PKR-eIF2alpha Phosphorylation Homology Domain Research Articles

Structural Modification in Hepatitis C Virus Envelope Protein; Potential Viral Strategy Against Interferon Therapy

Interferons (IFNs) are the main part of the immune system as a defensive response to viral infection. The phosphorylation domain of Hepatitis C virus (HCV) E2 envelope glycoprotein is homologous to the host key enzymes in IFN-mediated response. So, if host kinases show no preference on E2 protein phosphorylation over IFN triggering factors, the IFN pathway would be competently inhibited. The aim of this study was to see if the resistance of HCV infected patients to IFN-therapy has something to do with HCV E2 envelope glycoprotein. For this purpose, the partially sequenced HCV E2 proteins obtained from 15 patients that were confirmed to be sustainable, semi-resistant (patients with relapsing infection) and resistant to IFN-therapy. The peptide sequences from 15 patients were analysed as a matter of homology, physicochemical properties and predicted structures using MEGA4 software, ProtParam, PSIPRED and I-TASSER tools, respectively. Then, certain point mutations introduced to sequences that acquired from patients resistant to IFN-therapy. Replacement of Asp 653 with Asn 653 in R1 at C-terminal of the E2 protein changes secondary and tertiary structures of downstream regions of E2 protein. Although the sequence of PKR-eIF2alpha phosphorylation homology domain in E2 protein remains intact during IFN therapy, its structure modifies in patients resistant to IFN-therapy, which then puts its phosphorylation potential in jeopardy. Therefore, structural modification in HCV E2 proteins, acquired form patients resistant to IFN-therapy, suggests a mechanism that shows how HCV gets resistant toward IFN-therapy.

Analysis of the PKR-eIF2alpha phosphorylation homology domain (PePHD) of hepatitis C virus genotype 1 in HIV-coinfected patients by ultra-deep pyrosequencing and its relationship to responses to pegylated interferon-ribavirin treatment

Chronic coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is among the greatest challenges facing public health worldwide. In this population, the response to hepatitis C therapy by treatment with pegylated interferon plus ribavirin (PEG-IFN+RBV) is lower than in HCV-monoinfected patients, particularly in those infected by HCV genotype 1. A PKR/eIF-2α phosphorylation homology domain (PePHD) within the E2 protein has been found to interact with PKR and inhibit PKR in vitro, suggesting a possible mechanism for HCV to evade the antiviral effects of IFN. The aim of this work was to analyze the amino acid conservation in the HCV-E2-PePHD and quasispecies diversity among HCV-HIV-coinfected patients exhibiting sustained virological response, non-response, or partial response with viral relapse to PEG-IFN+RBV by ultra-deep pyrosequencing. For this purpose, HCV-E2-PePHD PCR products were generated and sequenced directly for four patients with a sustained response, seven patients with no virological response, and four patients with viral relapse before and after treatment with PEG-IFN+RBV. HCV-E2-PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment (24h, 4weeks, and 12weeks). Quasispecies analysis of the HCV-E2-PePHD and flanking genomic regions was performed using 454/Roche pyrosequencing, analyzing 39,364 sequence reads in total. The HCV-E2-PePHD sequence at the amino acid and nucleotide level was highly conserved among HCV genotype 1 strains, irrespective of the PEG-IFN+RBV response. This high degree of amino acid conservation and sporadic mutations in the HCV-E2-PePHD domain do not appear to be associated with treatment outcome. The HCV-E2-PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in patients coinfected with HCV genotype 1 and HIV.

Predictive factors for interferon and ribavirin combination therapy in patients with chronic hepatitis C

To confirm the predictive factors for interferon (IFN)-alpha and ribavirin combination therapy for chronic hepatitis patients with hepatitis C virus (HCV) genotype 1b. HCV RNA from 50 patients infected with HCV genotype 1b was studied by cloning and sequencing of interferon sensitivity determining region (ISDR), PKR-eIF2alpha phosphorylation homology domain (PePHD). Patients were treated with IFN-alpha and ribavirin for 6 mo and grouped by effectiveness of the therapy. A variety of factors were analyzed. Our data showed that age, HCV RNA titer, and ISDR type could be used as the predictive factors for combined IFN-alpha and ribavirin efficacy. Characteristically, mutations in PePHD appeared only when the combination therapy was effective. Other factors, such as sex and alanine aminotransferase (ALT) level, were not related to its efficacy. Adjusting for age and HCV RNA titer indicated that the ISDR type was the most potent predictive factor. HCV RNA ISDR type is an important factor for predicting efficacy of IFN-alpha and ribavirin combination therapy in Korean patients.

Mutations in carboxy-terminal part of E2 including PKR/eIF2α phosphorylation homology domain and interferon sensitivity determining region of nonstructural 5A of hepatitis C virus 1b: Their correlation with response to interferon monotherapy and viral load

To study the amino acid substitutions in the carboxy (C)-terminal part of E2 protein and in the interferon (IFN) sensitivity determining region (ISDR) and their correlation with response to IFN and viral load in 85 hepatitis C virus (HCV)-1b-infected patients treated with IFN. The C-terminal part of E2 (codons 617-711) including PKR/eIF2alpha phosphorylation homology domain (PePHD) and ISDR was sequenced in 85 HCV-1b-infected patients treated by IFN monotherapy. The amino acid substitutions in PePHD detected only in 4 of 85 patients were not correlated either with response to IFN or with viral load. The presence of substitutions in a N-terminal variable region (codons 617-641) in the C-terminal part of E2 was significantly correlated with both small viral load (33.9% vs 13.8%, P = 0.0394) and sustained response to IFN (25.0% vs 6.9%, P = 0.0429). Four or more substitutions in ISDR were significantly correlated with both small viral load (78.6% vs 16.2%, P < 0.0001) and sustained response to IFN (85.7% vs 2.9%, P < 0.0001). In multivariate analysis, ISDR in nonstructural (NS) 5A (OR = 0.39, P < 0.0001) and N-terminal variable region (OR = 0.51, P = 0.039) was selected as the independent predictors for small viral load, and ISDR (OR = 39.0, P < 0.0001) was selected as the only independent predictor for sustained response. The N-terminal variable region in the C-terminal part of E2 correlates with both response to IFN monotherapy and viral load and is one of the factors independently associated with a small viral load.

Mutations in the E2–PePHD region of hepatitis C virus type 1b in patients with hepatocellular carcinoma

An interaction between the protein kinase (PKR)-eIF2-alpha phosphorylation homology domain (PePHD) within the E2 protein of hepatitis C virus (HCV) and cell protein kinase (PKR) may affect the control of protein synthesis and cell growth. In an attempt to investigate the genetic variability of the E2-PePHD domain in hepatocellular carcinoma (HCC), we studied sera and liver tissues from HCC patients. The partial E2-PePHD region was analysed by direct sequencing of the sera of 47 HCCs in cirrhotic livers and 31 cases of chronic active hepatitis (CAH), and tumoral and non-tumoral liver tissues from 13 HCC patients. A similar number of mutations was detected within the E2 domain in the HCC and CAH cases, but nine of the 47 HCCs (19%) showed an amino acid (aa) mutation at position 660, eight of which involved a change in the same aa (alanine instead of serine; A/S). No such mutation was detected in any of the PePHD sequences from the CAH patients: this difference was statistically significant (P = 0.008). The aa change at position 660 was also found in two sequences from tumoral but not non-tumoral tissue from the same liver. The analysis of 461 sequences obtained from GenBank supports the conclusion that the observed aa change is an infrequent event in HCV-infected patients, thus suggesting that it could be associated with HCC.

Mutations in the NS5A and E2-PePHD regions of hepatitis C virus genotype 1b and response to combination therapy of interferon plus ribavirin.

Combination therapy with interferon (IFN) and ribavirin is the current standard treatment for chronic hepatitis C, but the efficacy is still not satisfactory, especially for genotype 1b. NS5A and E2 proteins of hepatitis C virus (HCV) may repress the IFN-induced RNA-dependent protein kinase (PKR), and thus have the potential to influence the response of HCV to IFN therapy; however, this issue remains controversial. Nucleotide sequences of the PKR-eIF2alpha phosphorylation homology domain (E2-PePHD) and PKR-binding domain (NS5A-PKR bd) of the HCV genome were analyzed by amplification and direct sequencing in 30 HCV genotype 1b patients who had been treated with IFN and ribavirin. Nine (30%) patients achieved a sustained virological response (SVR) to combination therapy. Pretreatment variables and amino acid substitutions were compared between responders and non-responders. The responders were younger than non-responders (37.2 +/- 10.4 vs. 45.4 +/- 9.5 years, P = 0.017), whereas no significant statistical differences were found in the number of amino acid substitutions in NS5A and E2-PePHD regions between the two groups. Genetic heterogeneity in NS5A and E2-PePHD regions of the HCV genome may not serve as a predictor for treatment outcome with combination therapy in Taiwanese patients with chronic HCV genotype 1b infection.

Sequence analysis of PePHD within HCV E2 region and correlation with resistance of interferon therapy in Japanese patients infected with HCV genotypes 2a and 2b.

Hepatitis C virus (HCV) E2 protein was recently reported to have a double-stranded RNA-activated protein kinase-eukaryotic initiation factor 2alpha (PKR-eIF2alpha) phosphorylation homology domain (PePHD); PKR is induced by interferon (IFN). PePHD interacts with PKR and inactivates it. PePHD could be a predictor for IFN response, like the interferon sensitivity determination region (ISDR) of HCV NS5A. Several groups reported that PePHD is conserved, and mutations in this region do not correlate with IFN response. In this study, we further investigated the amino acid variation of PePHD among four major genotypes and its correlation with IFN response. We enrolled 74 patients for this study and determined PePHD sequence of HCV derived from sera of patients infected with HCV genotype 1a (1 patient; nonresponder [NR]), 1b (36 patients; 4 complete responders [CR], 32 NR), 2a (29 patients; 17 CR, 12 NR), and 2b (8 patients; 3 CR, 5 NR). We also analyzed mutations in ISDR of HCV genotype 1b in 31 patients. PePHD had several variations among four genotypes investigated. In patients infected with HCV genotype 1b, PePHD sequence was well conserved and seemed to have no correlation with IFN response. Mutations in ISDR were correlated with IFN response. In patients with HCV genotypes 2a and 2b, PePHD had multiple variations, and one particular motif, "RGQQ-" at the N-terminus, showed a close correlation with IFN resistance. All eight patients with HCV containing this motif were IFN nonresponders. IFN resistance of HCV correlates with its "RGQQ-" motif at the N-terminus of PePHD in HCV genotype 2a and 2b. PePHD of HCV could be a predictor of IFN resistance in patients infected with HCV genotype 2a and 2b.

Mutations in the NS5A and E2-PePHD region of hepatitis C virus type 1b and correlation with the response to combination therapy with interferon and ribavirin.

Nonstructural 5A (NS5A) and the second envelope (E2) proteins of hepatitis C virus (HCV) have the potential to block interferon (IFN)-induced RNA-dependent protein kinase (PKR) and may therefore interfere with the response to IFN therapy, but controversy still exists regarding the relevance of this. This study aimed to assess whether mutations in these regions correlated with the response to combination therapy, IFN and ribavirin. Pretreatment parameters were analysed in 57 HCV-1b patients who had received IFN-alpha2b (3 or 5 MU three times weekly) and ribavirin (800-1200 mg per day) for 24 weeks. The amino acid sequences of the NS5A and PKR-eIF2alpha phosphorylation homology domain (E2-PePHD) were deduced from the corresponding coding sequence, which were determinated by direct sequencing of the HCV genome amplified by the polymerase chain reaction. Twenty (36%) patients achieved a sustained virological response (SVR). The mean number of amino acid substitutions in the NS5A-PKR binding domain (2209-2274), interferon sensitivity-determining region (ISDR) (2209-2248), and E2-PePHD sequence (659-670) in patients with and without SVR were 4.53 +/- 3.31 vs 2.83 +/- 1.78 (P = 0.094), 2.45 +/- 2.74 vs 1.03 +/- 1.32 (P = 0.042) and 0.25 +/- 0.70 vs 0.03 +/- 0.17 (P = 0.109), respectively. Patients with a mutant-type (>/= 4) NS5A-ISDR had a higher rate of SVR (six of nine, 67%) than those with wild-type (five of 22, 23%) (P = 0.038). Stepwise multiple logistic regression analysis of the factors (age, gender, viral load, cirrhosis rate, IFN dosage and amino acid substitutions) revealed that the mutation in NS5A-ISDR (>/= 4 vs < 4) was the only independent variable of treatment outcome. Our study showed that NS5A-ISDR mutations were correlated with the SVR to combination therapy in chronic HCV-1b patients in Taiwan.

Analysis of sequence configurations of the ISDR, PKR-binding domain, and V3 region as predictors of response to induction interferon-alpha and ribavirin therapy in chronic hepatitis C infection.

Interferon (IFN) and ribavirin combination therapy for chronic hepatitis C virus (HCV) infection yields a sustained response rate of only approximately 40%. Previous studies have linked IFN responsiveness to viral sequence variation in parts of the E2 and NS5A genes, but this remains controversial. We studied pretreatment sera from 28 subjects (23 with HCV genotype 1a) who received high-dose IFN induction followed by IFN-ribavirin combination therapy. Serum HCV sequences were amplified and compared from 14 responders with undetectable HCV RNA 24 weeks after therapy and 11 nonresponders (excluding three who dropped out of the study). Analysis included the E2 PKR eIF-2alpha phosphorylation homology domain (PePHD, codons 659-670), where the sequence was well conserved, and codons 2001-2420 of NS5A. In NS5A, the proposed PKR binding domain (codons 2209-2274), containing the putative IFN sensitivity determining region (ISDR, codons 2209-2248), showed too little variation among subjects to differentiate responders and nonresponders. NS5A codons 2356-2385 (which includes the V3 region) exhibited more variation. Here, six of 12 genotype 1a responders showed four or more amino acid changes from the prototype HCV-1 sequence, as compared with one of eight nonresponders, but this fell short of statistical significance (P = 0.16). NS5A sequences from posttreatment sera were examined in six nonresponders to look for selection of treatment-resistant viral subpopulations, but no consistent change was detected. In conclusion, our results indicate that the sequences of the ISDR, the PKR-binding domain, and the PePHD are unlikely to have predictive value for IFN treatment success in those infected with HCV genotype 1a. However, the finding of greater variability among treatment responders in the carboxy end of NS5A suggests that the V3 region merits further investigation.

Quasispecies heterogeneity of the carboxy-terminal part of the E2 gene including the PePHD and sensitivity of hepatitis C virus 1b isolates to antiviral therapy.

Two regions within the HCV genome, hypervariable region 1 (HVR1) within the envelope (E)2 region and the PKR-binding domain (PKRbD) comprising the interferon sensitivity determining region (ISDR) within the nonstructural (NS)5A protein, have been reported to correlate with the outcome of antiviral treatment. Recently, a PKR/eIF2alpha phosphorylation homology domain (PePHD) within the E2 protein of HCV-1 isolates was described to inhibit PKR in vitro. PePHD deleted HCV-1 mutants remain capable of binding PKR to some extent while inhibition of PKR was found to be abolished by carboxy-terminal truncated E2 protein. The importance of mutations and quasispecies heterogeneity within the carboxy-terminal part of the E2 protein comprising the PePHD of HCV-1b is unknown. Therefore, the carboxy-terminal part of the HCV E2 gene (codons 618-746) including the PePHD was analyzed by sequencing of PCR products and individual clones of 41 HCV-1b-infected patients with sustained (SR, n = 12), end-of-treatment (ETR, n = 10), or no virological (NR, n = 19) response to antiviral therapy. Two highly conserved regions (codons 658-673 comprising the PePHD and codons 675-704) and one variable region (codons 705-720) were detected within the carboxy-terminal part of E2. No significant correlation of specific mutations or number of mutations with treatment response was observed for the PePHD and the carboxy-terminal part of the E2 protein. Phylogenetic and conformational analyses showed no specific clusters related to treatment outcome. Calculation of genetic complexity and diversity based on nucleotide sequence analyses of 20 individual clones per patient showed no differences between matched SR, ETR, and NR patients. However, calculation of genetic complexity and diversity on the basis of amino acid sequences showed significantly lower normalized Shannon entropy as well as mean Hamming distances for SR patients than in ETR and NR patients (P = 0.029 and P = 0.027, respectively). This indicates that patients achieving a sustained virological response to interferon-alpha-based antiviral therapy may elicit more effective immunological pressure, resulting in continuous clearing of individual variants of HCV quasispecies.