Genetic determinants of coxsackievirus B3 pathogenesis.
ABSTRACT The development of high throughput genomic and bioinformatic analysis tools, coupled with established molecular techniques, has allowed new insights into the pathogenesis of infectious diseases. In humans, coxasackievirus B3 (CVB3) is the primary etiological agent of viral myocarditis, an inflammatory disease process involving the heart muscle. Early host cellular survival and apoptotic mechanisms during viral infections, as well as immune events, affect myocarditis progression and outcome. Therefore, our laboratory has been keenly interested in infectomics, defined here as the transcriptional events of both virus and host. We first elucidated up- or downregulated transcriptional activities in CVB3-infected hearts by mRNA differential display. Further characterization of these regulated genes including Nip21, IP10, and IGTPase, and study of their role in CVB3-infection are underway. In further dissection of the stages of myocarditis-peak viremia, inflammatory infiltration and tissue repair-we used cDNA microarrays to probe differential gene expression in the myocardium following virus infection. Following virus infection, there are global decreases in metabolic and mitochondrial genes, increases in signaling genes and distinctive patterns in other functional groups. To establish early gene expression profiles in infected cells by themselves, we also used oligonucleotide arrays in an in vitro model of CVB3 infection. Notably, we have found increased expression of transcription factors c-fos and c-jun down-stream of extracellular signal-related kinase, a pathway which is crucial for virus replication and pathogenesis. Our investigations based on gene profiling following CVB3 infection have thus far been fruitful in providing new experimental leads. High throughput genetic analysis has allowed us to simultaneously try on greater than 12,000 potential genetic "glass slippers." Our in vitro experimental plan has enabled us to chart prominent patterns of gene expression, analyzed by novel bioinformatic approaches, and to separate varied and potentially significant gene expression events.
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ABSTRACT: The lengthy 5' untranslated region (5'UTR) of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure, which plays an important role in controlling viral transcription and translation. Our previous work has delineated the internal ribosome entry site (IRES) by mutation of mono- and bicistronic plasmids containing the 5'UTR and subsequent cell- free translation in rabbit reticular lysate (D. Yang, J. E. Wilson, D. R. Anderson, L. Bohunek, C. Cordeiro, R. Kandolf, and B. M. McManus. (1997). Virology 228, 63-73). To further identify the sequence elements responsible for viral translation and infectivity in tissue culture cells, >30 full-length mutants of CVB3 were constructed by mutations of the IRES and its flanking regions. Viral RNAs were transcribed from these constructs and transfected into HeLa cells. When the stem-loops G and H in the putative IRES were deleted, viral infectivity was abolished and viral protein translation was also undetectable by immunoblot analysis. However, when stem-loops A and B were deleted or stem-loop E was partially deleted, viral protein translation could be detected although cytopathic effect could not be observed. The data suggest that the crucial sequence of the IRES is located at stem-loops G and H. Further serial deletion mapping up and down stream of the crucial sequence defined more accurately the 5' and 3' boundaries of the IRES, located at nucleotides (nts) 309-432 and 639-670, respectively. These results indicate that the core sequence of the IRES should be located at nts 432-639. This IRES segment is much shorter and located closer to the initiation codon than that of poliovirus. To further define critical nucleotides within the IRES core, site-directed mutagenesis was conducted at the IRES core sequence by PCR. A 46-nt deletion in the pyrimidine-rich tract of stem-loop G abolished viral translation and infectivity. Interestingly, five single-nt substitutions in the pyrimidine-rich tract aimed at destabilizing the base pairing between the viral IRES and host 18S rRNA did not abolish CVB3 infectivity although viral protein translation was significantly reduced. This finding suggests that ribosomal internal initiation of translation and viral infectivity not only may require RNA secondary structure but also may need tertiary structure and perhaps the assistance of host protein factors.Virology 01/2000; 265(2):206-17. · 3.37 Impact Factor
Article: ATF3 and stress responses.[show abstract] [hide abstract]
ABSTRACT: The purpose of this review is to discuss ATF3, a member of the ATF/CREB family of transcription factors, and its roles in stress responses. In the introduction, we briefly describe the ATF/CREB family, which contains more than 10 proteins with the basic region-leucine zipper (bZip) DNA binding domain. We summarize their DNA binding and heterodimer formation with other bZip proteins, and discuss the nomenclature of these proteins. Over the years, identical or homologous cDNA clones have been isolated by different laboratories and given different names. We group these proteins into subgroups according to their amino acid similarity; we also list the alternative names for each member, and clarify some potential confusion in the nomenclature of this family of proteins. We then focus on ATF3 and its potential roles in stress responses. We review the evidence that the mRNA level of ATF3 greatly increases when the cells are exposed to stress signals. In animal experiments, the signals include ischemia, ischemia coupled with reperfusion, wounding, axotomy, toxicity, and seizure; in cultured cells, the signals include serum factors, cytokines, genotoxic agents, cell death-inducing agents, and the adenoviral protein E1A. Despite the overwhelming evidence for its induction by stress signals, not much else is known about ATF3. Preliminary results suggest that the JNK/SAPK pathway is involved in the induction of ATF3 by stress signals; in addition, IL-6 and p53 have been demonstrated to be required for the induction of ATF3 under certain conditions. The consequences of inducing ATF3 during stress responses are not clear. Transient transfection and in vitro transcription assays indicate that ATF3 represses transcription as a homodimer; however, ATF3 can activate transcription when coexpressed with its heterodimeric partners or other proteins. Therefore, it is possible that, when induced during stress responses, ATF3 activates some target genes but represses others, depending on the promoter context and cellular context. Even less is understood about the physiological significance of inducing ATF3. We will discuss our preliminary results and some reports by other investigators in this regard.Gene Expression 02/1999; 7(4-6):321-35. · 2.24 Impact Factor
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ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) infection alters the expression of host cell genes at both the mRNA and protein levels. To obtain a more comprehensive view of the global effects of HIV infection of CD4-positive T-cells at the mRNA level, we performed cDNA microarray analysis on approximately 1500 cellular cDNAs at 2 and 3 days postinfection (p.i.) with HIV-1. Host cell gene expression changed little at 2 days p.i., but at 3 days p.i. 20 cellular genes were identified as differentially expressed. Genes involved in T-cell signaling, subcellular trafficking, and transcriptional regulation, as well as several uncharacterized genes, were among those whose mRNAs were differentially regulated. These results support the hypothesis that HIV-1 infection alters expression of a broad array of cellular genes and provides a framework for future functional studies on the differentially expressed mRNA products.Virology 01/2000; · 3.37 Impact Factor