An Immunological Analysis of Ty1 Virus-like Particle Structure

Brookman, Jayne L.; Stott, Andrew J.; Cheeseman, Philip J.; Burns, Nigel R.; Adams, Sally E.; Kingsman, Alan J.; Gull, Keith

doi:10.1006/viro.1995.1051

Cited by 48 publications

(55 citation statements)

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“…We further assayed T-body contents by determining the cellular location of the Tya protein, the main structural component of Ty1 VLPs. For this purpose, we used an antibody raised against the Tya N-terminal amino acids 27 to 32 (8). In some cells devoid of T bodies, this reagent revealed a punctuated cytoplasmic Tya distribution, in agreement with the reported speckled cytoplasmic VLP distribution (9).…”

Section: Vol 28 2008 Determinants For T-body Formation 6025supporting

confidence: 79%

“…Nop1p, Nsp1p, GFP (green fluorescent protein), and Tya proteins were visualized using the following mouse monoclonal antibodies: mAb28F2 (EnCor), mAb32D6 (EnCor), GFP (B-2; Santa Cruz Biotechnology), and Tya (BB2, serum; [8]). These antibodies were used in dilutions of 1/1,000, 1/500, 1/250, and 1/100, respectively.…”

Section: Colocalization Experiments With Ty1 and Poly(a)mentioning

confidence: 99%

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The T Body, a New Cytoplasmic RNA Granule in Saccharomyces cerevisiae

Malagón

Jensen

2008

Molecular and Cellular Biology

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A large share of mRNA processing and packaging events occurs cotranscriptionally. To explore the hypothesis that transcription defects may affect mRNA fate, we analyzed poly(A) ؉ RNA distribution in Saccharomyces cerevisiae strains harboring mutations in Rpb1p, the largest subunit of RNA polymerase II. In certain rpb1 mutants, a poly(A) ؉ RNA granule, distinct from any known structure, strongly accumulated in a confined space of the cytoplasm. RNA and protein expressed from Ty1 retrovirus-like elements colocalized with this new granule, which we have consequently named the T body. A visual screen revealed that the deletion of most genes with proposed functions in Ty1 biology unexpectedly does not alter T-body levels. In contrast, the deletion of genes encoding the Mediator transcription initiation factor subunits Srb2p and Srb5p as well as the Ty1 transcriptional regulator Spt21p greatly enhances T-body formation. Our data disclose a new cellular body putatively involved in the assembly of Ty1 particles and suggest that the cytoplasmic fate of mRNA can be affected by transcription initiation events.In eukaryotic cells, mRNA-protein particles (mRNPs) are assembled in the nucleus and are normally transported to cytoplasmic ribosomes for translation. The protein composition of mRNPs is highly dynamic and changes in both a temporal and a spatial fashion, consistent with a changing need for associated factors at different stages of the mRNP life cycle. The initial mRNA-protein association occurs as soon as the 5Ј end of the nascent RNA extrudes from the exit channel of the RNA polymerase II (RNAPII) complex (4, 63). A number of factors coat the mRNA cotranscriptionally in order to effectively process the molecule into a mature length as well as to make the new mRNP competent for nuclear export (12). Importantly, RNAPII itself is an active player in these early mRNP assembly processes. This is perhaps best illustrated by the physical interactions of mRNA processing factors with the carboxy-terminal domain (CTD) of Rpb1p (50). However, additional levels of RNAPII/mRNP intimacy exist, exemplified by the suggestion that the RNAPII subunits Rpb4p and Rpb7p, which both affect the cytoplasmic decay rate of certain mRNAs, might be integral mRNP components (35).Although mRNA ultimately functions as a template for translation, the characteristics of mRNPs made from different genes are highly variable at the levels of cellular localization, mRNA stability, and ribosome availability. Combined with a high level of mRNP maturation control, this allows for ample plasticity of gene expression at posttranscriptional stages. For example, controlling the spatial distribution of mRNPs regulates developmental processes in eukaryotes, e.g., the location of Vg1 transcripts at the vegetal pole of Xenopus oocytes (38, 64) and the asymmetric distribution of ASH1 RNA to the daughter cell during budding in Saccharomyces cerevisiae (34,59). Variability between mRNPs produced from the same gene also occurs. This may be caused by transcription or ...

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Section: Vol 28 2008 Determinants For T-body Formation 6025supporting

confidence: 79%

Section: Colocalization Experiments With Ty1 and Poly(a)mentioning

confidence: 99%

The T Body, a New Cytoplasmic RNA Granule in Saccharomyces cerevisiae

Malagón

Jensen

2008

Molecular and Cellular Biology

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“…We also constructed a truncation of RetS lacking both the periplasmic domain and six of the seven transmembrane domains (RetS ⌬37-360). Both constructs were tagged with the BB2 epitope, allowing us to measure steady-state protein levels and subcellular localization of each mutant (7). The tagged proteins were expressed in a ⌬retS strain from a high-copy-number plasmid under the control of the native retS promoter.…”

Section: Vol 74 2006 Analysis Of a Sensor Kinase-response Regulatormentioning

confidence: 99%

Mutational Analysis of RetS, an Unusual Sensor Kinase-Response Regulator Hybrid Required for Pseudomonas aeruginosa Virulence

Laskowski

Kazmierczak

2006

Infect Immun

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Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in a wide range of hosts. Expression of the type III secretion system (T3SS) proteins is correlated with virulence in models of acute infection, while downregulation of the T3SS and upregulation of genes important for biofilm formation are observed during chronic infections. RetS, a hybrid sensor kinase-response regulator protein of P. aeruginosa, plays a key role in the reciprocal regulation of virulence factors required for acute versus chronic infection and is postulated to act in concert with two other sensor kinase-response regulator hybrids, GacS and LadS. This work examines the roles of the putative sensing and signal transduction domains of RetS in induction of the T3SS in vitro and in a murine model of acute pneumonia. We identify distinct signaling roles for the tandem receiver domains of RetS and present evidence suggesting that RetS may serve as a substrate for another sensor kinase. Phenotypes associated with RetS alleles lacking periplasmic and/or transmembrane domains further indicate that the periplasmic domain of RetS may transmit a signal that inhibits RetS activity during acute infections.

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“…VLPs might enter the nucleus intact. However, VLPs are estimated to be 600 Å in diameter and thus seem too large to be transported through the nuclear pore complex (NPC) (13,19,34,50). Maximal pore size estimates range from 90 Å (for proteins that enter the nucleus passively) to 260 Å (for NLS-bearing proteins of Ͼϳ40 kDa) (23, 29).…”

mentioning

confidence: 99%

Invading the Yeast Nucleus: a Nuclear Localization Signal at the C Terminus of Ty1 Integrase Is Required for Transposition In Vivo

Kenna

Brachmann

Devine

et al. 1998

Molecular and Cellular Biology

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Retrotransposon Ty1 faces a formidable cell barrier during transposition-the yeast nuclear membrane which remains intact throughout the cell cycle. We investigated the mechanism by which transposition intermediates are transported from the cytoplasm (the presumed site of Ty1 DNA synthesis) to the nucleus, where they are integrated into the genome. Ty1 integrase has a nuclear localization signal (NLS) at its C terminus. Both full-length integrase and a C-terminal fragment localize to the nucleus. C-terminal deletion mutants in Ty1 integrase were used to map the putative NLS to the last 74 amino acid residues of integrase. Mutations in basic segments within this region decreased retrotransposition at least 50-fold in vivo. Furthermore, these mutant integrase proteins failed to localize to the nucleus. Production of virus-like particles, reverse transcriptase activity, and complete in vitro Ty1 integration resembled wild-type levels, consistent with failure of the mutant integrases to enter the nucleus.Yeast retrotransposon Ty1 is similar in many aspects to retroviruses (7). Ty1 must generate more copies of itself for insertion into the genome of the host cell. Ty1 mRNA is transcribed and processed in the nucleus and then transported to the cytoplasm, where it is translated into Gag and Gag-Pol proteins. These are assembled into virus-like particles (VLPs) resembling retroviral core particles (34, 50). Reverse transcription of Ty1 RNA into Ty1 cDNA occurs in the VLPs, which also contain integrase (IN) (30). Unlike retroviruses, in which the viral core becomes enveloped and leaves the producer cell to infect a new cell and integrate into the target genome, Ty1 cDNA reenters the producer cell nucleus to integrate at a new target site. How extrachromosomal Ty1 cDNA enters the nucleus where it can access its target site DNA remains unresolved.The question of how Ty1 enters the nucleus is especially important because in all fungi, including the Ty1 host Saccharomyces cerevisiae, the nuclear envelope (NE) does not break down during mitosis (43). This significant intracellular barrier prevents Ty1 DNA from accessing the genome via mitotic NE dissociation, a cellular process apparently exploited by many retroviruses to gain nuclear access (55). However, this opportunity exists only in cells that are undergoing growth and division. Lentiviruses like human immunodeficiency virus type 1 (HIV-1) are able to infect nondividing cells (15, 17), which, like growing yeast cells, do not offer a mitotic window of opportunity to the nucleus. Thus, an understanding of the mechanism used by Ty1 to enter the nucleus should shed light on the analogous process in lentiviruses.Several possible mechanisms for nuclear entry can be envisioned. VLPs might enter the nucleus intact. However, VLPs are estimated to be 600 Å in diameter and thus seem too large to be transported through the nuclear pore complex (NPC) (13,19,34,50). Maximal pore size estimates range from 90 Å (for proteins that enter the nucleus passively) to 260 Å (for NLS-bearing pr...

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An Immunological Analysis of Ty1 Virus-like Particle Structure

Cited by 48 publications

References 0 publications

The T Body, a New Cytoplasmic RNA Granule in Saccharomyces cerevisiae

The T Body, a New Cytoplasmic RNA Granule in Saccharomyces cerevisiae

Mutational Analysis of RetS, an Unusual Sensor Kinase-Response Regulator Hybrid Required for Pseudomonas aeruginosa Virulence

Invading the Yeast Nucleus: a Nuclear Localization Signal at the C Terminus of Ty1 Integrase Is Required for Transposition In Vivo

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