Further characterization of the gapped DNA intermediates of human spumavirus: evidence for a dual initiation of plus-strand DNA synthesis

Tobaly-Tapiero, Joelle; Kupiec, Jean-Jacques; Santillana-Hayat, Maud; Canivet, M; Périès, J.; Emanoil-Ravier, Rodica

doi:10.1099/0022-1317-72-3-605

Cited by 46 publications

(38 citation statements)

References 15 publications

(14 reference statements)

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“…Another explanation is that Pol binds to the upstream PES, followed by minus strong stop cDNA synthesis, while the downstream PES is involved in second-strand cDNA synthesis. This would be in accordance with the presence of the second polypurine tract (PPT) within CASII (14,19,36). The role of this internal PPT has yet to be experimentally addressed.…”

Section: Protein Requirements For the Encapsidation Of Pol Proteinmentioning

confidence: 61%

RNA and Protein Requirements for Incorporation of the Pol Protein into Foamy Virus Particles

Peters

Wiktorowicz

Heinkelein

et al. 2005

J Virol

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Foamy viruses (FVs) generate their Pol protein precursor molecule independently of the Gag protein from a spliced mRNA. This mode of expression raises the question of the mechanism of Pol protein incorporation into the viral particle (capsid). We previously showed that the packaging of (pre)genomic RNA is essential for Pol encapsidation (M. Heinkelein, C. Leurs, M. Rammling, K. Peters, H. Hanenberg, and A. Rethwilm, J. Virol. 76: [10069][10070][10071][10072][10073] 2002). Here, we demonstrate that distinct sequences in the RNA, which we termed Pol encapsidation sequences (PES), are required to incorporate Pol protein into the FV capsid. Two PES were found, which are contained in the previously identified cis-acting sequences necessary to transfer an FV vector. One PES is located in the U5 region of the 5 long terminal repeat and one at the 3 end of the pol gene region. Neither element has any significant effect on RNA packaging. However, deletion of either PES resulted in a significant reduction in Pol encapsidation. On the protein level, we show that only the Pol precursor, but not the individual reverse transcriptase (RT) and integrase (IN) subunits, is incorporated into FV particles. However, enzymatic activities of the protease (PR), RT, or IN are not required. Our results strengthen the view that in FVs, (pre)genomic RNA functions as a bridging molecule between Gag and Pol precursor proteins.Spumaretroviruses, or foamy viruses (FVs), are one of two subfamilies of retroviruses (30). A hallmark of the replication strategy of FVs is the Gag-independent expression of a Pol precursor protein from a spliced RNA (3,6,18,25,40). The consequences of this highly unusual strategy for the generation of a retroviral Pol protein are poorly understood. It has been shown that FV reverse transcriptase (RT) is much more processive than that of orthoretroviruses (31). Furthermore, it has been suggested that a very few molecules of Pol precursor are packaged into the FV capsid (4, 31). The FV means of expressing and encapsidating Pol protein has to be viewed in the context of reverse transcription, which appears to occur to a large extent in the virus-producing cell prior to budding (26,32,41). This leads to linear DNA, which is probably the virion nucleic acid and more relevant for infection than RNA (30).The actual mechanism of FV Pol protein particle incorporation despite lack of a Gag-Pol precursor has remained obscure. It is evident that FVs must have found a means of Pol encapsidation that acknowledges that a Gag-Pol precursor protein, which in orthoretroviruses facilitates Pol incorporation into the viral particle, is not generated (35). We previously showed that the incorporation of (pre)genomic RNA is essential for Pol protein encapsidation (12). Here, we took the experiments a step further and asked whether specific sequences on the RNA which allow Pol protein incorporation can be identified and whether such sequences are different from signals needed to package RNA. Furthermore, we wanted to know what requirements e...

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Section: Protein Requirements For the Encapsidation Of Pol Proteinmentioning

confidence: 61%

RNA and Protein Requirements for Incorporation of the Pol Protein into Foamy Virus Particles

Peters

Wiktorowicz

Heinkelein

et al. 2005

J Virol

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“…Central polypurine tracts (cPPT) have mainly been described for lentiviruses, including visna virus, feline immunodeficiency virus, bovine immunodeficiency virus, equine infectious anemia virus, simian immunodeficiency virus (SIV), human immunodeficiency virus type 1 (HIV-1), and HIV-2, but they have also been found in spumaretroviruses and in the yeast retrotransposons Ty1 and Ty3 (3,12,29,32,33). Due to the presence of the cPPT, the plus strands of these viruses are synthesized as two distinct segments separated by a unique discontinuity (6,7,10,15,16,18,31,33). The downstream segment, initiated at the cPPT, covers the 3Ј half of the genome, including the whole 3Ј LTR.…”

mentioning

confidence: 99%

Functional Central Polypurine Tract Provides Downstream Protection of the Human Immunodeficiency Virus Type 1 Genome from Editing by APOBEC3G and APOBEC3B

Wurtzer

Goubard

Mammano

et al. 2006

J Virol

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Lentiviruses utilize two polypurine tracts for initiation of plus-strand viral DNA synthesis. We have examined to what extent human immunodeficiency virus type 1 plus-strand initiation at the central polypurine tract (cPPT) could protect the viral genome from DNA editing by APOBEC3G and APOBEC3B. The presence of a functional cPPT, but not of a mutated cPPT, extensively reduced editing by both APOBEC3G and APOBEC3B of sequences downstream, but not upstream, of the cPPT, with significant protection observed as far as 400 bp downstream. Thus, in addition to other potential functions, the cPPT could help protect lentiviruses from editing by cytidine deaminases of the APOBEC family.Retroviral replication requires reverse transcription of particle-associated genomic viral RNA into double-stranded DNA. Reverse transcription follows sequential steps, during which discrete segments of DNA are synthesized by reverse transcriptase, with each of these segments being initiated at a precise site by a specific primer (30). Synthesis of the first (minus) strand of DNA uses genomic viral RNA as a template and is initiated by a tRNA primer that defines the 3Ј boundary of the U5 region of the long terminal repeat (LTR). Synthesis of the second (plus) strand of DNA uses minus-strand DNA as a template and is initiated by a short RNA primer corresponding to a purine-rich sequence, the polypurine tract (PPT) (24,27). While all retroviruses carry a PPT near the 3Ј end of the retroviral genome, defining the 5Ј boundary of the U3 region of the LTR, several retroviruses, including lentiviruses, carry a second PPT near the center of the genome. Central polypurine tracts (cPPT) have mainly been described for lentiviruses, including visna virus, feline immunodeficiency virus, bovine immunodeficiency virus, equine infectious anemia virus, simian immunodeficiency virus

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“…The products were separated in a 20% sequencing gel. 4,6,7,9,10,12,13,15, and 16) to allow cleavage or RNase H Ϫ RT as a control (lanes 2, 5, 8, 11, and 14), either ddCTP-labeling mix (see "Experimental Procedures"), lanes 2-10, or ddTTP-labeling mix, lanes 11-16, was added. Following a 30-min extension reaction, stalled products were chased by the addition of excess cold dTTP (lanes 2-10) or dCTP (lanes 11-16).…”

Section: Resultsmentioning

confidence: 99%

“…Accurate plus strand initiation from the PPT is required to generate an integrationcompetent final DNA product. The finding that some retroviruses possess a second copy of the PPT (cPPT) near the middle of the genome that efficiently primes synthesis, as well as evidence that plus strands are discontinuous in a number of retroviral systems, indicates that priming from regions upstream of the PPT in addition to PPT priming may not be detrimental to the virus (12)(13)(14)(15)(16)(17)(18)(19). By contrast, current models predict that plus strands initiated from primer RNAs downstream of the PPT will generate dead-end products (6).…”

mentioning

confidence: 99%

RNA Degradation and Primer Selection by Moloney Murine Leukemia Virus Reverse Transcriptase Contribute to the Accuracy of Plus Strand Initiation

Kelleher

Champoux

2000

Journal of Biological Chemistry

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During reverse transcription, plus strand DNA synthesis is initiated at a purine-rich RNA primer generated by the RNase H activity of reverse transcriptase (RT). Specific initiation of plus strand synthesis from this polypurine tract (PPT) RNA is essential for the subsequent integration of the linear viral DNA product. Based on current models, it is predicted that priming from sites upstream of the PPT may be tolerated by the virus, whereas efficient extension from RNA primers located downstream from the PPT is predicted to generate dead-end products. By using hybrid duplex substrates derived from the Moloney murine leukemia virus long terminal repeat, we investigated the extent to which RNase H degrades the viral RNA during time course cleavage assays, and we tested the capacity of the polymerase activity of RT to use the resulting cleavage products as primers. We find that the majority of the RNA fragments generated by RNase H are 2-25 nucleotides in length, and only following extensive degradation are most fragments reduced to 10 nucleotides or smaller. Although extensive RNA degradation by RNase H likely eliminates many potential RNA primers, based on thermostability predictions it appears that some RNA fragments remain stably annealed to the DNA template. RNA primers generated by RNase H within the long terminal repeat sequence are found to have the capacity to initiate DNA synthesis by RT; however, the priming efficiency is significantly less than that observed with the PPT primer. We find that Moloney murine leukemia virus nucleocapsid protein reduces RNase H degradation and slightly alters the cleavage specificity of RT; however, nucleocapsid protein does not appear to enhance PPT primer utilization or suppress extension from non-PPT RNA primers.

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Further characterization of the gapped DNA intermediates of human spumavirus: evidence for a dual initiation of plus-strand DNA synthesis

Cited by 46 publications

References 15 publications

RNA and Protein Requirements for Incorporation of the Pol Protein into Foamy Virus Particles

RNA and Protein Requirements for Incorporation of the Pol Protein into Foamy Virus Particles

Functional Central Polypurine Tract Provides Downstream Protection of the Human Immunodeficiency Virus Type 1 Genome from Editing by APOBEC3G and APOBEC3B

RNA Degradation and Primer Selection by Moloney Murine Leukemia Virus Reverse Transcriptase Contribute to the Accuracy of Plus Strand Initiation

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