Cooperation between Reverse Transcriptase and Integrase during Reverse Transcription and Formation of the Preintegrative Complex of Ty1

Wilhelm, M.L.; Wilhelm, F.X.

doi:10.1128/ec.00159-06

Cited by 20 publications

(22 citation statements)

References 48 publications

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“…This may further allow for Gag processing and reverse transcription. Close interaction of IN with RT is critical for the correct folding and activity of RT (61). This could explain why the concentration of Ty1 products in foci may support cDNA accumulation, which is reduced in P-body component mutants.…”

Section: Discussionmentioning

confidence: 99%

P-Body Components Are Required for Ty1 Retrotransposition during Assembly of Retrotransposition-Competent Virus-Like Particles

Checkley

Nagashima

Lockett

et al. 2010

Molecular and Cellular Biology

157

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Ty1 is a retrovirus-like retrotransposon whose replication is influenced by diverse cellular processes in Saccharomyces cerevisiae. We have identified cytoplasmic P-body components encoded by DHH1, KEM1, LSM1, and PAT1 as cofactors that posttranscriptionally enhance Ty1 retrotransposition. Using fluorescent in situ hybridization and immunofluorescence microscopy, we found that Ty1 mRNA and Gag colocalize to discrete cytoplasmic foci in wild-type cells. These foci, which are distinct from P-bodies, do not form in P-body component mutants or under conditions suboptimal for retrotransposition. Our immunoelectron microscopy (IEM) data suggest that mRNA/Gag foci are sites where virus-like particles (VLPs) cluster. Overexpression of Ty1 leads to a large increase in retrotransposition in wild-type cells, which allows VLPs to be detected by IEM. However, retrotransposition is still reduced in P-body component mutants under these conditions. Moreover, the percentage of Ty1 mRNA/Gag foci and VLP clusters and levels of integrase and reverse transcriptase are reduced in these mutants. Ty1 antisense RNAs, which have been reported to inhibit Ty1 transposition, are more abundant in the kem1⌬ mutant and colocalize with Ty1 mRNA in the cytoplasm. Therefore, Kem1p may prevent the aggregation of Ty1 antisense and mRNAs. Overall, our results suggest that P-body components enhance the formation of retrotransposition-competent Ty1 VLPs.

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Section: Discussionmentioning

confidence: 99%

P-Body Components Are Required for Ty1 Retrotransposition during Assembly of Retrotransposition-Competent Virus-Like Particles

Checkley

Nagashima

Lockett

et al. 2010

Molecular and Cellular Biology

157

View full text Add to dashboard Cite

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“…The RT of Ty3 has a similar alpha-beta configuration (18,25). In addition, the interaction of Ty1 IN and RT has been shown to be necessary to activate RT (44). Likewise, the RT and IN of Tf1 have been shown to interact with each other in two-hybrid experiments (43).…”

Section: Resultsmentioning

confidence: 99%

The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection

Chatterjee

Leem

Kelly

et al. 2009

J Virol

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The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHD⌬, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHD⌬ caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting.Long terminal repeat (LTR) retrotransposons are close relatives of retroviruses that propagate by inserting their DNA into the genome of the host. Both retroviruses and LTR retrotransposons encode an integrase (IN) protein that mediates this process. A wealth of information is available about the function of IN from in vitro studies conducted with recombinant proteins (9); however, much less is known about how each domain of IN functions in vivo.IN proteins of retroviruses and LTR retrotransposons contain three distinct protease-resistant domains. The N-terminal domain has the conserved amino acid motif HX 3-7 HX [23][24][25][26][27][28][29][30][31][32]

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“…While our assays do not permit us to directly address the influence of IN on RT stability, a required postproteolytic interaction between Ty1 IN and RT has been reported (51,62,61) as well as several observations of retroviral IN-RT interactions (31,56,64,68). Wilhelm and Wilhelm (62) have proposed that Ty1 RT is a component of a heteromeric complex that includes IN and element cDNA in a preintegration complex and that RT remains associated with IN even after nuclear import of the preintegration complex.…”

Section: Discussionmentioning

confidence: 99%

Functional Analysis of N-Terminal Residues of Ty1 Integrase

Moore

Garfinkel

2009

J Virol

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The Ty1 retrotransposon of Saccharomyces cerevisiae is comprised of structural and enzymatic proteins that are functionally similar to those of retroviruses. Despite overall sequence divergence, certain motifs are highly conserved. We have examined the Ty1 integrase (IN) zinc binding domain by mutating the definitive histidine and cysteine residues and thirteen residues in the intervening (X 32 ) sequence between IN-H22 and IN-C55. Mutation of the zinc-coordinating histidine or cysteine residues reduced transposition by more than 4,000-fold and led to IN and reverse transcriptase (RT) instability as well as inefficient proteolytic processing. Alanine substitution of the hydrophobic residues I28, L32, I37 and V45 in the X 32 region reduced transposition 85-to 688-fold. Three of these residues, L32, I37, and V45, are highly conserved among retroviruses, although their effects on integration or viral infectivity have not been characterized. In contrast to the HHCC mutants, all the X 32 mutants exhibited stable IN and RT, and protein processing and cDNA production were unaffected. However, glutathione S-transferase pulldowns and intragenic complementation analysis of selected transposition-defective X 32 mutants revealed decreased IN-IN interactions. Furthermore, virus-like particles with in-L32A and in-V45A mutations did not exhibit substantial levels of concerted integration products in vitro. Our results suggest that the histidine/cysteine residues are important for steps in transposition prior to integration, while the hydrophobic residues function in IN multimerization.The structural and functional similarities of the Ty retrotransposons of Saccharomyces to mammalian retroviruses suggest similar mechanisms for Ty transposition and retroviral integration. Like retroviruses, Ty elements are bound by long terminal repeats (LTR), contain two overlapping open reading frames analogous to retroviral gag and pol, and undergo proteolytic processing of a Gag/Pol fusion protein. The similarities between retrotransposons and retroviruses extend to the highly conserved sequence homologies in the catalytic active sites of the Pol proteins, protease (PR), RT,and IN (1,36,19,55,60). A noncatalytic sequence, the zinc binding domain (ZBD), is also conserved in the N-terminal regions of retroviral and retrotransposon INs (36). This domain, characterized by the sequence H-X 3-7 H-X 23-32 C-X 2 C, binds a single zinc ion (10, 37, 42, 67), which stabilizes the N-terminal region of IN and promotes formation of IN tetramers, thereby enhancing the enzyme's catalytic activity (37, 67).The IN catalytic domain has been well characterized in retroviruses (7, 34) and to a lesser extent in Ty1 (45, 54). The retroviral IN ZBD has also been studied extensively, initially by evaluating the effects of N-terminal deletions (53,8,11,12,16,23,59) or mutations of the histidine or cysteine residues (24, 57, 58) in assays which recapitulate biochemical activities of IN, i.e., 3Ј dinucleotide processing and integration of donor molecules representing v...

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Cooperation between Reverse Transcriptase and Integrase during Reverse Transcription and Formation of the Preintegrative Complex of Ty1

Cited by 20 publications

References 48 publications

P-Body Components Are Required for Ty1 Retrotransposition during Assembly of Retrotransposition-Competent Virus-Like Particles

P-Body Components Are Required for Ty1 Retrotransposition during Assembly of Retrotransposition-Competent Virus-Like Particles

The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection

Functional Analysis of N-Terminal Residues of Ty1 Integrase

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