AIM: To search for the presence of cis elements in hepatitis D virus(HDV) genomic and antigenomic RNA capable of promoting nuclear export. METHODS: We made use of a well characterized chloramphenicol acetyl-transferas...AIM: To search for the presence of cis elements in hepatitis D virus(HDV) genomic and antigenomic RNA capable of promoting nuclear export. METHODS: We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid p DM138. Twenty c DNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pD M138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by realtime polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export.RESULTS: Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter m RNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. CONCLUSION: A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.展开更多
MicroRNAs (miRNAs) are small, noncoding RNA molecules that play important roles in the regulation of gene expression of the cell. Recent studies have described cytoplasmic RNA virus genome- derived miRNAs. Moreover, m...MicroRNAs (miRNAs) are small, noncoding RNA molecules that play important roles in the regulation of gene expression of the cell. Recent studies have described cytoplasmic RNA virus genome- derived miRNAs. Moreover, miRNAs have also been encountered in the reverse strand of the viral mRNA, revealing the presence of miRNAs in replication intermediaries. In order to get insight into the possible role of Hepatitis C Virus (HCV) antigenome in relation to miRNA coding, we computationally identified potential miRNAs on the antigenome of HCV reference strain H77. By utilizing a series of bioinformatics tools, we identified a miRNA present in the antigenomeof HCV H77 strain. This miRNA maps in the 5’non-translated region (5’UTR) of the HCV genome and is found to be conserved among HCV genotypes and sub-types. In silico target prediction generated 17 cellular genes. These potential targets are involved in apoptosis as well as immune response pathways, suggesting that they could play a role in the pathogenesis caused by viral infection. The results of these studies revealed the presence of a viral miRNA in the negative-sense RNA strand used as a replication template for the HCV genome, as observed for other RNA viruses.展开更多
基金Supported by A grant from Fundao para a Ciência e Tecnologia,Portugal to Freitas N
文摘AIM: To search for the presence of cis elements in hepatitis D virus(HDV) genomic and antigenomic RNA capable of promoting nuclear export. METHODS: We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid p DM138. Twenty c DNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pD M138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by realtime polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export.RESULTS: Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter m RNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. CONCLUSION: A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.
文摘MicroRNAs (miRNAs) are small, noncoding RNA molecules that play important roles in the regulation of gene expression of the cell. Recent studies have described cytoplasmic RNA virus genome- derived miRNAs. Moreover, miRNAs have also been encountered in the reverse strand of the viral mRNA, revealing the presence of miRNAs in replication intermediaries. In order to get insight into the possible role of Hepatitis C Virus (HCV) antigenome in relation to miRNA coding, we computationally identified potential miRNAs on the antigenome of HCV reference strain H77. By utilizing a series of bioinformatics tools, we identified a miRNA present in the antigenomeof HCV H77 strain. This miRNA maps in the 5’non-translated region (5’UTR) of the HCV genome and is found to be conserved among HCV genotypes and sub-types. In silico target prediction generated 17 cellular genes. These potential targets are involved in apoptosis as well as immune response pathways, suggesting that they could play a role in the pathogenesis caused by viral infection. The results of these studies revealed the presence of a viral miRNA in the negative-sense RNA strand used as a replication template for the HCV genome, as observed for other RNA viruses.
