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The yeast Ty1 retrotransposon requires components of the nuclear pore complex for transcription and genomic integration.

ABSTRACT: Nuclear pore complexes (NPCs) orchestrate cargo between the cytoplasm and nucleus and regulate chromatin organization. NPC proteins, or nucleoporins (Nups), are required for human immunodeficiency virus type 1 (HIV-1) gene expression and genomic integration of viral DNA. We utilize the Ty1 retrotransposon of Saccharomyces cerevisiae (S. cerevisiae) to study retroviral integration because retrotransposons are the progenitors of retroviruses and have conserved integrase (IN) enzymes. Ty1-IN targets Ty1 elements into the genome upstream of RNA polymerase (Pol) III transcribed genes such as transfer RNA (tRNA) genes. Evidence that S. cerevisiae tRNA genes are recruited to NPCs prompted our investigation of a functional role for the NPC in Ty1 targeting into the genome. We find that Ty1 mobility is reduced in multiple Nup mutants that cannot be accounted for by defects in Ty1 gene expression, cDNA production or Ty1-IN nuclear entry. Instead, we find that Ty1 insertion upstream of tRNA genes is impaired. We also identify Nup mutants with wild type Ty1 mobility but impaired Ty1 targeting. The NPC nuclear basket, which interacts with chromatin, is required for both Ty1 expression and nucleosome targeting. Deletion of components of the NPC nuclear basket causes mis-targeting of Ty1 elements to the ends of chromosomes.

SUBMITTER: Manhas S

PROVIDER: S-EPMC5909446 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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The yeast Ty1 retrotransposon requires components of the nuclear pore complex for transcription and genomic integration.

Manhas Savrina S Ma Lina L Measday Vivien V

Nucleic acids research 20180401 7

Nuclear pore complexes (NPCs) orchestrate cargo between the cytoplasm and nucleus and regulate chromatin organization. NPC proteins, or nucleoporins (Nups), are required for human immunodeficiency virus type 1 (HIV-1) gene expression and genomic integration of viral DNA. We utilize the Ty1 retrotransposon of Saccharomyces cerevisiae (S. cerevisiae) to study retroviral integration because retrotransposons are the progenitors of retroviruses and have conserved integrase (IN) enzymes. Ty1-IN target ...[more]

PMID: 29514267

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Project description:The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels in cycling cells. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

Project description:Ty1 is one of the many transposons in the budding yeast Saccharomyces cerevisiae. The life-cycle of Ty1 shows numerous similarities with that of retroviruses, e.g. the initially synthesized polyprotein precursor undergoes proteolytic processing by the protease. The retroviral proteases have become important targets of current antiretroviral therapies due to the critical role of the limited proteolysis of Gag-Pol polyprotein in the replication cycle and they therefore belong to the most well-studied enzymes. Comparative analyses of retroviral and retroviral-like proteases can help to explore the key similarities and differences which may help understanding how resistance is developed against protease inhibitors, but the available information about the structural and biochemical characteristics of retroviral-like, and especially retrotransposon, proteases is limited. To investigate the main characteristics of Ty1 retrotransposon protease of Saccharomyces cerevisiae, untagged and His6-tagged forms of Ty1 protease were expressed in E. coli. After purification of the recombinant proteins, activity measurements were performed using synthetic oligopeptide and fluorescent recombinant protein substrates, which represented the wild-type and the modified forms of naturally occurring cleavage sites of the protease. We investigated the dependence of enzyme activity on different reaction conditions (pH, temperature, ionic strength, and urea concentration), and determined enzyme kinetic parameters for the studied substrates. Inhibitory potentials of 10 different protease inhibitors were also tested. Ty1 protease was not inhibited by the inhibitors which have been designed against human immunodeficiency virus type 1 protease and are approved as antiretroviral therapeutics. A quaternary structure of homodimeric Ty1 protease was proposed based on homology modeling, and this structure was used to support interpretation of experimental results and to correlate some structural and biochemical characteristics with that of other retroviral proteases.

Dataset Information

The yeast Ty1 retrotransposon requires components of the nuclear pore complex for transcription and genomic integration.

Publications

The yeast Ty1 retrotransposon requires components of the nuclear pore complex for transcription and genomic integration.

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