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Open reading frame 3 of genotype 1 hepatitis E virus inhibits nuclear factor-κappa B signaling induced by tumor necrosis factor-α in human A549 lung epithelial cells.

ABSTRACT: Hepatitis E virus (HEV) is one of the primary causative agents of acute hepatitis, and represents a major cause of severe public health problems in developing countries. The pathogenesis of HEV is not well characterized, however, primarily due to the lack of well-defined cell and animal models. Here, we investigated the effects of genotype 1 HEV open reading frame 3 (ORF3) on TNF-α-induced nucleus factor-κappa B (NF-κB) signaling. Human lung epithelial cells (A549) were transiently transfected with ORF3 containing plasmids. These cells were then stimulated with TNF-α and the nucleus translocation of the p65 NF-κB subunit was assessed using western blot and laser confocal microscopy. DNA-binding activity of p65 was also examined using electrophoretic mobility shift assay (EMSA), and the suppression of NF-κB target genes were detected using real-time RT-PCR and ELISA. These results enabled us to identify the decreased phosphorylation levels of IKBα. We focused on the gene of negative regulation of NF-κB, represented by TNF-α-induced protein 3 (TNFAIP3, also known as A20). Reducing the levels of A20 with siRNAs significantly enhances luciferase activation of NF-κB. Furthermore, HEV ORF3 regulated A20 primarily via activating transcription factor 6 (ATF6), involved in unfolded protein response (UPR), resulting in the degradation or inactivation of the receptor interacting protein 1 (RIP1), a major upstream activator of IKB kinase compounds (IKKs). Consequently, the phosphorylation of IKBα and the nucleus translocation of p65 are blocked, which contributes to diminished NF-κB DNA-binding activation and NF-κB-dependent gene expression. The findings suggest that genotype 1 HEV, through ORF3, may transiently activate NF-κB through UPR in early stage, and subsequently inhibit TNF-α-induced NF-κB signaling in late phase so as to create a favorable virus replication environment.

SUBMITTER: Xu J

PROVIDER: S-EPMC4069097 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pegylated interferon plus ribavirin therapy for hepatitis C virus (HCV) fails in approximately half of genotype 1 patients. Treatment failure occurs either by nonresponse (minimal declines in viral titer) or relapse (robust initial responses followed by rebounds of viral titers during or after therapy). HCV is highly variable genetically. To determine if viral genetic differences contribute to the difference between response and relapse, we examined the inter-patient genetic diversity and mutation pattern in the full open reading frame HCV genotype 1a consensus sequences.Pre- and post-therapy sequences were analyzed for 10 nonresponders and 10 relapsers from the Virahep-C clinical study. Pre-therapy interpatient diversity among the relapsers was higher than in the nonresponders in the viral NS2 and NS3 genes, and post-therapy diversity was higher in the relapsers for most of HCV's ten genes. Pre-therapy diversity among the relapsers was intermediate between that of the non-responders and responders to therapy. The average mutation rate was just 0.9% at the amino acid level and similar numbers of mutations occurred in the nonresponder and relapser sequences, but the mutations in NS2 of relapsers were less conservative than in nonresponders. Finally, the number and distribution of regions under positive selection was similar between the two groups, although the nonresponders had more foci of positive selection in E2.The HCV sequences were unexpectedly stable during failed antiviral therapy, both nonresponder and relapser sequences were under selective pressure during therapy, and variation in NS2 may have contributed to the difference in response between the nonresponder and relapser groups. These data support a role for viral genetic variability in determining the outcome of anti-HCV therapy, with those sequences that are more distant from an optimal sequence being less able to resist the pressures of interferon-based therapy.ClinicalTrials.gov NCT00038974.

Project description:Translation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as UUG, ACG and AUU are also used as start sites at low levels in S. cerevisiae. Initiation from NCCs or AUGs in the 5’-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 ˚C relative to 30 ˚C and decreased efficiency at 20 ˚C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5’-leader relative to the 5’-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. Overall, our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

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Open reading frame 3 of genotype 1 hepatitis E virus inhibits nuclear factor-κappa B signaling induced by tumor necrosis factor-α in human A549 lung epithelial cells.

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