Biochemical and Structural Properties of Entecavir-Resistant Hepatitis B Virus Polymerase with L180M/M204V Mutations.

Entecavir (ETV) is a widely used anti-hepatitis B virus (HBV) drug. However, the emergence of resistant mutations in HBV reverse transcriptase (RT) results in treatment failure. To understand the mechanism underlying the development of ETV resistance by HBV RT, we analyzed the L180M, M204V, and L180M/M204V mutants using a combination of biochemical and structural techniques. ETV-triphosphate (ETV-TP) exhibited competitive inhibition with dGTP in both wild-type (wt) RT and M204V RT, as observed using Lineweaver-Burk plots. In contrast, RT L180M or L180M/M204V did not fit either competitive, uncompetitive, noncompetitive, or typical mixed inhibition, although ETV-TP was a competitive inhibitor of dGTP. Crystallography of HIV RTY115F/F116Y/Q151M/F160M/M184V, mimicking HBV RT L180M/M204V, showed that the F115 bulge (F88 in HBV RT) caused by the F160M mutation induced deviated binding of dCTP from its normal tight binding position. Modeling of ETV-TP on the deviated dCTP indicated that a steric clash could occur between ETV-TP methylene and the 3'-end nucleoside ribose. ETV-TP is likely to interact primarily with HBV RT M171 prior to final accommodation at the deoxynucleoside triphosphate (dNTP) binding site (Y. Yasutake, S. Hattori, H. Hayashi, K. Matsuda, et al., Sci Rep 8:1624, 2018, https://doi.org/10.1038/s41598-018-19602-9). Therefore, in HBV RT L180M/M204V, ETV-TP may be stuck at M171, a residue that is conserved in almost all HBV isolates, leading to the strange inhibition pattern observed in the kinetic analysis. Collectively, our results provide novel insights into the mechanism of ETV resistance of HBV RT caused by L180M and M204V mutations. IMPORTANCE HBV infects 257 million people in the world, who suffer from elevated risks of liver cirrhosis and cancer. ETV is one of the most potent anti-HBV drugs, and ETV resistance mutations in HBV RT have been extensively studied. Nevertheless, the mechanisms underlying ETV resistance have remained elusive. We propose an attractive hypothesis to explain ETV resistance and effectiveness using a combination of kinetic and structural analyses. ETV is likely to have an additional interaction site, M171, beside the dNTP pocket of HBV RT; this finding indicates that nucleos(t)ide analogues (NAs) recognizing multiple interaction sites within RT may effectively inhibit the enzyme. Modification of ETV may render it more effective and enable the rational design of efficient NA inhibitors.

Fluorescence-based biochemical analysis of human hepatitis B virus reverse transcriptase activity.

Although the unique mechanism by which hepatitis B virus (HBV) polymerase primes reverse transcription is now well-characterized, the subsequent elongation process remains poorly understood. Reverse transcriptase (RT)-RNase H sequences from polymerase amino acid 304 (the C-terminal part of spacer domain) to 843 were expressed in Escherichia coli and purified partially. RT elongation activity was investigated using the fluorescent-tagged primer and homopolymeric RNA templates. RT elongation activity depended on both Mg2+ and Mn2+, and had low affinity for purine deoxynucleotides, which may be related with the success of adefovir, tenofovir, and entecavir. However, the polymerization rate was lower than that of human immunodeficiency virus RT. All HBV genotypes displayed similar RT activity, except for genotype B, which demonstrated increased elongation activity.

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Two mutations in the C-terminal domain of influenza virus RNA polymerase PB2 enhance transcription by enhancing cap-1 RNA binding activity.

Influenza virus RNA polymerase (RdRp) PB2 is the cap-1 binding subunit and determines host range and pathogenicity. The mutant human influenza virus RdRp containing PB2 D701N and D701N/S714R demonstrated enhanced replicon activity in mammalian cells. We investigated the influence of these mutations on RdRp activity. Cap-1-dependent transcription activities of D701N/S714R, D701N, and S714R were 348.1±6.2%, 146.4±11%, and 250.1±0.8% of that of the wild type (wt), respectively. Replication activity of these mutants for complimentary RNA to viral RNA ranged from 44% to 53% of that of the wt. Cap-1 RNA-binding activities of D701N/S714R, D701N, and S714R were 262±25%, 257±34%, and 315±9.6% of that of the wt, respectively, and their cap-dependent endonuclease activities were similar to that of the wt. These mutations did not affect template RNA-binding activities. D701N and S714R mutations enhanced transcription by enhancing cap-1 RNA-binding activity, but they may exhibit decreased efficiency of priming by the cap-1 primer. These mutations at the C-terminal domain of PB2 may affect its cap-binding domain.

Different mechanisms of hepatitis C virus RNA polymerase activation by cyclophilin A and B in vitro.

BACKGROUND: Cyclophilins (CyPs) are cellular proteins that are essential to hepatitis C virus (HCV) replication. Since cyclosporine A was discovered to inhibit HCV infection, the CyP pathway contributing to HCV replication is a potential attractive stratagem for controlling HCV infection. Among them, CyPA is accepted to interact with HCV nonstructural protein (NS) 5A, although interaction of CyPB and NS5B, an RNA-dependent RNA polymerase (RdRp), was proposed first. METHODS: CyPA, CyPB, and HCV RdRp were expressed in bacteria and purified using combination column chromatography. HCV RdRp activity was analyzed in vitro with purified CyPA and CyPB. RESULTS: CyPA at a high concentration (50× higher than that of RdRp) but not at low concentration activated HCV RdRp. CyPB had an allosteric effect on genotype 1b RdRp activation. CyPB showed genotype specificity and activated genotype 1b and J6CF (2a) RdRps but not genotype 1a or JFH1 (2a) RdRps. CyPA activated RdRps of genotypes 1a, 1b, and 2a. CyPB may also support HCV genotype 1b replication within the infected cells, although its knockdown effect on HCV 1b replicon activity was controversial in earlier reports. CONCLUSIONS: CyPA activated HCV RdRp at the early stages of transcription, including template RNA binding. CyPB also activated genotype 1b RdRp. However, their activation mechanisms are different. GENERAL SIGNIFICANCE: These data suggest that both CyPA and CyPB are excellent targets for the treatment of HCV 1b, which shows the greatest resistance to interferon and ribavirin combination therapy.

Effect of the methyltransferase domain of Japanese encephalitis virus NS5 on the polymerase activity.

Japanese encephalitis virus (JEV) NS5 consists of an N-terminal guanylyltransferase/methyltransferase (MTase) domain and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. We purified JEV NS5 from bacteria and examined its RdRp activity in vitro. It showed exclusive specificity for Mn(2+) and alkaline conditions (pH 8-10) for RdRp activity. It showed strong RdRp activity with dinucleotide primers, and the order of template strength was poly(U)>(I)>(A)>(C). It showed weak transcription activity without primers, but could not transcribe poly(I) without primers. It bound homopolymeric RNA templates, but weakly bound poly(C). The Km (µM) values were 22.13±1.11 (ATP), 21.94±3.88 (CTP), 21.27±1.23 (GTP), and 9.91±0.30 (UTP), indicating low substrate affinity. Vmax (/min) values were 0.216±0.017 (ATP), 0.781±0.020 (CTP), 0.597±0.049 (GTP), and 0.347±0.022 (UTP), indicating high polymerization activity. The RdRp domain alone did not show RdRp activity; a structural and functional interaction between the MTase and RdRp domains via 299-EHPYRTWTYH-308 (MTase domain) and 739-LIGRARISPG-748 (RdRp domain) was predicted, because mutations in the MTase domain affected RdRp activity.

Fluorescent primer-based in vitro transcription system of viral RNA-dependent RNA polymerases.

Viral infection is a leading cause of disease and death. Although vaccines are the most effective method of controlling viral infections, antiviral drugs are also important. Here, we established an in vitro transcription system by using fluorescein isothiocyanate-conjugated primers for RNA polymerases of viruses that are important disease-causing human pathogens (influenza, hepatitis C, Japanese encephalitis viruses, and enterovirus 71). This technology will allow us to analyze RNA polymerase activity without using radioisotopes.

Hepatitis C virus NS5B protein delays s phase progression in human hepatocyte-derived cells by relocalizing cyclin-dependent kinase 2-interacting protein (CINP).

Cell cycle dysregulation is a critical event in virus infection-associated tumorigenesis. Previous studies have suggested that hepatitis C virus NS5B modulates cell cycle progression in addition to participating in RNA synthesis as an RNA-dependent RNA polymerase. However, the molecular mechanisms have thus far remained unclear. In this study, a HepG2 Tet-On NS5B stable cell line was generated to confirm the effect of NS5B on the cell cycle. To better understand the role of NS5B in cell cycle regulation, yeast two-hybrid assays were performed using a human liver cDNA library. The cyclin-dependent kinase 2-interacting protein (CINP) was identified. The interaction between NS5B and CINP was further demonstrated by in vivo and in vitro assays, and their association was found to be indispensable for S phase delay and cell proliferation suppression. Further experiments indicated that NS5B relocalized CINP from the nucleus to the cytoplasm. Directly knocking down CINP by specific siRNA resulted in a significant alteration in the DNA damage response and expression of cell cycle checkpoint proteins, including an increase in p21 and a decrease in phosphorylated Retinoblastoma and Chk1. Similar results were observed in cells expressing NS5B, and the effects were partially reversed upon ectopic overexpression of CINP. These studies suggest that the DNA damage response might be exploited by NS5B to hinder cell cycle progression. Taken together, our data demonstrate that NS5B delays cells in S phase through interaction with CINP and relocalization of the protein from the nucleus to the cytoplasm. Such effects might contribute to hepatitis C virus persistence and pathogenesis.

Biochemical characterization of enterovirus 71 3D RNA polymerase.

An unusual enterovirus 71 (EV71) epidemic has begun in China since 2008. EV71 RNA polymerases (3D(pol)) showed polymerase activity with an Mn(2+). Little activity was detected with Co(2+), and no activity was detected with Mg(2+), Ca(2+), Cu(2+), Ni(2+), Cd(2+), or Zn(2+). It is a primer-dependent polymerase, and the enzyme functioned with both di- and 10-nucleotide RNA primers. DNA primer, dT15, increased primer activity, similar to other enterovirus 3D(pol). However, EV71 3D(pol) initiated de novo transcription with a poly(C) template and genome RNA. Its RNA binding activity was weak. Terminal nucleotidyl transferase and reverse transcriptase activity were not detected. The Km and Vmax for EV71 3D(pol) were calculated from classic Lineweaver-Burk plots. The Km values were 2.35±0.05 (ATP), 5.40±0.93 (CTP), 1.12±0.10 (GTP) and 2.81±0.31 (UTP), and the Vmax values were 0.00078±0.00005/min (ATP), 0.011±0.0017/min (CTP), 0.050±0.0043/min (GTP) and 0.0027±0.0005/min (UTP). The Km of EV71 3D(pol) was similar to that of foot and mouth disease virus and rhinovirus. Polymerase activity of BrCr-TR strain and a strain from a clinical isolate in Beijing, 2008 were similar, indicating the potential for 3D(pol) as an antiviral drug target.

RNA polymerase activity and specific RNA structure are required for efficient HCV replication in cultured cells.

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3'X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3' untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3'X region. We next analyzed the 3' structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3'UTR were required for J6CF replication in cultured cells.

PA from an H5N1 highly pathogenic avian influenza virus activates viral transcription and replication and induces apoptosis and interferon expression at an early stage of infection.

BACKGROUND: Although gene exchange is not likely to occur freely, reassortment between the H5N1 highly pathogenic avian influenza virus (HPAIV) and currently circulating human viruses is a serious concern. The PA polymerase subunit of H5N1 HPAIV was recently reported to activate the influenza replicon activity. METHODS: The replicon activities of PR8 and WSN strains (H1N1) of influenza containing PA from HPAIV A/Cambodia/P0322095/2005 (H5N1) and the activity of the chimeric RNA polymerase were analyzed. A reassortant WSN virus containing the H5N1 Cambodia PA (C-PA) was then reconstituted and its growth in cells and pathogenicity in mice examined. The interferon promoter, TUNEL, and caspase 3, 8, and 9 activities of C-PA-infected cells were compared with those of WSN-infected cells. RESULTS: The activity of the chimeric RNA polymerase was slightly higher than that of WSN, and C-PA replicated better than WSN in cells. However, the multi-step growth of C-PA and its pathogenicity in mice were lower than those of WSN. The interferon promoter, TUNEL, and caspase 3, 8, and 9 activities were strongly induced in early infection in C-PA-infected cells but not in WSN-infected cells. CONCLUSIONS: Apoptosis and interferon were strongly induced early in C-PA infection, which protected the uninfected cells from expansion of viral infection. In this case, these classical host-virus interactions contributed to the attenuation of this strongly replicating virus.

Internal initiation of influenza virus replication of viral RNA and complementary RNA in vitro.

Influenza virus transcription is a prototype of primer-dependent initiation. Its replication mechanism is thought to be primer-independent. The internal initiation and realignment model for influenza virus genome replication has been recently proposed (Deng, T., Vreede, F. T., and Brownlee, G. G. (2006) J. Virol. 80, 2337-2348). We obtained new results, which led us to propose a novel model for the initiation of viral RNA (vRNA) replication. In our study, we analyzed the initiation mechanisms of influenza virus vRNA and complementary RNA (cRNA) synthesis in vitro, using purified RNA polymerase (RdRp) and 84-nt model RNA templates. We found that, for vRNA → cRNA →, RdRp initiated replication from the second nucleotide of the 3'-end. Therefore, host RNA-specific ribonucleotidyltransferases are required to add one nucleotide (purine residues are preferred) to the 3'-end of vRNA to make the complete copy of vRNA. This hypothesis was experimentally proven using poly(A) polymerase. For cRNA → vRNA, the dinucleotide primer AG was synthesized from UC (fourth and fifth from the 3'-end) by RdRp pausing at the sixth U of UUU and realigning at the 3'-end of cRNA template; then RdRp was able to read through the entire template RNA. The RdRp initiation complex was not stable until it had read through the UUU of cRNA and the UUUU of vRNA at their respective 3'-ends. This was because primers overlapping with the first U of the clusters did not initiate transcription efficiently, and the initiation product of v84+G (the v84 template with an extra G at its 3'-end), AGC, realigned to the 3'-end.

Sphingomyelin activates hepatitis C virus RNA polymerase in a genotype-specific manner.

Hepatitis C virus (HCV) replication and infection depend on the lipid components of the cell, and replication is inhibited by inhibitors of sphingomyelin biosynthesis. We found that sphingomyelin bound to and activated genotype 1b RNA-dependent RNA polymerase (RdRp) by enhancing its template binding activity. Sphingomyelin also bound to 1a and JFH1 (genotype 2a) RdRps but did not activate them. Sphingomyelin did not bind to or activate J6CF (2a) RdRp. The sphingomyelin binding domain (SBD) of HCV RdRp was mapped to the helix-turn-helix structure (residues 231 to 260), which was essential for sphingomyelin binding and activation. Helix structures (residues 231 to 241 and 247 to 260) are important for RdRp activation, and 238S and 248E are important for maintaining the helix structures for template binding and RdRp activation by sphingomyelin. 241Q in helix 1 and the negatively charged 244D at the apex of the turn are important for sphingomyelin binding. Both amino acids are on the surface of the RdRp molecule. The polarity of the phosphocholine of sphingomyelin is important for HCV RdRp activation. However, phosphocholine did not activate RdRp. Twenty sphingomyelin molecules activated one RdRp molecule. The biochemical effect of sphingomyelin on HCV RdRp activity was virologically confirmed by the HCV replicon system. We also found that the SBD was the lipid raft membrane localization domain of HCV NS5B because JFH1 (2a) replicon cells harboring NS5B with the mutation A242C/S244D moved to the lipid raft while the wild type did not localize there. This agreed with the myriocin sensitivity of the mutant replicon. This sphingomyelin interaction is a target for HCV infection because most HCV RdRps have 241Q.

Host-specific genetic variation of highly pathogenic avian influenza viruses (H5N1).

The complete genome sequences of two isolates A/chicken/Egypt/CL6/07 (CL6/07) and A/duck/Egypt/D2br10/07 (D2br10/07) of highly pathogenic avian influenza virus (HPAI) H5N1 isolated at the beginning of 2007 outbreak in Egypt were determined and compared with all Egyptian HPAI H5N1 sequences available in the GenBank. Sequence analysis utilizing the RNA from the original tissue homogenate showed amino acid substitutions in seven of the viral segments in both samples. Interestingly, these changes were different between the CL6/07 and D2br10/07 when compared to other Egyptian isolates. Moreover, phylogenetic analysis showed independent sub-clustering of the two viruses within the Egyptian sequences signifying a possible differential adaptation in the two hosts. Further, pre-amplification analysis of H5N1 might be necessary for accurate data interpretation and identification of distinct factor(s) influencing the evolution of the virus in different poultry species.

Biochemical and kinetic analysis of the influenza virus RNA polymerase purified from insect cells.

The influenza virus RNA polymerase (RdRp) was purified from insect cells (around 0.2mg/l). The RdRp catalyzed all the biochemical reactions of influenza virus transcription and replication in vitro; dinucleotide ApG and globin mRNA-primed transcription, de novo initiation (replication), and polyadenylation. The optimal Mg concentration, pH and temperature were 8mM, 8.0 and 25 degrees C, respectively, which were slightly different from those measured for RdRp of virions. This system is a single-round transcription system. K(m) (microM) were 10.74+/-0.26 (GTP), 33.22+/-3.37 (ATP), 28.93+/-0.48 (CTP) and 22.01+/-1.48 (UTP), and V(max) (fmol nucleotide/pmol RdRp/min) were 2.40+/-0.032 (GTP), 1.95+/-0.17 (ATP), 2.07+/-0.17 (CTP), and 1.52+/-0.38 (UTP), which agreed with high mutation of influenza viruses.

Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay.

BACKGROUND: Hepatitis B virus (HBV) polymerase is the only virus-encoded enzyme essential for producing the HBV genome and is regarded as an attractive drug target. However, the difficulty of synthesizing and purifying recombinant HBV polymerase protein has hampered the development of new drugs targeting this enzyme, especially compounds unrelated to the nucleoside structure. We recently have developed a technique for the synthesis and purification of recombinant HBV polymerase containing the reverse transcriptase (RT) domain that carried DNA elongation activity in vitro. METHODS: We used the overproduced protein to establish an in vitro high-throughput screening system to identify compounds that inhibit the elongation activity of HBV polymerase. RESULTS: We screened 1120 compounds and identified a stilbene derivative, piceatannol, as a potential anti-HBV agent. Derivative analysis identified another stilbene derivative, PDM2, that was able to inhibit HBV replication with an IC50 of 14.4 ± 7.7 µM. An infection experiment suggested that the compounds inhibit the replication of HBV rather than the entry process, as expected. Surface plasmon resonance analysis demonstrated a specific interaction between PDM2 and the RT domain. Importantly, PDM2 showed similar inhibitory activity against the replication of both wild-type HBV and a lamivudine/entecavir-resistant HBV variant. Furthermore, PDM2 showed an additive effect in combination with clinically used nucleos(t)ide analogs. CONCLUSIONS: We report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.

Monoclonal antibody recognizing SLLTEVET epitope of M2 protein potently inhibited the replication of influenza A viruses in MDCK cells.

The ectodomain of influenza A virus M2 protein (M2e) is composed of 24 amino acids and induces antibodies with inhibitory effect against a broad spectrum of influenza A subtypes in vitro and in vivo. Although relatively conserved, 21 M2e variants emerged in recent influenza A strains, most of the mutations appeared in the middle part of M2e domain. In this study, we characterized the in vitro inhibition efficacy of a monoclonal antibody (mAb) M2e8-7 recognizing the N terminus highly conserved epitope SLLTEVET (aa 2-9) which is common for both M1 and M2 proteins. Peptide binding assay showed that mAb M2e8-7 reacted strongly with M2e and 19 M2e variant peptides. The mAb M2e8-7 potently inhibited the replication of influenza A virus H1 and H3 subtypes in MDCK cells. Two important amino acids in M2e epitope, Threonine at position five and the Glutamic acid at position six, were identified to lead antibody-escaping variants. These results brought new insight in developing vaccine and therapeutic agents against influenza A virus infections.

design of multiplexed detection assays for identification of avian influenza a virus subtypes pathogenic to humans by SmartCycler real-time reverse transcription-PCR.

Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.

Modification of hepatitis C virus 1b RNA polymerase to make a highly active JFH1-type polymerase by mutation of the thumb domain.

Hepatitis C virus (HCV) JFH1 efficiently replicates and produces infectious virus particles in cultured cells. We compared polymerase activity between JFH1 and 1b strains in vitro. The RNA polymerase activity of 1b was 6.4% of that of JFH1. In order to study the mechanism and identify domains responsible for the high polymerase activity of JFH1, we converted the amino acids of 1b RdRp to those of JFH1, and compared their Km, Vmax and template binding activity. Four amino acid mutations in the thumb domain of 1b RdRp, S377R, A450S, E455N and Y561F increased 1b polymerase activity, and their activity was 23.1, 45.8, 28.9, and 36.1% of JFH1, respectively. Vmax and RNA binding activity of JFH1, 1bwt and 1bA450S was JFH1 > 1bA450S > 1b, which indicated both high processivity and slightly higher template binding activity contributed to the high polymerase activity of JFH1.