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ABSTRACT: We sought to determine the frequency and profile of HIV-1 BF recombinants in vitro and in vivo. Laboratory HIV-1 strains from subtypes B and F were cocultured and evaluated. Clinical samples from the city of Santos, Brazil, where the first HIV-1 B/F circulating recombinant forms (CRF) were described, were also assessed. Five real-time PCR assays were developed to equally amplify subtypes B and F, and subtype-specific probes were developed and optimized. To validate the PCR systems, clinical samples from Santos were sequenced and phylogenetically analyzed. The real-time PCR assays were performed on these samples and on the supernatant of an in vitro competition assay to assess emergent recombinant strains. Out of 157 clinical samples, 62.1% were defined as subtype B, 3.0% were subtype F, 16.7% presented the CRF28_BF profile, and 13.6% of the samples presented the CRF29_BF profile. The specificity and sensitivity in the discrimination assay for this sample panel were 93% and 92%, respectively. The HIV that emerged from the coinfected cell culture closely resembled the CRF28_BF profile. The first-described CRFs are still fixed in this geographic region of Brazil, and the in vitro emerging strains detected by real-time PCR suggest that in addition to the shaping of recombinant strains by immune selection, viral structures may also play an important role in emerging CRFs.
AIDS research and human retroviruses 09/2010; 26(9):981-90. · 2.18 Impact Factor
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ABSTRACT: Processing bodies (P-bodies) are cytoplasmic RNA granules that contain translationally repressed messenger ribonucleoproteins (mRNPs) and messenger RNA (mRNA) decay factors. The physical interactions that form the individual mRNPs within P-bodies and how those mRNPs assemble into larger P-bodies are unresolved. We identify direct protein interactions that could contribute to the formation of an mRNP complex that consists of core P-body components. Additionally, we demonstrate that the formation of P-bodies that are visible by light microscopy occurs either through Edc3p, which acts as a scaffold and cross-bridging protein, or via the "prionlike" domain in Lsm4p. Analysis of cells defective in P-body formation indicates that the concentration of translationally repressed mRNPs and decay factors into microscopically visible P-bodies is not necessary for basal control of translation repression and mRNA decay. These results suggest a stepwise model for P-body assembly with the initial formation of a core mRNA-protein complex that then aggregates through multiple specific mechanisms.
The Journal of Cell Biology 12/2007; 179(3):437-49. · 10.26 Impact Factor
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ABSTRACT: Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), that contain untranslating mRNAs in conjunction with proteins involved in translation repression and mRNA decapping and degradation. However, the order of protein assembly into P-bodies and the interactions that promote P-body assembly are unknown. To gain insight into how yeast P-bodies assemble, we examined the P-body accumulation of Dcp1p, Dcp2p, Edc3p, Dhh1p, Pat1p, Lsm1p, Xrn1p, Ccr4p, and Pop2p in deletion mutants lacking one or more P-body component. These experiments revealed that Dcp2p and Pat1p are required for recruitment of Dcp1p and of the Lsm1-7p complex to P-bodies, respectively. We also demonstrate that P-body assembly is redundant and no single known component of P-bodies is required for P-body assembly, although both Dcp2p and Pat1p contribute to P-body assembly. In addition, our results indicate that Pat1p can be a nuclear-cytoplasmic shuttling protein and acts early in P-body assembly. In contrast, the Lsm1-7p complex appears to primarily function in a rate limiting step after P-body assembly in triggering decapping. Taken together, these results provide insight both into the function of individual proteins involved in mRNA degradation and the mechanisms by which yeast P-bodies assemble.
Molecular Biology of the Cell 07/2007; 18(6):2274-87. · 4.94 Impact Factor
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ABSTRACT: Post-transcriptional control mechanisms play an important role in regulating gene expression during cellular responses to stress. For example, many stresses inhibit translation, and at least some stresses inhibit mRNA turnover in yeast and mammalian cells. We show that hyperosmolarity, heat shock, and glucose deprivation stabilize multiple mRNAs in yeast, primarily through inhibition of deadenylation. Although these stresses inhibit translation and promote the movement of mRNAs into P-bodies, we also observed inhibition of deadenylation in cycloheximide-treated cells as well as in a mutant strain where translation initiation is impaired. This argues that inhibition of poly(A)-shortening is independent of the translational state of the mRNAs and can occur when mRNAs are localized in polysomes or are not engaged in translation. Analysis of pan2Delta or ccr4Delta strains indicates that stress inhibits the function of both the Ccr4p/Pop2p/Notp and the Pan2p/Pan3p deadenylases. We suggest that under stress, simultaneous repression of translation and deadenylation allows cells to selectively translate mRNAs specific to the stress response, while retaining the majority of the cytoplasmic pool of mRNAs for later reuse and recovery from stress. Moreover, because various cellular stresses also inhibit deadenylation in mammalian cells, this mechanism is likely to be a conserved aspect of the stress response.
RNA 11/2006; 12(10):1835-45. · 5.09 Impact Factor
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ABSTRACT: Eukaryotic cells contain nontranslating messenger RNA concentrated in P-bodies, which are sites where the mRNA can be decapped and degraded. We present evidence that mRNA molecules within yeast P-bodies can also return to translation. First, inhibiting delivery of new mRNAs to P-bodies leads to their disassembly independent of mRNA decay. Second, P-bodies decline in a translation initiation-dependent manner during stress recovery. Third, reporter mRNAs concentrate in P-bodies when translation initiation is blocked and resume translation and exit P-bodies when translation is restored. Fourth, stationary phase yeast have large P-bodies containing mRNAs that reenter translation when growth resumes. The reciprocal movement of mRNAs between polysomes and P-bodies is likely to be important in the control of mRNA translation and degradation. Moreover, the presence of related proteins in P-bodies and maternal mRNA storage granules suggests this mechanism is widely adapted for mRNA storage.
Science 11/2005; 310(5747):486-9. · 31.20 Impact Factor
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ABSTRACT: Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), wherein mRNA decay factors are concentrated and where mRNA decay can occur. However, the physical nature of P-bodies, their relationship to translation, and possible roles of P-bodies in cellular responses remain unclear. We describe four properties of yeast P-bodies that indicate that P-bodies are dynamic structures that contain nontranslating mRNAs and function during cellular responses to stress. First, in vivo and in vitro analysis indicates that P-bodies are dependent on RNA for their formation. Second, the number and size of P-bodies vary in response to glucose deprivation, osmotic stress, exposure to ultraviolet light, and the stage of cell growth. Third, P-bodies vary with the status of the cellular translation machinery. Inhibition of translation initiation by mutations, or cellular stress, results in increased P-bodies. In contrast, inhibition of translation elongation, thereby trapping the mRNA in polysomes, leads to dissociation of P-bodies. Fourth, multiple translation factors and ribosomal proteins are lacking from P-bodies. These results suggest additional biological roles of P-bodies in addition to being sites of mRNA degradation.
RNA 05/2005; 11(4):371-82. · 5.09 Impact Factor
