A computational approach identifies two regions of Hepatitis C Virus E1 protein as interacting domains involved in viral fusion process

<p>Abstract</p> <p>Background</p> <p>The E1 protein of Hepatitis C Virus (HCV) can be dissected into two distinct hydrophobic regions: a central domain containing an hypothetical fusion peptide (FP), and a C-terminal domain (CT) comprising two segments, a pre-anchor and a trans-membrane (TM) region. In the currently accepted model of the viral fusion process, the FP and the TM regions are considered to be closely juxtaposed in the post-fusion structure and their physical interaction cannot be excluded. In the present study, we took advantage of the natural sequence variability present among HCV strains to test, by purely sequence-based computational tools, the hypothesis that in this virus the fusion process involves the physical interaction of the FP and CT regions of E1.</p> <p>Results</p> <p>Two computational approaches were applied. The first one is based on the co-evolution paradigm of interacting peptides and consequently on the correlation between the distance matrices generated by the sequence alignment method applied to FP and CT primary structures, respectively. In spite of the relatively low random genetic drift between genotypes, co-evolution analysis of sequences from five HCV genotypes revealed a greater correlation between the FP and CT domains than respect to a control HCV sequence from Core protein, so giving a clear, albeit still inconclusive, support to the physical interaction hypothesis.</p> <p>The second approach relies upon a non-linear signal analysis method widely used in protein science called Recurrence Quantification Analysis (RQA). This method allows for a direct comparison of domains for the presence of common hydrophobicity patterns, on which the physical interaction is based upon. RQA greatly strengthened the reliability of the hypothesis by the scoring of a lot of cross-recurrences between FP and CT peptides hydrophobicity patterning largely outnumbering chance expectations and pointing to putative interaction sites. Intriguingly, mutations in the CT region of E1, reducing the fusion process <it>in vitro</it>, strongly reduced the amount of cross-recurrence further supporting interaction between this region and FP.</p> <p>Conclusion</p> <p>Our results support a fusion model for HCV in which the FP and the C-terminal region of E1 are juxtaposed and interact in the post-fusion structure. These findings have general implications for viruses, as any visualization of the post-fusion FP-TM complex has been precluded by the impossibility to obtain crystallised viral fusion proteins containing the trans-membrane region. This limitation gives to sequence based modelling efforts a crucial role in the sketching of a molecular interpretation of the fusion process. Moreover, our data also have a more general relevance for cell biology as the mechanism of intracellular fusion showed remarkable similarities with viral fusion</p

Microarray analysis identifies a common set of cellular genes modulated by different HCV replicon clones

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) RNA synthesis and protein expression affect cell homeostasis by modulation of gene expression. The impact of HCV replication on global cell transcription has not been fully evaluated. Thus, we analysed the expression profiles of different clones of human hepatoma-derived Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system).</p> <p>Results</p> <p>First, we compared the expression profile of HCV replicon clone 21-5 with both the Huh-7 parental cells and the 21-5 cured (21-5c) cells. In these latter, the HCV RNA has been eliminated by IFN-α treatment. To confirm data, we also analyzed microarray results from both the 21-5 and two other HCV replicon clones, 22-6 and 21-7, compared to the Huh-7 cells. The study was carried out by using the Applied Biosystems (AB) Human Genome Survey Microarray v1.0 which provides 31,700 probes that correspond to 27,868 human genes. Microarray analysis revealed a specific transcriptional program induced by HCV in replicon cells respect to both IFN-α-cured and Huh-7 cells. From the original datasets of differentially expressed genes, we selected by Venn diagrams a final list of 38 genes modulated by HCV in all clones. Most of the 38 genes have never been described before and showed high fold-change associated with significant p-value, strongly supporting data reliability. Classification of the 38 genes by Panther System identified functional categories that were significantly enriched in this gene set, such as histones and ribosomal proteins as well as extracellular matrix and intracellular protein traffic. The dataset also included new genes involved in lipid metabolism, extracellular matrix and cytoskeletal network, which may be critical for HCV replication and pathogenesis.</p> <p>Conclusion</p> <p>Our data provide a comprehensive analysis of alterations in gene expression induced by HCV replication and reveal modulation of new genes potentially useful for selection of antiviral targets.</p

A computational approach to identify point mutations associated with occult hepatitis B: significant mutations affect coding regions but not regulative elements of HBV

<p>Abstract</p> <p>Background</p> <p>Occult Hepatitis B Infection (OBI) is characterized by absence of serum HBsAg and persistence of HBV-DNA in liver tissue, with low to undetectable serum HBV-DNA. The mechanisms underlying OBI remain to be clarified. To evaluate if specific point mutations of HBV genome may be associated with OBI, we applied an approach based on bioinformatics analysis of complete genome HBV sequences. In addition, the feasibility of bioinformatics prediction models to classify HBV infections into OBI and non-OBI by molecular data was evaluated.</p> <p>Methods</p> <p>41 OBI and 162 non-OBI complete genome sequences were retrieved from GenBank, aligned and subjected to univariable analysis including statistical evaluation. Their S coding region was analyzed for Stop codon mutations too, while S amino acid variability could be evaluated for genotype D only, due to the too small number of available complete genome OBI sequences from other genotypes.</p> <p>Prediction models were derived by multivariable analysis using Logistic Regression, Rule Induction and Random Forest approaches, with extra-sample error estimation by Multiple ten-fold Cross-Validation (MCV). Models were compared by t-test on the Area Under the Receiver Operating Characteristic curve (AUC) distributions obtained from the MCV runs for each model against the best-performing model.</p> <p>Results</p> <p>Variations in seven nucleotide positions were significantly associated with OBI, and occurred in 11 out of 41 OBI sequences (26.8%): likely, other mutations did not reach statistical significance due to the small size of OBI dataset. All variations affected at least one HBV coding region, but none of them mapped to regulative elements. All viral proteins, with the only exception of the X, were affected. Stop codons in the S, that might account for absence of serum HBsAg, were not significantly enriched in OBI sequences. In genotype D, amino acid variability in the S was higher in OBI than non-OBI, particularly in the immunodominant region. A Random Forest prediction model showed the best performance, but all models were not satisfactory in terms of specificity, due to the small sample size of OBIs; however results are promising in the perspective of a broader dataset of complete genome OBI sequences.</p> <p>Conclusions</p> <p>Data suggest that point mutations rarely occur in regulative elements of HBV, if ever, and contribute to OBI by affecting different viral proteins, suggesting heterogeneous mechanisms may be responsible for OBI, including, at least in genotype D, an escape mutation mechanism due to imperfect immune control. It appears possible to derive prediction models based on molecular data when a larger set of complete genome OBI sequences will become available.</p

How future surgery will benefit from SARS-COV-2-related measures: a SPIGC survey conveying the perspective of Italian surgeons

COVID-19 negatively affected surgical activity, but the potential benefits resulting from adopted measures remain unclear. The aim of this study was to evaluate the change in surgical activity and potential benefit from COVID-19 measures in perspective of Italian surgeons on behalf of SPIGC. A nationwide online survey on surgical practice before, during, and after COVID-19 pandemic was conducted in March-April 2022 (NCT:05323851). Effects of COVID-19 hospital-related measures on surgical patients' management and personal professional development across surgical specialties were explored. Data on demographics, pre-operative/peri-operative/post-operative management, and professional development were collected. Outcomes were matched with the corresponding volume. Four hundred and seventy-three respondents were included in final analysis across 14 surgical specialties. Since SARS-CoV-2 pandemic, application of telematic consultations (4.1% vs. 21.6%; p &lt; 0.0001) and diagnostic evaluations (16.4% vs. 42.2%; p &lt; 0.0001) increased. Elective surgical activities significantly reduced and surgeons opted more frequently for conservative management with a possible indication for elective (26.3% vs. 35.7%; p &lt; 0.0001) or urgent (20.4% vs. 38.5%; p &lt; 0.0001) surgery. All new COVID-related measures are perceived to be maintained in the future. Surgeons' personal education online increased from 12.6% (pre-COVID) to 86.6% (post-COVID; p &lt; 0.0001). Online educational activities are considered a beneficial effect from COVID pandemic (56.4%). COVID-19 had a great impact on surgical specialties, with significant reduction of operation volume. However, some forced changes turned out to be benefits. Isolation measures pushed the use of telemedicine and telemetric devices for outpatient practice and favored communication for educational purposes and surgeon-patient/family communication. From the Italian surgeons' perspective, COVID-related measures will continue to influence future surgical clinical practice

Repression of Interferon Regulatory Factor 1 by Hepatitis C Virus Core Protein Results in Inhibition of Antiviral and Immunomodulatory Genes

Hepatitis C virus (HCV) proteins are known to interfere at several levels with both innate and adaptive responses of the host. A key target in these effects is the interferon (IFN) signaling pathway. While the effects of nonstructural proteins are well established, the role of structural proteins remains controversial. We investigated the effect of HCV structural proteins on the expression of interferon regulatory factor 1 (IRF-1), a secondary transcription factor of the IFN system responsible for inducing several key antiviral and immunomodulatory genes. We found substantial inhibition of IRF-1 expression in cells expressing the entire HCV replicon. Suppression of IRF-1 synthesis was mainly mediated by the core structural protein and occurred at the transcriptional level. The core protein in turn exerted a transcriptional repression of several interferon-stimulated genes, targets of IRF-1, including interleukin-15 (IL-15), IL-12, and low-molecular-mass polypeptide 2. These data recapitulate in a unifying mechanism, i.e., repression of IRF-1 expression, many previously described pathogenetic effects of HCV core protein and suggest that HCV core-induced IRF-1 repression may play a pivotal role in establishing persistent infection by dampening an effective immune response

An integrated approach identifies IFN-regulated microRNAs and targeted mRNAs modulated by different HCV replicon clones

Abstract Background Infections with hepatitis C virus (HCV) progress to chronic phase in 80% of patients. To date, the effect produced by HCV on the expression of microRNAs (miRs) involved in the interferon-β (IFN-β) antiviral pathway has not been explored in details. Thus, we compared the expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in three different clones of Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system). Methods The expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in HCV replicon 21-5 clone with respect to Huh-7 parental cells was analysed by real-time PCR. To exclude clone specific variations, the level of 16 out of 24 miRs, found to be modulated in 21-5 clone, was evaluated in two other HCV replicon clones, 22-6 and 21-7. Prediction of target genes of 3 miRs, confirmed in all HCV clones, was performed by means of miRGator program. The gene dataset obtained from microarray analysis of HCV clones was farther used to validate target prediction. Results The expression profile revealed that 16 out of 24 miRs were modulated in HCV replicon clone 21-5. Analysis in HCV replicon clones 22-6 and 21-7 indicated that 3 out of 16 miRs, (miR-128a, miR-196a and miR-142-3p) were modulated in a concerted fashion in all three HCV clones. Microarray analysis revealed that 37 out of 1981 genes, predicted targets of the 3 miRs, showed an inverse expression relationship with the corresponding miR in HCV clones, as expected for true targets. Classification of the 37 genes by Panther System indicated that the dataset contains genes involved in biological processes that sustain HCV replication and/or in pathways potentially implicated in the control of antiviral response by HCV infection. Conclusions The present findings reveal that 3 IFN-β-regulated miRs and 37 genes, which are likely their functional targets, were commonly modulated by HCV in three replicon clones. The future use of miR inhibitors or mimics and/or siRNAs might be useful for the development of diagnostic and therapeutic strategies aimed at the recovering of protective innate responses in HCV infections.</p