RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential cis-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5 and 3 ends of the RNA genome should provide new insights into HCV RNA replication.The first molecular clones of hepatitis C virus (HCV) were reported in 1989 (11). Comparative sequence analysis revealed that HCV is related to flavi-and pestiviruses (12), and HCV was subsequently placed in the family Flaviviridae. The genomic RNA of viruses in this family has a long open reading frame (ORF) flanked by nontranslated regions (NTRs) at the 5Ј and 3Ј termini (48). HCV initiates translation of the ORF via an internal ribosome entry site (IRES) (25, 51). Translation of the ORF yields a polyprotein that is cleaved co-and posttranslationally by host and viral proteases. The HCV polyprotein gives rise to 10 viral proteins: core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (3). NS5B, the viral RNAdependent RNA polymerase, comprises the C-terminal portion of the polyprotein. The 5Ј and 3Ј NTRs of HCV have cis-acting replication elements (CREs) which are essential for the viral life cycle (14,32,33,36,70) and are known to bind cellular proteins (15,21,26,46,57,67).Coding regions of viral RNAs often have embedded nucleic acid signals and overlapping reading frames that encode proteins, as in X174 (52), hepatitis B virus (16), and Rous sarcoma virus (27). Embedded RNA signals include promoters (29), nucleation sites for encapsidation (2,30,35,49), and other types of CREs (5,18,40,43,44,53). Among the best characterized is the picornavirus CRE (17,18,40,43,45,71). The picornavirus CRE acts as a template for the uridylylation of VPg, the protein primer for genome replication, and thus plays a direct role in initiation of RNA replication. The exac...

Background Chronic liver disease (CLD) represents a major global health burden. We undertook this study to identify the factors associated with adverse outcomes in patients with CLD who acquire the novel coronavirus-2019 (COVID-19). Methods We conducted a multi-center, observational cohort study across 21 institutions in the United States (US) of adult patients with CLD and laboratory-confirmed diagnosis of COVID-19 between March 1, 2020 and May 30, 2020. We performed survival analysis to identify independent predictors of all-cause mortality and COVID-19 related mortality, and multivariate logistic regression to determine the risk of severe COVID-19 in patients with CLD. Results Of the 978 patients in our cohort, 867 patients (mean age 56.9±14.5 years, 55% male) met inclusion criteria. The overall all-cause mortality was 14.0% (n = 121), and 61.7% (n = 535) had severe COVID-19. Patients presenting with diarrhea or nausea/vomiting were more likely to have severe COVID-19. The liver-specific factors associated with independent risk of higher overall mortality were alcohol-related liver disease (ALD) (hazard ratio [HR] 2.42, 95% confidence interval [CI] 1.29-4.55), decompensated cirrhosis (HR 2.91 [1.70-5.00]) and hepatocellular carcinoma (HCC) (HR 3.31 [1.53-7.16]). Other factors were increasing age, diabetes, hypertension, chronic obstructive pulmonary disease and current smoker. Hispanic ethnicity (odds ratio [OR] 2.33 [1.47-3.70]) and decompensated cirrhosis (OR 2.50 [1.20-5.21]) were independently associated with risk for severe COVID-19. Conclusions The risk factors which predict higher overall mortality among patients with CLD and COVID-19 are ALD, decompensated cirrhosis and HCC. Hispanic ethnicity and decompensated cirrhosis are associated with severe COVID-19. Our results will enable risk stratification and personalization of the management of patients with CLD and COVID-19.

Experimental data concerning viroid-specific nucleic acids accumulating in tomato plants establish, together with earlier studies, the major features of a replication cycle for viroid RNA in plant cells. Many features of this pathway, which involves multimeric strands of both polarities, may be shared by other small infectious RNA's including certain satellite RNA's and "virusoid" RNA's which replicate in conjunction with conventional plant viruses. The presence, in host plans, of an elaborate machinery for replicating these disease agents suggests a role for endogenous small RNA's in cellular development.

Many viruses have overlapping genes and/or regions in which a nucleic acid signal is embedded in a coding sequence. To search for dual-use regions in the hepatitis C virus (HCV), we developed a facile computer-based sequence analysis method to map dual-use regions in coding sequences. Eight diverse full-length HCV RNA and polyprotein sequences were aligned and analyzed. A cluster of unusually conserved synonymous codons was found in the core-encoding region, indicating a potential overlapping open reading frame (ORF). Four peptides (A1, A2, A3, and A4) representing this alternate reading frame protein (ARFP), two others from the HCV core protein, and one from bovine serum albumin (BSA) were conjugated to BSA and used in western blots to test sera for specific antibodies from 100 chronic HCV patients, 44 healthy controls, and 60 patients with non-HCV liver disease. At a 1:20,000 dilution, specific IgGs to three of the four ARFP peptides were detected in chronic HCV sera. Reactivity to either the A1 or A3 peptides (both ARFP derived) was significantly associated with chronic HCV infection, when compared to non-HCV liver disease serum samples (10/100 versus 1/60; p , 0.025). Antibodies to A4 were not detected in any serum sample. Our western blot assays confirmed the presence of specific antibodies to a new HCV antigen encoded, at least in part, in an alternate reading frame (ARF) overlapping the core-encoding region. Because this novel HCV protein stimulates specific immune responses, it has potential value in diagnostic tests and as a component of vaccines. This protein is predicted to be highly basic and may play a role in HCV replication, pathogenesis, and carcinogenesis.

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Background: HMGB1 is a proinflammatory cytokine produced in response to tissue injury, but its role in ALD is unknown.Results: HMGB1 increases; translocates; and undergoes acetylation, phosphorylation, and oxidation in ALD. HMGB1 ablation in hepatocytes protects against steatosis and injury in ALD.Conclusion: HMGB1 plays a key role in ALD.Significance: Dissecting how the increase in HMGB1 causes hepatotoxicity is key for understanding the pathogenesis of ALD.

In the core protein-coding region of hepatitis C virus (HCV), evidence exists for both phylogenetically conserved RNA structures and a ؉1 alternative reading frame (ARF). To investigate its role in HCV infection, we introduced four stop codons into the ARF of a genotype 1a H77 molecular clone. The changes did not alter the core protein sequence, but were predicted to disrupt RNA secondary structures. An attenuated infection was established after inoculation of the mutant HCV RNA into an HCV naïve chimpanzee. The acute infection was atypical with low peak viremia, minimal alanine aminotransferase elevation, and early virus control by a diverse adaptive immune response. Sequencing circulating virus revealed progressive reversions at the third and then fourth stop codon. In cell culture, RNA replication of a genome with four stop codons was severely impaired. In contrast, the revertant genome exhibited only a 5-fold reduction in replication. Genomes harboring only the first two stop codons replicated to WT levels. Similarly, reversions at stop codons 3 and 4, which improved replication, were selected with recombinant, infectious HCV in cell culture. We conclude that ARF-encoded proteins initiating at the polyprotein AUG are not essential for HCV replication in cell culture or in vivo. Rather, our results provide evidence for a functionally important RNA element in the ARF region.alternative reading frame ͉ RNA replication ͉ RNA structure