The Effects Of Amino Acid Mutations In HBV RNase H Domain On The Replication Of Hepatitis B Virus

Part I. Effects of Mutation of Amino Acids in HBV RNase H Active Site on Viral ReplicationObjective: To explore position of key amino acids in HBV RNase H domain active site and effects of their mutation on the replication of Hepatitis B Virus. Methods: Adopting computer software to compare the amino acids sequences in RNase H domain from HBV(adw2),DHBV, Heron HBV and WHV, and referring to D-E-D-D motif structure of RNase H active site from both HIV and E.Coli, the four amino acids D702, H 728, E731 and D790 in HBV RNase H domain are site-directed, and thus the express plasmids of four RNase mutants D702A, H728V,E731A and D790A are constructed by means of Mega-PCR( Mega-Primer PCR). The constructed RNase H mutants, positive control D750 (RNase H deficient) and negative control pCMV-HBV-LE-(RNase H normal) are transfected respectively into Huh 7 cells. On the fifth day after the transfection, cytoplasmic core particles are prepared and purified. In order to identify viral nucleinic acid constituents within the particles, each sample is divided into two parts, one is digested by RNase H of E.Coli in vitro while the other is not. And then both the digested and undigested products are detected by Southern analysis. Results: Southern analysis shows that with the RNase H activity deficiency or decrease of RNase H mutants of D 702A, E731A, D 790A and D 750A in the transfected cells, RNA:DNA heleroduplexes produced during replicative cycle of HBV DNA can't be removed and therefore the replication of HBV DNA is affected whereas the activity of H 728V is normal and the replication of HBV DNA is not affected.Conclusion: HBV RNase H domain active site are composed of four amino acids of D702-E731-D750-D790, i.e. D-E-D-D motif , and what's more, any mutation of them may induce RNase H defects or decreased activity and consequently the affected replication of HBV.PartⅡ. Effect of Mutation of Amino Acid in HBV RNase H Primer Grip on Viral ReplicationObjective: To identify hypothesis that HBV is the same as HIV in that there exists a RNase H primer grip in RNase H domain, which is composed of a series of amino acid around its RNase H domain active site and to investigate effect of mutation of amino acid in RNase H primer grip region on viral replication. Methods:By comparing alignments of RNase H domain from E. coli, Bacillus halodurans and HIV, and referring to the composition of amino acid of HIV RNaseH primer grip and focusing on D-E-D-D motif of HBV RNase H active site, the following ten positions'amino acid R718, T729, S742, T749, S756, R757, K758, S761, S793 and R796 are selected for mutation, of which four positively-charged amino acid R (arginine) and K (lysine) are mutated as negatively-charged amino acid D (aspartic acid) and R718D, R757D, K758D and R796D mutants are constructed consequently, and the rest six positions amino acid are all mutated as alanine (A), i.e. Alanine-scanning mutations, with T729A, S742A, T749A, S756A, S761A and S793A mutants constructed as well. All ten mutants are built into an HBV expression vector, i.e. pCMV-HBV-LE–, positive control D750V(RNase H function-deficiency) , negative control pCMV-HBV-LE– (RNase H function-normal) are transfected into Huh 7 cells. On the fifth day after the transfection, HBV core particles are extracted from the cells and are purified, and the nucleinic acid extractor (a prototype of HBV DNA) within the particles are detected by Southern analysis. Results: Among the four RNase H mutants with a positively-charged amino acid replaced by a negatively-charged one, two mutants K758D and R796D have deficiency in polymerase activity, the former even loses its function. Whereas among the six RNase H mutants with an amino acid replaced by an A (alanine), only one mutant S793A shows its RNase H function deficiency, the other five mutants are normal in RNase H function. Conclusions: There exist some amino acids around the HBV RNase H active site, especially those with positive-charge or some polar amino acid and those amino acids constitute so-called HBV RNase H primer grip. The mutation of the amino acid in this region may lead to RNase H activity deficiency and decrease or diminishing of polymerase, which directly or indirectly affects the replication of HBV. Objective: To obtain a comprehensive understanding of the role of HCV QS in antiviral therapy. Methods: 189 patients were treated with peginterferon alpha-2a and are the subjects in present study. Patient recruitment was restricted to HCV genotype 1. Serum samples were collected at multiple time points during the early phase of antiviral therapy, including baseline (w00), week 4 (w04), week 8 (w08) and week 12 (w12). For each patient, HCV QS profiles were generated at two time points, the baseline and the latest time point during the early phase of antiviral therapy (≤week 12) with a minimum HCV viral load more than 1000 copies per milliliter. Based on this standard, we finally identified 110 patients with two time points and 43 patients with only one time point (baseline), which resulted in 263 serum samples to be studied. A total of 4,314 clones were generated and sequenced from these samples followed by detailed genetic analyses. Results: Among 153 patients, 104 (68%) achieved early virological response (EVR). With regard to pretreatment genetic diversity, subjects with EVR had overall higher values than non-EVR group in the most of parameters measured. However, only HVR1 dN reached statistical significance (p=0.039). Like genetic diversity, EVR group had a higher pretreatment genetic complexity than Non-EVR group at either nucleotide or amino acid level. The latter reached statistical significance (p = 0.0499). Our data showed an exponential distribution pattern of intra-patient QS diversity in this study population in which most patients (63%) had small QS diversity with genetic distance (d) less than 0.2. The group of patients with genetic distance located in the decay region (d>0.53) had a significantly higher early EVR rate (89.5%), which contributed substantially to the overall association between EVR and increased baseline QS diversity. Conclusion: EVR is associated with elevated HCV QS diversity and complexity, especially in patients with significantly higher HCV genetic heterogeneity.