Utilization of Nonviral Sequences for Minus-Strand DNA Transfer and Gene Reconstitution during Retroviral Replication

Cheslock, Sara Rasmussen; Anderson, Jeffrey A.; Hwang, Carey; Pathak, Vinay K.; Hu, Wei-Shau

doi:10.1128/jvi.74.20.9571-9579.2000

Cited by 23 publications

(26 citation statements)

References 44 publications

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“…This agrees with the observations of others that a minimum homology is required (20 -22). Moreover, the ability to reconstitute minus strand transfers by substituting the viral R sequence with nonviral sequences supports the argument that it is the homology rather than the specific sequence that is important for transfer (79). The reason why some retroviruses have evolved a shorter homologous region for ϪsssDNA transfer is not known, but the effects of differences in homology length may be compensated by a myriad of factors.…”

Section: Discussionmentioning

confidence: 68%

Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

Chen¹,

Balakrishnan²,

Roques

et al. 2003

Journal of Biological Chemistry

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Retrovirus minus strand strong stop transfer (minus strand transfer) requires reverse transcriptase-associated RNase H, R sequence homology, and viral nucleocapsid protein. The minus strand transfer mechanism in human immunodeficiency virus-1 was examined in vitro with purified protein and substrates. Blocking donor RNA 5 -end cleavage inhibited transfers when template homology was 19 nucleotides (nt) or less. Cleavage of the donor 5 -end occurred prior to formation of transfer products. This suggests that when template homology is short, transfer occurs through a primer terminus switch-initiated mechanism, which requires cleavage of the donor 5 terminus. On templates with 26-nt and longer homology, transfer occurred before cleavage of the donor 5 terminus. Transfer was unaffected when donor 5 -end cleavages were blocked but was reduced when internal cleavages within the donor were restricted. Based on the overall data, we conclude that in human immunodeficiency virus-1, which contains a 97-nt R sequence, minus strand transfer occurs through an acceptor invasion-initiated mechanism. Transfer is initiated at internal regions of the homologous R sequence without requiring cleavage at the donor 5 -end. The acceptor invades at gaps created by reverse transcriptase-RNase H in the donor-cDNA hybrid. The fragmented donor is eventually strand-displaced by the acceptor, completing the transfer.Reverse transcription, during which the single-stranded viral genomic RNA is converted into the double-stranded DNA, is an essential step in viral replication (see Ref. 1 and reference therein). This process is catalyzed by the virus-encoded enzyme reverse transcriptase (RT). 1 RT has two enzymatic activities, the DNA polymerase and the RNase H activities. Reverse transcription is initiated by the partial annealing between the cellular tRNA Lys3 primer and the viral primer binding site, which is located near the 5Ј-end of the viral RNA. Synthesis proceeds to the 5Ј-end of the genomic RNA, creating the minus strand strong stop DNA (ϪsssDNA), which comprises all of the U5 and R sequences from the 5Ј-untranslated region. During minus strand synthesis, RT simultaneously cleaves the copied RNA using its RNase H activity. This frees the ϪsssDNA, enabling its transfer to the homologous R sequence in the 3Ј-untranslated region and continuation of minus strand synthesis. This step is called minus strand strong stop transfer (minus strand transfer), and it is obligatory for reverse transcription and viral replication. Extensive studies have identified the RT-RNase H activity, R sequence homology, and viral nucleocapsid (NC) protein as important components for minus strand transfer (see Ref. 2 and references therein).Viruses with defective RNase H fail to complete reverse transcription, particularly the minus strand transfer step, indicating that RNase H is required for the transfer event (3-8). HIV-1 RT has been shown to partially cleave the template RNA during polymerization, leaving RNA fragments annealed to the cDNA (9, 10). Since there a...

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Section: Discussionmentioning

confidence: 68%

Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

Chen¹,

Balakrishnan²,

Roques

et al. 2003

Journal of Biological Chemistry

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“…MLV-based vector pSR2 (8) contains all the cis-acting elements essential for replication; pSR2 does not express viral proteins, but expresses the hygromycin phosphotransferase B gene (hygro), which confers resistance to hygromycin (22). pSR2 also has two modified LTRs, each of which contains a copy of the green fluorescent protein gene.…”

Section: Methodsmentioning

confidence: 99%

“…D17 cells are dog osteosarcoma cells permissive to amphotropic MLV infection (33); 293T cells are human embryonic kidney cells (12). Both cell lines were maintained at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with serum and antibiotics (8,12). Using a CalPhos transfection kit (ClonTech) or MBS transfection kit (Stratagene) with a mixture of DNA containing the gag-pol expression construct, pSR2, and pSV-a-MLV-env at a 2:2:1 weight ratio, DNA transfection was performed by the calcium phosphate precipitation method (34).…”

Section: Methodsmentioning

confidence: 99%

Charged Assembly Helix Motif in Murine Leukemia Virus Capsid: an Important Region for Virus Assembly and Particle Size Determination

Cheslock

Poon

et al. 2003

J Virol

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We have identified a region near the C terminus of capsid (CA) of murine leukemia virus (MLV) that contains many charged residues. This motif is conserved in various lengths in most MLV-like viruses. One exception is that spleen necrosis virus (SNV) does not contain a well-defined domain of charged residues. When 33 amino acids of the MLV motif were deleted to mimic SNV CA, the resulting mutant produced drastically reduced amounts of virions and the virions were noninfectious. Furthermore, these viruses had abnormal sizes, often contained punctate structures resembling those in the cell cytoplasm, and packaged both ribosomal and viral RNA. When 11 or 15 amino acids were deleted to modify the MLV CA to resemble those from other gammaretroviruses, the deletion mutants produced virions at levels comparable to those of the wild-type virus and were able to complete one round of virus replication without detectable defects. We generated 10 more mutants that displayed either the wild-type or mutant phenotype. The distribution of the wild-type or mutant phenotype did not directly correlate with the number of amino acids deleted, suggesting that the function of the motif is determined not simply by its length but also by its structure. Structural modeling of the wild-type and mutant proteins suggested that this region forms ␣-helices; thus, we termed this motif the "charged assembly helix." This is the first description of the charged assembly helix motif in MLV CA and demonstration of its role in virus budding and assembly.

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“…1. The parent plasmid was pMP-1, which has been described previously (11). This vector possesses all of the cis genomic sequences necessary for RNA packaging and reverse transcription.…”

Section: Resultsmentioning

confidence: 99%

“…The parent vector for all retroviral constructs is the M-MLV-based pMP-1 (11). New proviral constructs were made as follows.…”

Section: Materials and Reagentsmentioning

confidence: 99%

Pausing during Reverse Transcription Increases the Rate of Retroviral Recombination

Lanciault

Champoux

2006

J Virol

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Retroviruses package two copies of genomic RNA into viral particles. During the minus-sense DNA synthesis step of reverse transcription, the nascent DNA can transfer multiple times between the two copies of the genome, resulting in recombination. The mechanism for this process is similar to the process of obligate strand transfers mediated by the repeat and primer binding site sequences. The location at which the DNA 3 terminus completely transfers to the second RNA strand defines the point of crossover. Previous work in vitro demonstrated that reverse transcriptase pausing has a significant impact on the location of the crossover, with a proportion of complete transfer events occurring very close to pause sites. The role of pausing in vivo, however, is not clearly understood. By employing a murine leukemia virusbased single-cycle infection assay, strong pausing was shown to increase the probability of recombination, as reflected in the reconstitution of green fluorescent protein expression. The infection assay results were directly correlated with the presence of strong pause sites in reverse transcriptase primer extension assays in vitro. Conversely, when pausing was diminished in vitro, without changing the sequence of the RNA template involved in recombination, there was a significant reduction in recombination in vivo. Together, these data demonstrate that reverse transcriptase pausing, as observed in vitro, directly correlates with recombination during minus-sense DNA synthesis in vivo.Retroviruses exhibit a high frequency of homologous recombination (7,29,35,36,37,43,58,59,61,65). Human immunodeficiency virus type 1 (HIV-1) recombination occurs at a rate of approximately 2.4 ϫ 10 Ϫ4 /bp per replication cycle or higher while the direct repeat deletion rate for murine leukemia virus (MLV) was reported at 1.09 ϫ 10 Ϫ5 /bp per replication cycle (29,46,50). The rate of nonhomologous recombination is much lower but is still mediated by short homologous sequences (62, 64). During one round of reverse transcription, multiple crossover events often appear to occur at a significantly higher rate than mathematically anticipated, suggesting that a second crossover is not an independent event (negative interference) (2, 3). There are conflicting reports concerning this phenomenon in HIV-1; early work suggested the existence of high negative interference (29), while a more recent report obtained the expected distribution of multiple crossover events (51).Recombination of two different retrovirus strains relies on coinfection of a host cell by two genetically distinct viruses and copackaging of one genomic RNA from each integrated provirus into a single virion (26,27,55). The heterozygous virion then infects a new target cell where a chimeric recombinant provirus can be generated during reverse transcription. Recently, copackaging of heterozygous virions was demonstrated to be strongly affected by the identity of the dimerization initiation sequence. Genomes with different dimerization initiation sequences were packaged...

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Utilization of Nonviral Sequences for Minus-Strand DNA Transfer and Gene Reconstitution during Retroviral Replication

Cited by 23 publications

References 44 publications

Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

Mechanism of Minus Strand Strong Stop Transfer in HIV-1 Reverse Transcription

Charged Assembly Helix Motif in Murine Leukemia Virus Capsid: an Important Region for Virus Assembly and Particle Size Determination

Pausing during Reverse Transcription Increases the Rate of Retroviral Recombination

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