou Pedersen
    Abstract:

    Retroviral reverse transcription is primed by a cellular tRNA molecule annealed to an 18-bp Primer Binding Site sequence. The sequence of the Primer Binding Site coincides with that of a negatively acting cis element that mediates transcriptional silencing of murine leukemia virus (MLV) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA Primers other than the cognate tRNAPro and analyze the effect of altering the Primer Binding Site sequence to match the 3′ end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary to findings from cell culture studies of Primer Binding Site-modified human immunodeficiency virus type 1 and avian retroviruses, our findings were that SL3-3 MLV may stably and efficiently replicate with tRNA Primers other than tRNAPro. Although lymphoma induction of the SL3-3 Lys3 mutant was significantly delayed relative to that of the wild-type virus, molecular tumor analysis indicated that all the Primer Binding Site-modified viruses induce T-cell lymphomas similar to those induced by the wild-type virus in terms of frequencies of genomic rearrangements within the T-cell receptor β-chain, the immunoglobulin κ light chain, and the c-myc locus. Whereas none of the mutants were found to revert to tRNAPro Primer utilization, in two tumors resulting from the injection of the SL3-3 Lys3 mutant the Primer Binding Site was altered to match that of a new Primer species, tRNA1,2Lys. In addition, recombination with endogenous viruses resulting in the generation of recombinant viruses carrying a glutamine Primer Binding Site was detected in the majority of the tumors induced by the SL3-3 Lys3 mutant as well as in two tumors induced by wild-type SL3-3 and the SL3-3 Arg1,2 mutant.

  • extended minus strand dna as template for r u5 mediated second strand transfer in recombinational rescue of Primer Binding Site modified retroviral vectors
    Journal of Virology, 1998
    Co-Authors: Jacob Giehm Mikkelsen, Anders H Lund, Mogens Duch, Karen Dybkaer, Finn Skou Pedersen
    Abstract:

    We have previously demonstrated recombinational rescue of Primer Binding Site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439–1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed.

  • complementation of a Primer Binding Site impaired murine leukemia virus derived retroviral vector by a genetically engineered trna like Primer
    Journal of Virology, 1997
    Co-Authors: Anders H Lund, M Duch, Jette Lovmand, Poul Jorgensen, Finn Skou Pedersen
    Abstract:

    Reverse transcription of retroviral genomes is primed by a tRNA annealed to an 18-nucleotide Primer Binding Site. Here, we present a Primer complementation system to study molecular interaction of the replication machinery with the Primer and Primer Binding Site in vivo. Introduction of eight base substitutions into the Primer Binding Site of a murine leukemia virus-based vector allowed efficient RNA encapsidation but resulted in severely reduced vector replication capacity. Replication was restored upon complementation with a synthetic gene designed to encode a complementary tRNA-like Primer, but not with a noncomplementary tRNA-like molecule. The engineered Primer was shown to be involved in both the initiation of first-strand synthesis and second-strand transfer. These results provide an in vivo demonstration that the retroviral replication machinery may recognize sequence complementarity rather than actual Primer Binding Site and 3' Primer sequences. Use of mutated Primer Binding Site vectors replicating via engineered Primers may add additional control features to retroviral gene transfer technology.

Mark A. Wainberg - One of the best experts on this subject based on the ideXlab platform.

  • leader sequences downstream of the Primer Binding Site are important for efficient replication of simian immunodeficiency virus
    Journal of Virology, 2000
    Co-Authors: Yongjun Guan, Mark A. Wainberg, James B Whitney, Karidia Diallo
    Abstract:

    Simian immunodeficiency virus (SIV) infection of macaques is remarkably similar to that of human immunodeficiency virus type 1 (HIV-1) in humans, and the SIV-macaque system is a good model for AIDS research. We have constructed an SIV proviral DNA clone that is deleted of 97 nucleotides (nt), i.e., construct SD, at positions (+322 to +418) immediately downstream of the Primer Binding Site (PBS) of SIVmac239. When this construct was transfected into COS-7 cells, the resultant viral progeny were severely impaired with regard to their ability to replicate in C8166 cells. Further deletion analysis showed that a virus termed SD1, containing a deletion of 23 nt (+322 to +344), was able to replicate with wild-type kinetics, while viruses containing deletions of 21 nt (+398 to +418) (construct SD2) or 53 nt (+345 to +397) (construct SD3) displayed diminished capacity in this regard. Both the SD2 and SD3 viruses were also impaired with regard to ability to package viral RNA, while SD1 viruses were not. The SD and SD3 constructs did not revert to increased replication ability in C8166 cells over 6 months in culture. In contrast, long-term passage of the SD2 mutated virus resulted in a restoration of replication capacity, due to the appearance of four separate point mutations. Two of these substitutions were located in leader sequences of viral RNA within the PBS and the dimerization initiation Site (DIS), while the other two were located within two distinct Gag proteins, i.e., CA and p6. The biological relevance of three of these point mutations was confirmed by Site-directed mutagenesis studies that showed that SD2 viruses containing each of these substitutions had regained a significant degree of viral replication capacity. Thus, leader sequences downstream of the PBS, especially the U5-leader stem and the DIS stem-loop, are important for SIV replication and for packaging of the viral genome.

  • 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
    Journal of Virology, 1997
    Co-Authors: Chen Liang, Liwei Rong, Elana Cherry, Lawrence Kleiman, Yue Huang, N. Morin, Mark A. Wainberg
    Abstract:

    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.

  • identification of sequences downstream of the Primer Binding Site that are important for efficient replication of human immunodeficiency virus type 1
    Journal of Virology, 1997
    Co-Authors: Chen Liang, Lawrence Kleiman, Michael A Parniak, Yudong Quan, R Chandok, Michael Laughrea, Mark A. Wainberg
    Abstract:

    Reverse transcription of retroviruses is initiated from an 18-nucleotide (nt) Primer Binding Site (PBS), located within the 5' region of viral genomic RNA, to which the host cell-derived tRNA Primer is annealed and also involves viral genomic sequences outside the PBS. We constructed proviral DNA clones of human immunodeficiency virus (HIV) that had selective deletions of either a 7-nt segment found immediately downstream of the PBS or an extended nontranslated 54-nt stretch located immediately downstream of the PBS and containing the aforementioned 7-nt segment. Synthesis of minus-strand strong-stop DNA was assessed with MT-4 cells infected with viruses derived from COS-7 cells that had been transfected with these various constructs. We found that similar levels of minus-strand strong-stop DNA as well as DNA produced after template switching were expressed in MT-4 cells infected with COS-7-derived wild-type viruses or with viruses that had the 7-nt segment deleted. In contrast, significantly lower levels of viral DNA were detected in MT-4 cells after infection with viruses that had deletions of the 54-nt stretch. Furthermore, the molecular clone containing the 7-nt deletion was able to replicate with wild-type kinetics, while that containing the 54-nt deletion displayed a significantly diminished capacity in this regard. Further deletion analysis showed that a 16-nt segment at the 3' end of this 54-nt segment was largely responsible for these effects. We also conducted studies to determine levels of viral mRNA in COS-7 cells that had been transfected with equivalent amounts of DNA derived from either a wild-type HIV construct or our various deletion mutants. In the case of transfections performed with the 7-nt deletion mutant and wild-type HIV DNA, high levels of viral mRNA transcripts were detected, which was not the case for the 54 nt-deletion mutant. However, these various mRNAs possessed similar stabilities, as shown through studies in which transcript formation was arrested by treatment of cells with actinomycin D. Thus, the 54-nt segment of 5' nontranslated RNA, located downstream of the PBS, is involved in efficient expression of each of viral DNA, mRNA, and infectious virus.

  • Primer trna3lys on the viral genome exists in unextended and two base extended forms within mature human immunodeficiency virus type 1
    Journal of Virology, 1997
    Co-Authors: Yue Huang, Mark A. Wainberg, J Wang, A Shalom, Ahmad Khorchid, Lawrence Kleiman
    Abstract:

    In human immunodeficiency virus type 1, tRNALys3 is placed upon the Primer Binding Site, where it serves as the Primer for reverse transcription. We show herein that genomic placement occurs independently of precursor protein processing and that the placed tRNALys3 exists in two major forms: unextended or in a form extended by the first two DNA bases incorporated, C and T. The two-base extended form of the tRNALys3 is absent in a protease-negative mutant, indicating a requirement for mature viral proteins to initiate reverse transcription within the virus.

  • 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
    Journal of Virology, 1996
    Co-Authors: Yudong Quan, Lawrence Kleiman, J L Darlix, Eric J Arts, Michael A Parniak, B D Preston, H De Rocquigny, B P Roques, Mark A. Wainberg
    Abstract:

    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.

Virendra N Pandey - One of the best experts on this subject based on the ideXlab platform.

  • destabilization of trna3lys from the Primer Binding Site of hiv 1 genome by anti a loop polyamide nucleotide analog
    Nucleic Acids Research, 2001
    Co-Authors: Neerja Kaushik, Tanaji T Talele, Raymond Monel, Paul Palumbo, Virendra N Pandey
    Abstract:

    Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by extension of the cellular tRNA 3 L y s which anneals to the Primer-Binding Site (PBS) on the 5' non-translated region of the viral RNA genome. The A-rich sequence (A-loop) upstream of the PBS interacts with the anticodon loop of tRNA 3 L y s and has been proposed to be essential for conferring specificity to tRNA 3 L y s for priming the initiation of HIV-1 reverse transcription. We observed that polyamide nucleic acid targeted to the A-loop sequence (PNAAL) exhibits high Binding specificity for its target sequence. The PNAAL pre-bound to the A-loop sequence prevents tRNA 3 L y s priming on the viral RNA consequently blocking in vitro initiation of reverse transcription. Further, PNAAL can efficiently disrupt the preformed [tRNA 3 L y s -viral RNA] complex thereby rendering it non-functional for reverse transcription. The endogenous reverse transcription in disrupted HIV-1 virions containing packaged tRNA 3 L y s and its replicating enzyme RT was significantly inhibited by PNAAL, thus providing direct evidence of the involvement of the A-loop region of viral RNA genome in tRNA 3 L y s priming process. These findings suggest the potential of the A-loop region as a critical target for blocking HIV-1 replication.

  • polyamide nucleic acid targeted to the Primer Binding Site of the hiv 1 rna genome blocks in vitro hiv 1 reverse transcription
    Biochemistry, 1998
    Co-Authors: Reaching Lee, Neerja Kaushik, Mukund J Modak, Ravi Vinayak, Virendra N Pandey
    Abstract:

    ABSTRACT : We report here that polyamide nucleic acid (PNA) as well as a polyamide nucleic acid-DNAchimera complementary to the Primer Binding Site of the HIV-1 genome can completely block primingby tRNA 3Lys and consequently the in Vitro initiation of reverse transcription by HIV-1 RT. Conventionalheating and cooling is not required for annealing PNA analogs to the complementary nucleotide sequenceas effective blockage of reverse transcription results from their invasion in the duplex region of preprimedU5-PBS HIV-1 RNA template-Primer and was seen even at ambient temperature. Further, the extensionof the initiated nascent (-) strand DNA can also be blocked by inclusion of another PNA, targeted toupstream sequences in the U5 region of the viral RNA. Interestingly, a PNA chimera having only twoDNA nucleotides annealed with the U5-PBS RNA is recognized as a bonafide Primer by HIV-1 RT, asthe 3′OH end of the chimeric molecule is extended by the enzyme in the presence of dNTPs. A significantobservation was that RNA/PNA or RNA/(PNA-DNA) hybrids were entirely resistant to the RNase Hactivity of HIV-1 RT. Furthermore, PNA invasion into the RNA/DNA hybrid completely prevented thecleavage of the RNA strand, suggesting that the RNase H activity of HIV-1 RT which was required inreverse transcription may also be inhibited by the PNA oligomer. These observations suggest thatoligomeric PNAs targeted to various critical regions of the viral genome are likely to have strong therapeuticpotential for interrupting multiple steps involved in the replication of HIV-1 and warrant seriousinvestigation especially in the area of an effective delivery system.One of the major currently utilized therapies for AIDS isthe use of inhibitors which block reverse transcription of viralRNA into double-stranded DNA. Several nucleoside analogsand non-nucleoside inhibitors exhibiting inhibitory activityon the viral reverse transcriptase have been deployed aschemotherapeutic agents (1). Recently, protease inhibitorshave been developed which appear to block the process ofviral maturation (2, 3). However, the rapid emergence ofmutant viral strains resistant to these inhibitors continues topose a constant threat to containing this disease. Anotherpotentially important approach is the use of antisense nucleicacid complementary to the target sequence of HIV-1 genomicRNA to block both mRNA translation and viral replication(4). Although this approach is very promising, the uptakeand the instability of the antisense oligonucleotides in thecells are problems yet to be overcome (5). We haveemployed a new class of antisense nucleic acid analog,polyamide nucleic acid (PNA),

  • polyamide nucleic acid targeted to the Primer Binding Site of the hiv 1 rna genome blocks in vitro hiv 1 reverse transcription
    Biochemistry, 1998
    Co-Authors: Reaching Lee, Neerja Kaushik, Mukund J Modak, Ravi Vinayak, Virendra N Pandey
    Abstract:

    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.

  • photoaffinity labeling of dna template Primer Binding Site in escherichia coli dna polymerase i identification of involved amino acids
    Journal of Biological Chemistry, 1994
    Co-Authors: Virendra N Pandey, Neerja Kaushik, Mukund J Modak
    Abstract:

    We have used two self-annealing template-Primers (TPs) to covalently cross-link the Klenow fragment of Escherichia coli DNA polymerase I in its polymerase mode. The specificity of cross-linking is demonstrated by the observation that other template-Primers, but not the template or Primer alone, readily compete with self-annealing TPs. The enzyme-TP covalent complex is catalytically active and can incorporate one nucleotide on the Primer terminus of the immobilized template-Primer. Using a peptide mapping approach, we have identified a 17-amino acid tryptic peptide spanning residues 759-775 as a major constituent of the TP Binding domain. Amino acid sequence analysis further revealed that Ile-765, Tyr-766 in the O-helix and Ser-769, Phe-771 in the O1-helix of the three-dimensional crystal structure of the Klenow fragment constitute the attachment Site for TP.

John K Wakefield - One of the best experts on this subject based on the ideXlab platform.

  • nucleotide substitutions within u5 are critical for efficient reverse transcription of human immunodeficiency virus type 1 with a Primer Binding Site complementary to trna his
    Journal of Virology, 1997
    Co-Authors: Zhijun Zhang, John K Wakefield, Sangmoo Kang, Casey D Morrow
    Abstract:

    Sequence analysis of integrated proviruses of human immunodeficiency virus type 1 (HIV-1) which utilize tRNA(His) to initiate reverse transcription [virus derived from pHXB2(His-AC-TGT)] revealed five additional nucleotide substitutions in the U5 and Primer Binding Site (PBS) regions (ATGAC for CCTGT at nucleotides 152, 160, 174, 181, and 200, respectively) (Z. Zhang et al., Virology 226:306-317, 1996). We constructed a mutant proviral genome [pHXB2(His-AC-GAC)] which contained the ATGAC substitutions to test if they represented a necessary adaptation by the virus for use of tRNA(His) to initiate reverse transcription. Viruses from pHXB2(His-AC-TGT) and pHXB2(His-AC-GAC) were infectious. Sequence analysis of the U5 and PBS regions of integrated provirus from a cell culture infected with virus derived from pHXB2(His-AC-TGT) revealed a G-to-A change in CCTGT at nucleotide 181 after limited in vitro culture, suggesting that this nucleotide change represented an adaptation by the virus to efficiently utilize tRNA(His) to initiate reverse transcription. To further address this possibility, we used a specific mutation in reverse transcriptase (RT), a methionine-to-valine change in the highly conserved YMDD amino acid motif of HIV-1 RT (M184V), which has been shown in previous studies to influence the fidelity and activity of the enzyme. The M184V RT mutation was cloned into pHXB2(His-AC-GAC) and pHXB2(His-AC-TGT). Virus derived from pHXB2(His-AC-GAC) with M184V RT had slightly delayed replication compared to the virus from pHXB2(His-AC-GAC) with wild-type RT; in contrast, virus from pHXB2(His-AC-TGT) with M184V RT was severely compromised in replication. Using an endogenous reverse transcription-PCR assay to analyze the reverse transcription of viruses obtained after transfection, we found that viruses derived from pHXB2(His-AC-GAC) with the wildtype RT were slightly faster in the initiation of reverse transcription than viruses with M184V RT. The initiation of reverse transcription was delayed in viruses derived from pHXB2(His-AC-TGT) with wild-type RT and M184V RT compared to viruses derived from pHXB2(His-AC-GAC). Finally, sequence analysis of U5 and PBS regions of proviruses from pHXB2(His-AC-GAC) with wild-type RT revealed considerably more nucleotide substitutions than in viruses derived from pHXB2(His-AC-GAC) containing the M184V mutation in RT after extended in vitro culture. Our studies point to a role for these additional nucleotide substitutions in U5 as an adaptation by the virus to utilize an alternative tRNA to initiate reverse transcription.

  • 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
    Virology, 1996
    Co-Authors: Sangmoo Kang, John K Wakefield, Casey D Morrow
    Abstract:

    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.

  • mutations within the Primer Binding Site of the human immunodeficiency virus type 1 define sequence requirements essential for reverse transcription
    Virology, 1996
    Co-Authors: John K Wakefield, Casey D Morrow
    Abstract:

    Abstract The Primer Binding Site (PBS) is involved in two stages during the reverse transcription of the retroviral RNA genome. In the early stage, the PBS provides complementary sequences through which tRNA Lys,3 binds the viral RNA genome to initiate minus-strand DNA synthesis; in the later stages, complementarity between the plus- and minus-strand copies of the PBS is required to facilitate the second template transfer needed to complete reverse transcription. We previously constructed a mutant HIV-1 proviral genome, designated as pHXB2PBS(pheC + 5) (now referred to as pheC + 5), which was used to identify regions of the PBS involved in the initiation and second template transfer steps of reverse transcription. To further define the sequence requirements of the PBS for the initiation of reverse transcription, we have made single nucleotide substitutions within the first six nucleotides of the pheC + 5 PBS. Our results demonstrate that mutations within the first five nucleotides of the PBS which disrupt base paring with tRNA Lys,3 -PBS results in an noninfectious virus; a G-U base pair at position six of the tRNA Lys,3 -PBS complex was tolerated. In contrast to the requirements for initiation, we found that complementary Binding between only three base pairs of the plus- and minus-strand PBSs was required for the extension of plus-strand DNA during the second template transfer. Furthermore, regions of the minus-strand DNA of up to 24 nucleotides could be looped-out to facilitate the complementarity required for the completion of plus-strand DNA synthesis. Taken together, the results of our studies demonstrate that different features of the PBS with respect to RNA:RNA and DNA:DNA interactions are required for initiation of reverse transcription and the completion of plus-strand DNA synthesis, respectively.

  • construction of a type 1 human immunodeficiency virus that maintains a Primer Binding Site complementary to trna his
    Journal of Virology, 1996
    Co-Authors: John K Wakefield, Sangmoo Kang, Casey D Morrow
    Abstract:

    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.

  • human immunodeficiency virus type 1 can use different trnas as Primers for reverse transcription but selectively maintains a Primer Binding Site complementary to trna 3lys
    Journal of Virology, 1995
    Co-Authors: John K Wakefield, A G Wolf, Casey D Morrow
    Abstract:

    The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at a Site in the viral RNA genome which is designated the Primer-Binding Site (PBS). The HIV-1 PBS is an 18-nucleotide sequence that is complementary to the 3'-terminal 18 nucleotides of tRNA(3Lys), which is used as the Primer for reverse transcription. All HIV-1 isolates sequenced to date contain a PBS complementary to tRNA(3Lys), suggesting that other cellular tRNAs might not function as Primers for reverse transcription. To investigate this possibility, we have substituted the HIV-1 PBS with sequences predicted to be complementary to the 3'-terminal nucleotides of tRNA(1,2Lys), tRNA(Ile), and tRNA(His), which previous studies have identified to be packaged into HIV-1 virions along with tRNA(3Lys). We demonstrate that infectious viruses which utilized tRNA(1,2Lys), tRNA(Ile), and tRNA(His) in reverse transcription can be recovered. However, the appearances of viruses with PBSs complementary to these alternate tRNAs were delayed compared with the wild type. After extended in vitro culture, viruses containing the PBSs complementary to these different tRNAs reverted back to the wild-type PBS complementary to tRNA3(Lys). Furthermore, only the first 9 nucleotides of the 18 nucleotide PBSs were sufficient for HIV-1 to utilize the alternate tRNA Primers in reverse transcription, demonstrating that HIV-1 does not require the complete 18-nucleotide PBS to utilize these tRNA Primers for reverse transcription. These results suggest that factors other than complementarity between the PBS and the Primer tRNA contribute to the selectivity of tRNA3(Lys) to initiate HIV-1 reverse transcription.