Abstract:Sam68 has been implicated in a variety of important cellular processes such as RNA metabolism and intracellular signaling. We have recently shown that Sam68 cytoplasmic mutants induces stress granules (SG) and inhibit HIV-1 nef mRNA translation [1]. These findings prompted us to investigate the possibility and the underlying mechanisms of the wild-type counterpart Sam68 SG recruitment. Herein, we revealed that Sam68 was significantly recruited into cytoplasmic SG under oxidative stress. We then demonstrated th… Show more
“…Therefore, virus-induced SGs are likely to be compositionally unique. The co-localization of Sam68 with TIA-1 in transient cytoplasmic granular structures in FMDV-infected cells resembles earlier observations in poliovirus and retroviruses [ 16 , 17 , 32 , 60 , 61 ]. Fig.…”
BackgroundThe nuclear protein Src-associated protein of 68 kDa in mitosis (Sam68) is known to bind RNA and be involved in cellular processes triggered in response to environmental stresses, including virus infection. Interestingly, Sam68 is a multi-functional protein implicated in the life cycle of retroviruses and picornaviruses and is also considered a marker of virus-induced stress granules (SGs). Recently, we demonstrated the partial redistribution of Sam68 to the cytoplasm in FMDV infected cells, its interaction with viral protease 3Cpro, and found a significant reduction in viral titers as consequence of Sam68-specific siRNA knockdowns. Despite of that, details of how it benefits FMDV remains to be elucidated.MethodsSam68 cytoplasmic localization was examined by immunofluorescent microscopy, counterstaining with antibodies against Sam68, a viral capsid protein and markers of SGs. The relevance of RAAA motifs in the IRES was investigated using electromobility shift assays with Sam68 protein and parental and mutant FMDV RNAs. In addition, full genome WT and mutant or G-luc replicon RNAs were tested following transfection in mammalian cells. The impact of Sam68 depletion to virus protein and RNA synthesis was investigated in a cell-free system. Lastly, through co-immunoprecipitation, structural modeling, and subcellular fractionation, viral protein interactions with Sam68 were explored.ResultsFMDV-induced cytoplasmic redistribution of Sam68 resulted in it temporarily co-localizing with SG marker: TIA-1. Mutations that disrupted FMDV IRES RAAA motifs, with putative affinity to Sam68 in domain 3 and 4 cause a reduction on the formation of ribonucleoprotein complexes with this protein and resulted in non-viable progeny viruses and replication-impaired replicons. Furthermore, depletion of Sam68 in cell-free extracts greatly diminished FMDV RNA replication, which was restored by addition of recombinant Sam68. The results here demonstrated that Sam68 specifically co-precipitates with both FMDV 3Dpol and 3Cpro consistent with early observations of FMDV 3Cpro-induced cleavage of Sam68.ConclusionWe have found that Sam68 is a specific binding partner for FMDV non-structural proteins 3Cpro and 3Dpol and showed that mutations at RAAA motifs in IRES domains 3 and 4 cause a decrease in Sam68 affinity to these RNA elements and rendered the mutant RNA non-viable. Interestingly, in FMDV infected cells re-localized Sam68 was transiently detected along with SG markers in the cytoplasm. These results support the importance of Sam68 as a host factor co-opted by FMDV during infection and demonstrate that Sam68 interact with both, FMDV RNA motifs in the IRES and viral non-structural proteins 3Cpro and 3Dpol.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0452-8) contains supplementary material, which is available to authorized users.
“…Therefore, virus-induced SGs are likely to be compositionally unique. The co-localization of Sam68 with TIA-1 in transient cytoplasmic granular structures in FMDV-infected cells resembles earlier observations in poliovirus and retroviruses [ 16 , 17 , 32 , 60 , 61 ]. Fig.…”
BackgroundThe nuclear protein Src-associated protein of 68 kDa in mitosis (Sam68) is known to bind RNA and be involved in cellular processes triggered in response to environmental stresses, including virus infection. Interestingly, Sam68 is a multi-functional protein implicated in the life cycle of retroviruses and picornaviruses and is also considered a marker of virus-induced stress granules (SGs). Recently, we demonstrated the partial redistribution of Sam68 to the cytoplasm in FMDV infected cells, its interaction with viral protease 3Cpro, and found a significant reduction in viral titers as consequence of Sam68-specific siRNA knockdowns. Despite of that, details of how it benefits FMDV remains to be elucidated.MethodsSam68 cytoplasmic localization was examined by immunofluorescent microscopy, counterstaining with antibodies against Sam68, a viral capsid protein and markers of SGs. The relevance of RAAA motifs in the IRES was investigated using electromobility shift assays with Sam68 protein and parental and mutant FMDV RNAs. In addition, full genome WT and mutant or G-luc replicon RNAs were tested following transfection in mammalian cells. The impact of Sam68 depletion to virus protein and RNA synthesis was investigated in a cell-free system. Lastly, through co-immunoprecipitation, structural modeling, and subcellular fractionation, viral protein interactions with Sam68 were explored.ResultsFMDV-induced cytoplasmic redistribution of Sam68 resulted in it temporarily co-localizing with SG marker: TIA-1. Mutations that disrupted FMDV IRES RAAA motifs, with putative affinity to Sam68 in domain 3 and 4 cause a reduction on the formation of ribonucleoprotein complexes with this protein and resulted in non-viable progeny viruses and replication-impaired replicons. Furthermore, depletion of Sam68 in cell-free extracts greatly diminished FMDV RNA replication, which was restored by addition of recombinant Sam68. The results here demonstrated that Sam68 specifically co-precipitates with both FMDV 3Dpol and 3Cpro consistent with early observations of FMDV 3Cpro-induced cleavage of Sam68.ConclusionWe have found that Sam68 is a specific binding partner for FMDV non-structural proteins 3Cpro and 3Dpol and showed that mutations at RAAA motifs in IRES domains 3 and 4 cause a decrease in Sam68 affinity to these RNA elements and rendered the mutant RNA non-viable. Interestingly, in FMDV infected cells re-localized Sam68 was transiently detected along with SG markers in the cytoplasm. These results support the importance of Sam68 as a host factor co-opted by FMDV during infection and demonstrate that Sam68 interact with both, FMDV RNA motifs in the IRES and viral non-structural proteins 3Cpro and 3Dpol.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0452-8) contains supplementary material, which is available to authorized users.
“…However, such on May 10, 2018 by guest http://jvi.asm.org/ differences might be more subtle than they appear since immunofluorescent labeling does not allow comparisons of the relative abundances of different proteins and since detection thresholds might vary according to the experimental set-up. For instance, conflicting data regarding Sam68 detection in oxidative stress-induced SG have been reported (14,26).…”
“…Materials-Cloning of the following EGFP fusion protein expression vectors is described elsewhere: Sp100 (21), coilin (22), Sam68 (23), Cbx4 (24,25), Bmi-1 (26), Sc35 (27). nalaskowski@uke.uni-hamburg.de.…”
Recent studies have shown that inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) possesses important roles in the development of immune cells. IP3KB can be targeted to multiple cellular compartments, among them nuclear localization and binding in close proximity to the plasma membrane. The B isoform is the only IP3K that is almost ubiquitously expressed in mammalian cells. Detailed mechanisms of its targeting regulation will be important in understanding the role of Ins(1,4,5)P 3 phosphorylation on subcellular calcium signaling and compartment-specific initiation of pathways leading to regulatory active higher phosphorylated inositol phosphates. Here, we identified an exportin 1-dependent nuclear export signal ( 134 LQRELQNVQV) and characterized the amino acids responsible for nuclear localization of IP3KB ( 129 RKLR). These two targeting domains regulate the amount of nuclear IP3KB in cells. We also demonstrated that the localization of IP3KB at the plasma membrane is due to its binding to cortical actin structures. Intriguingly, all three of these targeting activities reside in one small polypeptide segment (amino acids 104 -165), which acts as a multitargeting domain (MTD). Finally, a hitherto unknown subnuclear localization of IP3KB could be demonstrated in rapidly growing H1299 cells. IP3KB is specifically enriched at nuclear invaginations extending perpendicular between the apical and basal surface of the nucleus of these flat cells. Such nuclear invaginations are known to be involved in Ins(1,4,5)P 3 -mediated Ca 2؉ signaling of the nucleus. Our findings indicate that IP3KB not only regulates cytoplasmic Ca 2؉ signals by phosphorylation of subplasmalemmal and cytoplasmic Ins(1,4,5)P 3 but may also be involved in modulating nuclear Ca 2؉ signals generated from these nuclear envelope invaginations.It is well established that the highly inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) 2 -specific IP3Ks remove the Ca 2ϩ -releasing second messenger Ins(1,4,5)P 3 by phosphorylating it to Ins(1,3,4,5)P 4 (1) supporting the formation of higher phosphorylated inositols in the cell (2). So far, three isoforms of IP3K, termed A (3), B (4), and C (5), have been identified in mammals. Full-length cDNAs encoding IP3K isoform B (IP3KB) were cloned from man (6) and rat (7). IP3KB was mapped to the telomeric end of human chromosome 1 (8, 9) and identified as a candidate gene in the pathogenesis of multiple sclerosis (10), Alzheimer disease (11), and malignant melanoma (12). But most importantly, IP3KB is a promising new target in immune disorders (13). Transgenic disruption of the IP3KB gene in mice causes a significant decrease in Ins(1,3,4,5)P 4 levels in thymocytes leading to a block of positive selection and consequently to a severe T cell deficiency (14). Recent studies described additional functions of IP3KB in selection and survival of B cells (15, 16), signaling of neutrophils (17), and modulation of myelopoiesis (18). In mammalian cells, multiple intracellular localizations of IP3KB, namely to the plasma membr...
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