Ultrasensitive methods to measure very low levels of hepatitis C virus (HCV) RNA in biological samples may have diagnostic and prognostic significance and be useful to evaluate the response to antiviral treatment. A sensitive assay to quantify HCV RNA in peripheral blood mononuclear cells (PBMCs) was developed and validated using the iCycler iQ™ Detection System (Bio-Rad) coupled with TaqMan® chemistry. HCV was co-amplified with the endogenous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex reaction. Calculated PCR amplification efficiencies for both target and control genes were used in a mathematical model for relative quantitation of HCV RNA. A linear relationship between input RNA and CT values over 6 log dilutions was observed for both HCV- and GAPDH-specific products (R2 ≥ 0.99). As few as 1.5 IU/reaction could be detected, with high accuracy (CV ≤ 3.94%) and reproducibility (CV ≤ 2.20%). Quantitation of HCV RNA levels ranging from 103 to 107 IU/ml as measured in 47 plasma samples was highly correlated with values obtained by the COBAS Amplicor™ HCV Monitor test, v2.0 (Roche) (R2 = 0.977). In conclusion, this assay provides an excellent tool to determine accurately HCV kinetics in PBMCs during antiviral therapy and to assess the long-term significance of different patterns of response to treatment.

HepatologyVolume 35, Issue 1 p. 239-240 CorrespondenceFree Access The 4,977–base pair common deletion of mitochondrial DNA is not associated with steatosis in chronic hepatitis C patients Patrizia Latorre Ph.D., Patrizia Latorre Ph.D. Division of Gastroenterology and Hepatology, University Hospital, Geneva, SwitzerlandSearch for more papers by this authorLaura Rubbia-Brandt M.D, Ph.D., Laura Rubbia-Brandt M.D, Ph.D. Division of Clinical Pathology, University Hospital, Geneva, SwitzerlandSearch for more papers by this authorEmiliano Giostra M.D., Emiliano Giostra M.D. Division of Gastroenterology and Hepatology, University Hospital, Geneva, SwitzerlandSearch for more papers by this authorKarim Abid B.Sc., Karim Abid B.Sc. Division of Gastroenterology and Hepatology, University Hospital, Geneva, SwitzerlandSearch for more papers by this authorFrancesco Negro M.D., Francesco Negro M.D. Division of Gastroenterology and Hepatology, University Hospital, Geneva, Switzerland Division of Clinical Pathology, University Hospital, Geneva, SwitzerlandSearch for more papers by this author Patrizia Latorre Ph.D., Patrizia Latorre Ph.D. Division of Gastroenterology and Hepatology, University Hospital, Geneva, SwitzerlandSearch for more papers by this authorLaura Rubbia-Brandt M.D, Ph.D., Laura Rubbia-Brandt M.D, Ph.D. Division of Clinical Pathology, University Hospital, Geneva, SwitzerlandSearch for more papers by this authorEmiliano Giostra M.D., Emiliano Giostra M.D. Division of Gastroenterology and Hepatology, University Hospital, Geneva, SwitzerlandSearch for more papers by this authorKarim Abid B.Sc., Karim Abid B.Sc. Division of Gastroenterology and Hepatology, University Hospital, Geneva, SwitzerlandSearch for more papers by this authorFrancesco Negro M.D., Francesco Negro M.D. Division of Gastroenterology and Hepatology, University Hospital, Geneva, Switzerland Division of Clinical Pathology, University Hospital, Geneva, SwitzerlandSearch for more papers by this author First published: 30 December 2003 https://doi.org/10.1053/jhep.2002.30426Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume35, Issue1January 2002Pages 239-240 ReferencesRelatedInformation

The viral protein 2A of hepatitis A virus (HAV) lacks the conserved 18 aa sequence found in other picornavirus proteases; hence, it is unclear whether the induction of CPE by culture-adapted HAV strains is due to 2A-mediated activity. Moreover, the cleavage sites and actual borders of HAV 2A are not known. Accordingly, a nested series of cDNA sequences encoding the segment of the HAV polyprotein (aa 760-1087) were linked to the 5'-UTR of poliovirus type 2 (Lansing strain) and inserted downstream of the gene encoding human growth hormone (GH). Following transfection of COS-1 cells, levels of GH (translation of which was entirely cap dependent) were determined in culture supernatants. Expression of HAV peptides extending from aa 764, 776 or 791 to 981 strongly inhibited cap-dependent translation of GH, whereas cap-independent expression of a reporter gene (CAT) directed by the poliovirus RNA 5'-UTR was unaffected. The inhibitory effect was absent in constructs expressing either the short peptide encompassing aa 760-836 or proteins initiated downstream of the putative cleavage site 836-837, suggesting that the boundaries of a functional HAV 2A may extend from the Gln/Ser junction 791-792 to residue 981, while peptides initiated at the Gln/Ala pair 836-837 may result from alternative cleavage. Point mutations that substituted members of the triad Ser(916), His(927) and Asp(931) abolished the inhibitory effect on cap-dependent translation, suggesting that the HAV-induced CPE may be mediated by 2A protein.

ABSTRACTWe have studied the relationship between the Sendai virus (SeV) C proteins (a nested set of four proteins initiated at different start codons) and the interferon (IFN)-mediated antiviral response in IFN-competent cells in culture. SeV strains containing wild-type or various mutant C proteins were examined for their ability (i) to induce an antiviral state (i.e., to prevent the growth of vesicular stomatitis virus [VSV] following a period of SeV infection), (ii) to induce the elevation of Stat1 protein levels, and (iii) to prevent IFN added concomitant with the SeV infection from inducing an antiviral state. We find that expression of the wild-type C gene and, specifically, the AUG114-initiated C protein prevents the establishment of an antiviral state: i.e., cells infected with wild-type SeV exhibited little or no increase in Stat1 levels and were permissive for VSV replication, even in the presence of exogenous IFN. In contrast, in cells infected with SeV lacking the AUG114-initiated C protein or containing a single amino acid substitution in the C protein, the level of Stat1 increased and VSV replication was inhibited. The prevention of the cellular IFN-mediated antiviral response appears to be a key determinant of SeV pathogenicity.

The Sendai virus (SeV) P/C mRNA expresses eight different polypeptide chains using a combination of ribosomal choice and cotranscriptional editing (an internal open reading frame (ORF) is accessed by the addition of a single G residue after a short run of Gs at position 1053 on the mRNA). The longest ORF within the mRNA starts at ATG104 (the second initiation site) and encodes the 568-aa P protein, an essential viral structural protein which serves both as a cofactor for the RNA-dependant RNA polymerase (L protein) and as a part of the assembly complex. The first (ACG81), third (ATG114), fourth (ATG183) and fifth (ATG201) initiation sites are used to express a C-terminal nested set of polypeptides which are in the +1 ORF relative to P, namely C′, C, Y1, and Y2, respectively (collectively named the C proteins). Leaky scanning accounts for translational initiation at the first three start sites (a non-ATG followed by ATGs in progressively stronger contexts). Consistent with this, changing the C′ ACG to an ATG (GCCATG81G; ATG81/C′) ablates all expression from the downstream ATG104/P and ATG114/C initiation codons, whereas initiation from ATG183/Y1 and ATG201/Y2 remains normal in this background. Initiation from ATG183/Y1/ ATG201/Y2 probably takes place by discontinuous scanning via a ribosomal shunt. Scanning complexes appear to assemble at the 5′ cap and then scan the first ≈30 nt of the 5′ UTR before being translocated to an acceptor site close to the Y initiation codons. No specific 5′ UTR or donor site sequence elements are required, and translation of the Y proteins continues even when their start codons are changed to ACG.

The Sendai virus P/C mRNA expresses eight primary translation products by using a combination of ribosomal choice and cotranscriptional mRNA editing. The longest open reading frame (ORF) of the mRNA starts at AUG104 (the second initiation site) and encodes the 568-amino-acid P protein, an essential subunit of the viral polymerase. The first (ACG81), third (ATG114), fourth (ATG183), and fifth (ATG201) initiation sites are used to express a C-terminal nested set of polypeptides (collectively named the C proteins) in the +1 ORF relative to P, namely, C', C, Y1, and Y2, respectively. Leaky scanning accounts for translational initiation at the first three start sites (a non-ATG followed by ATGs in progressively stronger contexts). Consistent with this, changing ACG81/C' to ATG (GCCATG81G) abrogates expression from the downstream ATG104/P and ATG114/C initiation codons. However, expression of the Y1 and Y2 proteins remains normal in this background. We now have evidence that initiation from ATG183/Y1 and ATG201/Y2 takes place via a ribosomal shunt or discontinuous scanning. Scanning complexes appear to assemble at the 5' cap and then scan ca. 50 nucleotides (nt) of the 5' untranslated region before being translocated to an acceptor site at or close to the Y initiation codons. No specific donor site sequences are required, and translation of the Y proteins continues even when their start codons are changed to ACG. Curiously, ATG codons (in good contexts) in the P ORF, placed either 16 nt upstream of Y1, 29 nt downstream of Y2, or between the Y1 and Y2 codons, are not expressed even in the ACGY1/ACGY2 background. This indicates that ATG183/Y1 and ATG201/Y2 are privileged start sites within the acceptor site. Our observations suggest that the shunt delivers the scanning complex directly to the Y start codons.

ABSTRACT Recombinant Sendai viruses were prepared which cannot express their Cprime, C, or Cprime plus C proteins due to mutation of their respective start codons ([Cprime-minus], [C-minus] and [double mutant], respectively). The [Cprime-minus] and [C-minus] stocks were similar to that of wild-type (wt) virus in virus titer and plaque formation, whereas the double-mutant stock had a much-reduced PFU or 50% egg infective dose/particle ratio and produced very small plaques. Relative to the wt virus infection, the [Cprime-minus] and [C-minus] infections of BHK cells resulted in significantly greater accumulation of viral RNAs, consistent with the known inhibitory effects of the Cprime and C proteins. The double-mutant infection, in contrast, was delayed in its accumulation of viral RNAs; however, once accumulation started, overaccumulation quickly occurred, as in the single-mutant infections. Our results suggest that the Cprime and C proteins both provide a common positive function early in infection, so that only the double mutant undergoes delayed RNA accumulation and exhibits the highly debilitated phenotype. Later in infection, the same proteins appear to act as inhibitors of RNA accumulation. In infections of mice, [Cprime-minus] was found to be as virulent as wt virus whereas [C-minus] was highly attenuated. These results suggest that the Cprime and C proteins cannot be functionally equivalent, since C can replace Cprime for virulence in mice whereas Cprime cannot replace C.