HCV Defective Genomes Promote Persistent Infection by Modulating the Viral Life Cycle

Defective interfering (DI) RNAs have been detected in several human viruses. HCV in-frame deletions mutants (IFDMs), missing mainly the envelope proteins, have been found in patient sera and liver tissues. IFDMs replicate independently and can be trans-packaged into infectious virions in the presence of full length viral genome. So far, their biological role is unclear. In this study, we have isolated and cloned IFDMs from sera samples and liver tissues of patients infected with HCV genotypes 1b, 2a, and 3a. IFDMs were present in up to 26% of samples tested. Using the in vitro HCV cell culture system, co-expression of the wild type (wt) HCV replicon with HCV IFDMs RNA resulted in increased HCV replication. Additionally, co-transfection of the HCV full length genome RNA and a defective mutant missing the envelope region led to increased viral release, collectively suggesting an important biological role for IFDMs in the virus life cycle. Recently, exosomes, masters of intercellular communication, have been implicated in the transport of HCV viral genomes. We report for the first time that exosomal RNA isolated from HCV sera samples contains HCV defective genomes. We also demonstrate that inhibition of exosomal biogenesis and release influences HCV viral replication. Overall, we provide evidence that the presence of HCV IFDMs affects both viral replication and release. IFDMs exploit exosomes as means of transport, a way to evade the immune system, to spread more efficiently and possibly maintain persistent infection

Targeting the YXXΦ Motifs of the SARS Coronaviruses 1 and 2 ORF3a Peptides by In Silico Analysis to Predict Novel Virus—Host Interactions

The emerging SARS-CoV and SARS-CoV-2 belong to the family of “common cold” RNA coronaviruses, and they are responsible for the 2003 epidemic and the current pandemic with over 6.3 M deaths worldwide. The ORF3a gene is conserved in both viruses and codes for the accessory protein ORF3a, with unclear functions, possibly related to viral virulence and pathogenesis. The tyrosine-based YXXΦ motif (Φ: bulky hydrophobic residue—L/I/M/V/F) was originally discovered to mediate clathrin-dependent endocytosis of membrane-spanning proteins. Many viruses employ the YXXΦ motif to achieve efficient receptor-guided internalisation in host cells, maintain the structural integrity of their capsids and enhance viral replication. Importantly, this motif has been recently identified on the ORF3a proteins of SARS-CoV and SARS-CoV-2. Given that the ORF3a aa sequence is not fully conserved between the two SARS viruses, we aimed to map in silico structural differences and putative sequence-driven alterations of regulatory elements within and adjacently to the YXXΦ motifs that could predict variations in ORF3a functions. Using robust bioinformatics tools, we investigated the presence of relevant post-translational modifications and the YXXΦ motif involvement in protein-protein interactions. Our study suggests that the predicted YXXΦ-related features may confer specific—yet to be discovered—functions to ORF3a proteins, significant to the new virus and related to enhanced propagation, host immune regulation and virulence

Hepatitis C Core Protein Induces a Genotype-Specific Susceptibility of Hepatocytes to TNF-Induced Death In Vitro and In Vivo

Hepatitis C virus (HCV) core protein is a multifunctional protein that is involved in the proliferation, inflammation, and apoptosis mechanism of hepatocytes. HCV core protein genetic variability has been implicated in various outcomes of HCV pathology and treatment. In the present study, we aimed to analyze the role of the HCV core protein in tumor necrosis factor α (TNFα)-induced death under the viewpoint of HCV genetic variability. Immortalized hepatocytes (IHH), and not the Huh 7.5 hepatoma cell line, stably expressing HCV subtype 4a and HCV subtype 4f core proteins showed that only the HCV 4a core protein could increase sensitivity to TNFα-induced death. Development of two transgenic mice expressing the two different core proteins under the liver-specific promoter of transthyretin (TTR) allowed for the in vivo assessment of the role of the core in TNFα-induced death. Using the TNFα-dependent model of lipopolysaccharide/D-galactosamine (LPS/Dgal), we were able to recapitulate the in vitro results in IHH cells in vivo. Transgenic mice expressing the HCV 4a core protein were more susceptible to the LPS/Dgal model, while mice expressing the HCV 4f core protein had the same susceptibility as their littermate controls. Transcriptome analysis in liver biopsies from these transgenic mice gave insights into HCV core molecular pathogenesis while linking HCV core protein genetic variability to differential pathology in vivo

Correction: Synonymous Mutations in the Core Gene Are Linked to Unusual Serological Profile in Hepatitis C Virus Infection

International audienceThe biological role of the protein encoded by the alternative open reading frame (core+1/ARF) of the Hepatitis C virus (HCV) genome remains elusive, as does the significance of the production of corresponding antibodies in HCV infection. We investigated the prevalence of anti-core and anti-core+1/ARFP antibodies in HCV-positive blood donors from Cambodia, using peptide and recombinant protein-based ELISAs. We detected unusual serological profiles in 3 out of 58 HCV positive plasma of genotype 1a. These patients were negative for anti-core antibodies by commercial and peptide-based assays using C-terminal fragments of core but reacted by Western Blot with full-length core protein. All three patients had high levels of anti-core+1/ARFP antibodies. Cloning of the cDNA that corresponds to the core-coding region from these sera resulted in the expression of both core and core+1/ARFP in mammalian cells. The core protein exhibited high amino-acid homology with a consensus HCV1a sequence. However, 10 identical synonymous mutations were found, and 7 were located in the aa(99-124) region of core. All mutations concerned the third base of a codon, and 5/10 represented a T>C mutation. Prediction analyses of the RNA secondary structure revealed conformational changes within the stem-loop region that contains the core+1/ARFP internal AUG initiator at position 85/87. Using the luciferase tagging approach, we showed that core+1/ARFP expression is more efficient from such a sequence than from the prototype HCV1a RNA. We provide additional evidence of the existence of core+1/ARFP in vivo and new data concerning expression of HCV core protein. We show that HCV patients who do not produce normal anti-core antibodies have unusually high levels of antit-core+1/ARFP and harbour several identical synonymous mutations in the core and core+1/ARFP coding region that result in major changes in predicted RNA structure. Such HCV variants may favour core+1/ARFP production during HCV infection

Hepatitis C virus modulates lipid regulatory factor Angiopoietin-like 3 gene expression by repressing HNF-1 alpha activity

Background & Aims: HCV relies on host lipid metabolism to complete its life cycle and HCV core is crucial to this interaction. Liver secreted ANGPTL-3 is an LXR-and HNF-1 alpha-regulated protein, which plays a key role in lipid metabolism by increasing plasma lipids via inhibition of lipase enzymes. Here we aimed to investigate the modulation of ANGPTL-3 by HCV core and identify the molecular mechanisms involved. Methods: qRT-PCR and ELISA were used to assess ANGPTL-3 mRNA and protein levels in HCV patients, the JFH-1 infectious system and liver cell lines. Transfections, chromatin immunoprecipitation and immunofluorescence delineated parts of the molecular mechanisms implicated in the core-mediated regulation of ANGPTL-3 gene expression. Results: ANGPTL-3 gene expression was decreased in HCV-infected patients and the JFH-1 infectious system. mRNA and promoter activity levels were down-regulated by core. The response was lost when an HNF-1 alpha element in ANGPTL-3 promoter was mutated, while loss of HNF-1 alpha DNA binding to this site was recorded in the presence of HCV core. HNF-1 alpha mRNA and protein levels were not altered by core. However, trafficking between nucleus and cytoplasm was observed and then blocked by an inhibitor of the HNF-1 alpha-specific kinase Mirk/Dyrk1B. Transactivation of LXR/RXR signalling could not restore coremediated down-regulation of ANGPTL-3 promoter activity. Conclusions: ANGPTL-3 is negatively regulated by HCV in vivo and in vitro. HCV core represses ANGPTL-3 expression through loss of HNF-1 alpha binding activity and blockage of LXR/RXR transactivation. The putative ensuing increase in serum lipid clearance and uptake by the liver may sustain HCV virus replication and persistence. (C) 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

HCV Defective Genomes Promote Persistent Infection by Modulating the Viral Life Cycle

Defective interfering (DI) RNAs have been detected in several human viruses. HCV in-frame deletions mutants (IFDMs), missing mainly the envelope proteins, have been found in patient sera and liver tissues. IFDMs replicate independently and can be trans-packaged into infectious virions in the presence of full length viral genome. So far, their biological role is unclear. In this study, we have isolated and cloned IFDMs from sera samples and liver tissues of patients infected with HCV genotypes 1b, 2a, and 3a. IFDMs were present in up to 26% of samples tested. Using the in vitro HCV cell culture system, co-expression of the wild type (wt) HCV replicon with HCV IFDMs RNA resulted in increased HCV replication. Additionally, co-transfection of the HCV full length genome RNA and a defective mutant missing the envelope region led to increased viral release, collectively suggesting an important biological role for IFDMs in the virus life cycle. Recently, exosomes, masters of intercellular communication, have been implicated in the transport of HCV viral genomes. We report for the first time that exosomal RNA isolated from HCV sera samples contains HCV defective genomes. We also demonstrate that inhibition of exosomal biogenesis and release influences HCV viral replication. Overall, we provide evidence that the presence of HCV IFDMs affects both viral replication and release. IFDMs exploit exosomes as means of transport, a way to evade the immune system, to spread more efficiently and possibly maintain persistent infection

Amino-acid sequence alignment of the core region derived from three HCV-1a isolates positive for anti-core antibodies (p29, p30, p36) and those that were negative for anti-core antibodies (p31, p32, p34).

<p>cDNA fragments from the HCV core region were obtained and cloned into the TOPO TA vector as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015871#s2" target="_blank">Material and Methods</a>. Three clones were sequenced for each patient and all three were identical for each patient. Protein sequences were analyzed using the CLUSTAL W program. The amino-acid sequence of the HCV-1a H reference strain is shown.</p

Deduced core+1/ARFP amino-acid sequences from cDNA shown in <b>Figure 5</b>.

<p>The sequences relate to isolates positive for anti-core antibodies (p29, p30, p36) and the three HCV-1a isolates negative for anti-core antibodies (p31, p32, p34). The corresponding amino-acid sequence of the consensus genotype 1a HCV is also shown.</p

The prevalence of anti-core antibodies in a panel of 58 HCV-positive plasma samples from Cambodia, detected by ELISA based on core synthetic peptide aa(3–75).

<p>The panel included 28 plasma samples from individuals infected with HCV-6; 21 HCV-1b; 8 HCV-1a; and 1 HCV-2a samples. Twenty-nine HCV-negative samples from individuals living in Cambodia were included in the panel as negative controls. Three of 58 plasma samples (all from HCV-1a infected individuals, designated as p31, p32 and p34) were found negative for anti-core antibodies.</p

Serological profiles in Cambodian patients infected with genotype 1a HCV.

<p>The absence of antibodies directed to epitopes localized in the immunodominant region of core aa(1–120) in plasma samples designated as p31, p32, p34 was confirmed using Chiron RIBA HCV 3.0 Strip Immunoblot Assay (SIA) (Novartis Vaccines and Diagnostics, Inc., Emeryville, CA). Virus load in all these plasma samples of 1a genotype was determined by b-DNA assay (Versant HCV RNA 3.0, Siemmens Health Care Diagnostics St. Denis, France) and the presence of HCV core protein was determined by a commercial ELISA assay Ortho Diagnostics (Wako, Germany).</p