The control of mRNA degradation and translation are important aspects of gene regulation. Recent results suggest that translation repression and mRNA decapping can be intertwined and involve the formation of a quiescent mRNP, which can accumulate in cytoplasmic foci referred to as P bodies. The Pat1 protein is a key component of this complex and an important activator of decapping, yet little is known about its function. In this work, we analyze Pat1 in Saccharomyces cerevisiae function by deletion and functional analyses. Our results identify two primary functional domains in Pat1: one promoting translation repression and P-body assembly and a second domain promoting mRNA decapping after assembly of the mRNA into a P-body mRNP. In addition, we provide evidence that Pat1 binds RNA and has numerous domain-specific interactions with mRNA decapping factors. These results indicate that Pat1 is an RNA binding protein and a multidomain protein that functions at multiple stages in the process of translation repression and mRNA decapping.A critical aspect of the regulation of eukaryotic gene expression is the control of mRNA turnover. In eukaryotes, two major pathways for the decay of mRNAs exist, both of which are initiated by deadenylation, with the predominant nuclease being the Ccr4/Pop2/Not1-5 deadenylase complex (31, 35). Following deadenylation, the transcript is susceptible to one of the two pathways of decay. In the 3Ј-to-5Ј decay pathway, the deadenylated mRNA is degraded 3Ј to 5Ј by a complex of proteins known as the exosome (1). Alternatively, the mRNA is decapped by the decapping enzyme (Dcp1/Dcp2), the mechanism that predominates in Saccharomyces cerevisiae, making the mRNA susceptible to the 5Ј-to-3Ј exonuclease Xrn1 (4,18,19,24,32). Decapping is modulated by a set of proteins including Dhh1, the Lsm1-7 complex, and Pat1 (7,16,22,44).Decapping is a critical node in the control of the life of an mRNA. Moreover, the processes of mRNA decapping and translation are mechanistically intertwined and appear to compete with each other, at least in yeast (14). For example, decreasing translation initiation by a variety of means increases the rate of mRNA decapping (28,33,39). Conversely, inhibition of translation elongation leads to a significant decrease in the rate of decapping (3). Moreover, coimmunoprecipitation experiments suggested that prior to decapping, an mRNA exits translation and then assembles into a translationally repressed messenger ribonucleoprotein (mRNP) complex (45).Additional evidence for a discrete population of nontranslating mRNPs has been that nontranslating mRNAs and the decapping machinery accumulate in discrete cytoplasmic foci called P bodies (also referred as GW182 or Dcp bodies) (17,25,30,40). P bodies have now been observed in yeast, insect cells, nematodes, and mammalian cells and contain various proteins involved in mRNA decay, including the decapping enzyme (Dcp1/Dcp2); activators of decapping, Dhh1, Pat1, Lsm1-7, and Edc3; and the exonuclease Xrn1 (2, 20, 34). Moreover, P bodies...

BackgroundNone of the HIV T-cell vaccine candidates that have reached advanced clinical testing have been able to induce protective T cell immunity. A major reason for these failures may have been suboptimal T cell immunogen designs.MethodsTo overcome this problem, we used a novel immunogen design approach that is based on functional T cell response data from more than 1,000 HIV-1 clade B and C infected individuals and which aims to direct the T cell response to the most vulnerable sites of HIV-1.ResultsOur approach identified 16 regions in Gag, Pol, Vif and Nef that were relatively conserved and predominantly targeted by individuals with reduced viral loads. These regions formed the basis of the HIVACAT T-cell Immunogen (HTI) sequence which is 529 amino acids in length, includes more than 50 optimally defined CD4+ and CD8+ T-cell epitopes restricted by a wide range of HLA class I and II molecules and covers viral sites where mutations led to a dramatic reduction in viral replicative fitness. In both, C57BL/6 mice and Indian rhesus macaques immunized with an HTI-expressing DNA plasmid (DNA.HTI) induced broad and balanced T-cell responses to several segments within Gag, Pol, and Vif. DNA.HTI induced robust CD4+ and CD8+ T cell responses that were increased by a booster vaccination using modified virus Ankara (MVA.HTI), expanding the DNA.HTI induced response to up to 3.2% IFN-γ T-cells in macaques. HTI-specific T cells showed a central and effector memory phenotype with a significant fraction of the IFN-γ+ CD8+ T cells being Granzyme B+ and able to degranulate (CD107a+).ConclusionsThese data demonstrate the immunogenicity of a novel HIV-1 T cell vaccine concept that induced broadly balanced responses to vulnerable sites of HIV-1 while avoiding the induction of responses to potential decoy targets that may divert effective T-cell responses towards variable and less protective viral determinants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0392-5) contains supplementary material, which is available to authorized users.

Background: The biosynthesis of IL-12p70 depends on the intracellular interaction of its p35 and p40 subunits.Results: The p40 subunit stabilizes p35 and promotes its secretion.Conclusion: Understanding the regulatory steps of IL-12 biosynthesis led to the generation of optimized IL-12 plasmids.Significance: Availability of expression-optimized IL-12 DNA plasmids is important for practical applications as DNA vaccine adjuvants and in cancer immunotherapy.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 is a viral early protein. Like its homologues herpes simplex virus type 1 (HSV-1) ICP27 (38), Epstein-Barr virus (EBV) EB2 (40), human cytomegalovirus (HCMV) UL69 (47), and varicella-zoster virus (VZV) IE4/ORF4 (32), KSHV ORF57 regulates virus gene expression at the posttranscriptional level (23). The KSHV genome with a disrupted ORF57 gene cannot efficiently express a subset of viral lytic genes (20). ORF57 functions as a viral splicing factor and promotes viral RNA splicing (19,21,23 (22,29). In contrast, a recent study indicates that ORF57 appears to recruit the entire TREX through its interaction with Aly/REF to facilitate the export of a viral late transcript, ORF47 (2). Since ORF57 specifically binds to ORF59 RNA only in the presence of cellular proteins, it is thought that the cellular adaptors may act as cofactors. In support of this, the posttranscriptional regulator EB2 of EBV, a member of the gammaherpesvirus subfamily, was reported to interact with OTT3 (also referred to as RNA binding motif protein 15B [RBM15B]) and OTT3 was shown to have posttranscriptional regulatory function (6).OTT3 (6, 26), RNA binding motif protein 15 (RBM15 [OTT1]) (16,26), and SHARP (SMRT/HDAC1-associated repressor protein) (30, 41) are members of the SPEN (split ends) protein family. These nuclear proteins share a domain structure comprising three highly conserved N-terminal RNA-binding motifs (RNA recognition motif [RRM]) and a highly conserved C-terminal SPOC (Spen paralogue and orthologue C-terminal) domain, with 52%, 68%, a...