Relationship Between Amino Acid Substitutions Of Hepatitis C Virus Core Protein And The Antiviral Response To Pegylated Interferon

Hepatitis C Virus (HCV) infection in general maintains a fast development momentumworldwide, the scale of the global epidemic is estimated that about180million people,representing3%of the world’s population. In our country, about42million of patients aredirect involvement of chronic HCV carriers and are at risk of developing liver cirrhosis andhepatocellular carcinoma. The government notified of a sharp increase in the number ofannual new cases, from5.3million persons per year in2005to21.9million in2012, whichhas become a serious health problem. At present, treatments based on pegylated interferon(PEG-IFN), in combination with ribavirin (RBV), are mainstay regimen for combatingHCV infection. However, the rate of patients who achieve a sustained virological response(SVR) is different depending on multiple factors, such as HCV genotypes. Host-relatedfactors include the single nucleotide polymorphisms (SNPs) near theinterferon-gamma3-encoding interleukin-28B (IL-28B) gene yet cannot completely explainthis enormous antiviral response discrepancy, while IL28B rs12979860CC genotype,which is considered to be advantageous to antiviral treatment response, is dominated(94.16%) in Chinese people. Sustained virological response rates were17%～24%amongre-treatment of patients who are non-responders to initial therapy historically, meanwhile,patients relapse to initial responsive therapy in which an non-SVR rate of approximately44%～62%after the second course of standard therapy. The underlying mechanisms of thedifferent virological responses to treatment are still unclear. Previous data indicated thatvirus itself could contribute to resist the response of IFN therapy, on the other hand, itprompting us to analyze the genetic diversity of HCV genome to find the reasons for whythe same therapy regimen could never have the identical success, even when patientsinfected the same genotype of HCV. Core protein is an important component constitutes thenucleocapsid of HCV, which plays an important role in the interaction of virus protein and host cell protein. It participates in the regulation of different genes transcription orexpression and forms a complicated network together with other transcription factors,results in a series of immunological status changes in body, for instance, immune response,immune tolerance and autoimmune homeostasis. Because of the special properties, coreprotein has become a new research hot topic in recent years. The basal researchdemonstrated that core protein interacts with signal transducer and activator of transcription(STAT) signaling cascade, to decrease transcription and translation of the interferonstimulate genes (ISGs), suggesting that core protein may play an important r ole ininterferon resistance. Clinical proofs have shown that amino acid substitutions of site70and91in the core region were significantly discrepant between SVR and non-SVR patients,especially substitutions of70Glu and91Met in the core of genotype1b were independentpredictors of poor virological response to IFN therapy. Fusao et al. designed retroviralvectors expressing the HCV core proteins with substitutions of Arg/Leu, Arg/Met, Gln/Leu,Gln/Met at aa70/aa91, then transiently transfected or stably transducted into four types ofhepatocyte lines to evaluate antiviral responses to IFN-α, found the promoter activity levelsor the mRNA levels of IFN-stimulated genes, such as2’-5’-oligoadenylate synthetase, wereenhanced by the core transduction during IFN-α treatment, although there were nodifferences in antiviral responses among the cells expressing different core types. Kumthipet al. also could not find specific mutation(s) in core protein obtained from HCV-1andHCV-3infected patients that may be responsible for IFN-response failure. Taken together, itis controversial whether there is an association among amino acid substitutions in coreprotein and response to IFN-based treatment. In China, the most common genotypes ofHCV infection are1b,2a,3a/3b and6a, and the constituent ratio are51.9%,29.4%,8.9%,4.8%, respectively. The present study retrospectively built a subset of HCV RNA-positive,chronically HCV-infected patients from outpatients and inpatients, who received PEG-IFNtreatment for full course. Full-length core gene was amplified through nestedreverse-transcription polymerase chain reaction (RT-PCR), positions and profiles of aminoacid substitutions among the above four genotypes that differed relative to consensussequence were identified with Mutation Master software. The main aim of this study was toidentify whether any amino acid positions differed consistently with the differences inresponse to IFN therapy, to determine if the presence of core protein polymorphism was associated with the IFN-response which could be used as predictive therapeutic markersand would further guide the individualized therapy. A subaim was to identify if there werepotential genotype-specific aa mutational positions that might affect response of treatment,and if there were a certain degree of relevance executing in between them.The major results as follows:1. Hypermutated sites with mutation frequency exceeded10%occurred at positions70,75,91,106,110,147and187amino acid residues of HCV core protein among fourgenotypes, feeble but widely correlations arise between each site.2. Most amino acid mutational sites were genotype-specific bias and the hydrophilicor hydrophobic performance of a few active residues mutations were shifted that mightaffect response of treatment.3. The proportion of Glutamine(Q) and non-Q at position70, methionine(M) andnon-M at position91, threonine(T) and non-T at position110of the core protein weresignificantly different between SVR and non-SVR group (P=0.004,0.039and0.025,respectively).4. Gln(Q) at position70, Met(M) at position91and Thr(T) at position110, whichwere defined as substitutions of―sluggish and resistant‖to IFN response, seemed to becorrelated with non-SVR. Correspondingly, the odds ratio (OR) and the95%confidenceinterval (CI) were0.288(0.120～0.693),0.413(0.177～0.966) and0.346(0.133～0.896),respectively.5. Patients who carried two or more undesirable substitutions mentioned-abovesimultaneously in core protein tended to attain gentler and slower viral descent at earlieststages (week4and week8) of treatment (P=0.001).Conclusion:1. Amino acid substitutions of HCV core protein were not sporadic randomly, buthighly rife and unified, implying that the functions of core protein which counteract theeffects of IFN therapy can be impaired or repaired by amino acid substitutions at multiplepositions.2. This study identified amino acid substitutions at positions70,91and110of HCVcore protein could deem to be perferably predictors of VR to pegylated interferon. 3. In essence, mutations would correlate with clearance because there are only a fewpositions to optimize activity of core protein but many ways to interfere with its function.