Rodrigo A. Villanueva

miR-122 does not modulate the elongation phase of hepatitis C virus RNA synthesis in isolated replicase complexes

miR-122 is an abundant, liver-specific microRNA that is required for efficient amplification of hepatitis C virus (HCV) RNA. Recent studies with a miR-122-specific locked nucleic acid antagomir have shown it to be an important host target for therapeutic intervention. However, considerable controversy exists concerning the mechanisms underlying the dependence of HCV replication on miR-122. We studied the impact of miR-122 on the rate of [(32)P]-incorporation into positive-strand viral RNA by membrane-bound replicase complexes isolated from cells containing HCV RNA replicons. [(32)P]-incorporation in this cell-free system represents primarily the elongation phase of RNA synthesis, with little or no de novo initiation, and was not affected by the addition of either excess miR-122 or a miR-122-specific antisense oligonucleotide that suppresses replication in vivo. We also found no evidence that detectable quantities of miR-122 are specifically associated with replicase complexes in vivo. These results are consistent with miR-122 acting at an alternative step in the viral life cycle, promoting cap-independent viral translation, enhancing viral RNA stability, or facilitating de novo initiation of viral RNA synthesis.

IL28B polymorphisms associated with therapy response in Chilean chronic hepatitis C patients

AIM To analyze the association of three IL28B single nucleotide polymorphisms with response to therapy in Chilean patients infected with hepatitis C virus (HCV). METHODS We studied two groups of patients with chronic HCV infection (genotype 1), under standard combined treatment with pegylated interferon plus ribavirin. One group consisted of 50 patients with sustained virological response, whereas the second group consisted of 49 null responders. In order to analyze the IL28B single nucleotide polymorphisms rs12979860, rs12980275 and rs8099917, samples were used for polymerase chain reaction amplification, and the genotyping was performed by restriction fragment length polymorphism. RESULTS The IL28B rs12979860 CC, rs12980275 AA and rs8099917 TT genotypes were much more frequently found in patients with sustained virological response compared to null responders (38%, 44% and 50% vs 2%, 8.2% and 8.2%, respectively). These differences were highly significant in all three cases (P < 0.0001). CONCLUSION The three IL28B polymorphisms studied are strongly associated with sustained virological response to therapy in Chilean patients with chronic HCV (genotype 1).

Impaired Replication of Hepatitis C Virus Containing Mutations in a Conserved NS5B Retinoblastoma Protein-Binding Motif

ABSTRACT Hepatitis C virus (HCV) downregulates the retinoblastoma tumor suppressor protein (Rb), a central cell cycle regulator which is also targeted by oncoproteins expressed by DNA tumor viruses. HCV genome replication is also enhanced in proliferating cells. Thus, it is possible that HCV interactions with host cell cycle regulators, such as Rb, have evolved to modify the intracellular environment to promote viral replication. To test this hypothesis and to determine the impact of viral regulation of Rb on HCV replication, we constructed infectious viral genomes containing mutations in the Rb-binding motif of NS5B which ablate the ability of HCV to regulate Rb. These genomes underwent replication in transfected cells but produced variably reduced virus yields. One mutant, L314A, was severely compromised for replication and rapidly mutated to L314V, thereby restoring both Rb regulation and replication competence. Another mutant, C316A, also failed to downregulate Rb abundance and produced virus yields that were about one-third that of virus with the wild-type (wt) NS5B sequence. Despite this loss of replication competence, purified NS5B-C316A protein was two- to threefold more active than wt NS5B in cell-free polymerase and replicase assays. Although small interfering RNA knockdown of Rb did not rescue the replication fitness of these mutants, we conclude that the defect in replication fitness is not due to defective polymerase or replicase function and is more likely to result from the inability of the mutated NS5B to optimally regulate Rb abundance and thereby modulate host gene expression.

Phylogenetic analysis of hepatitis B virus genotype F complete genome sequences from Chilean patients with chronic infection

Molecular epidemiological data concerning the hepatitis B virus (HBV) in Chile are not known completely. Since the HBV genotype F is the most prevalent in the country, the goal of this study was to obtain full HBV genome sequences from patients infected chronically in order to determine their subgenotypes and the occurrence of resistance‐associated mutations. Twenty‐one serum samples from antiviral drug‐naive patients with chronic hepatitis B were subjected to full‐length PCR amplification, and both strands of the whole genomes were fully sequenced. Phylogenetic analyses were performed along with reference sequences available from GenBank (n = 290). The sequences were aligned using Clustal X and edited in the SE‐AL software. Bayesian phylogenetic analyses were conducted by Markov Chain Monte Carlo simulations (MCMC) for 10 million generations in order to obtain the substitution tree using BEAST. The sequences were also analyzed for the presence of primary drug resistance mutations using CodonCode Aligner Software. The phylogenetic analyses indicated that all sequences were found to be the HBV subgenotype F1b, clustered into four different groups, suggesting that diverse lineages of this subgenotype may be circulating within this population of Chilean patients. J. Med. Virol. 83:1530–1536, 2011.

Prevalence of hepatitis B virus genotypes in chronic carriers in Santiago, Chile

The eight genotypes (designated A–H) of hepatitis B virus (HBV) display distinctive geographical distribution worldwide, with genotypes A, D and F frequently detected in South America. To determine the prevalence of HBV genotypes in Santiago, Chile, 131 samples from chronic carriers were used for PCR amplification, and genotyping was performed by RFLP. The results indicated that genotype F was the most prevalent among HBV carries (84% of the cases), whereas genotypes A, B, C and D were found at a prevalence of 3.8, 3.8, 6.1, and 2.3%, respectively. We discuss these data in the complex scenario of HBV epidemiology.

Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small-molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing.

Genotype F of hepatitis B: response to interferon

BACKGROUND The relevance of HBV genotype diversity on interferon (IFN) therapy outcome in chronic hepatitis B patients has recently been highlighted. Data available for genotype F is poor. The aim of this work was to analyse the response of HBV genotype F to treatment with IFN. Additionally, response was analysed according to the role of single nucleotide polymorphisms (SNPs) near to the IL28B gene. METHODS A total of 29 HBeAg-positive patients with chronic infection were included with a median age 47 (18-68) years. Of them, 27 were male. One patient was treated with standard IFN-α for 16 weeks, 6 patients received PEG-IFN-α2a 180 μg weekly for 24 weeks and 22 patients for 48 weeks. Response to treatment was defined as loss of HBeAg, anti-HBe seroconversion and decline of HBV DNA level to below 3 log of baseline (IU/ml) at the 6-month of follow-up. The SNPs rs12979860, rs12980275 and rs8099917 were studied by PCR-RFLP. RESULTS The overall response was obtained in 18 (62%) patients, including one patient who was treated with standard IFN. Additionally, a total of 9 (31%) patients cleared HBsAg, with appearance of anti-HBs. The viral load was undetectable in all of these patients. The same IL28B variants associated with IFN response in HCV infections were also more frequently found in HBV patients compared with non-responders. CONCLUSIONS Our study indicates that treatment with IFN is effective in patients with HBV genotype F.