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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. 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. 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. We report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.

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 RT(Y115F/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.

In September 2015, the Japanese Diet enacted a series of laws – the Peace and Security Legislation – to enable the Japan Self-Defense Force to play an enhanced role in peace deployments overseas. The enactment of the new laws was made possible by a “new” interpretation of Article 9 of the Japanese Constitution. While the main objective of the introduction of the laws was aimed at strengthening the alliance with the United States, jsdf participation in peace operations was also an important consideration politically. This article suggests that the Japanese government will now need to convince the public that the new Peace and Security Legislation is compatible with Japan’s constitutionally mandated pacifism. In light of the prospect of an expanded participation of Japan in international peace operations this will be particularly important.

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.

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.

Recently, we found that sphingomyelin bound and activated hepatitis C virus (HCV) 1b RNA polymerase (RdRp), thereby recruiting the HCV replication complex into lipid raft structures. Detergents are commonly used for resolving lipids and purifying proteins, including HCV RdRp. Here, we tested the effect of detergents on HCV RdRp activity in vitro and found that non-ionic (Triton X-100, NP-40, Tween 20, Tween 80, and Brij 35) and twitterionic (CHAPS) detergents activated HCV 1b RdRps by 8-16.6 folds, but did not affect 1a or 2a RdRps. The maximum effect of these detergents was observed at around their critical micelle concentrations. On the other hand, ionic detergents (SDS and DOC) completely inactivated polymerase activity at 0.01%. In the presence of Triton X-100, HCV 1b RdRp did not form oligomers, but recruited more template RNA and increased the speed of polymerization. Comparison of polymerase and RNA-binding activity between JFH1 RdRp and Triton X-100-activated 1b RdRp indicated that monomer RdRp showed high activity because JFH1 RdRp was a monomer in physiological conditions of transcription. Besides, 502H plays a key role on oligomerization of 1b RdRp, while 2a RdRps which have the amino acid S at position 502 are monomers. This oligomer formed by 502H was disrupted both by high salt and Triton X-100. On the contrary, HCV 1b RdRp completely lost fidelity in the presence of 0.02% Triton X-100, which suggests that caution should be exercised while using Triton X-100 in anti-HCV RdRp drug screening tests.

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.

The N-terminal amphipathic helix α 0 of hepatitis C virus (HCV) NS3 protein is an essential structural determinant for the protein membrane association. Here, we performed functional analysis to probe the role of this helix α 0 in the HCV life cycle. A point mutation M21P in this region that destroyed the helix formation disrupted the membrane association of NS3 protein and completely abolished HCV replication. Mechanistically the mutation did not affect either protease or helicase/NTPase activities of NS3, but significantly reduced the stability of NS3 protein. Furthermore, the membrane association and stability of NS3 protein can be restored by replacing the helix α 0 with an amphipathic helix of the HCV NS5A protein. In summary, our data demonstrated that the amphipathic helix α 0 of NS3 protein determines the proper membrane association of NS3, and this subcellular localization dictates the functional role of NS3 in the HCV life cycle.

Background: Because of high mutation rates, new drug-resistant viruses are rapidly evolving, thus making the necessary control of influenza virus infection difficult. Methods: We screened a constrained cysteine-rich peptide library mimicking μ-conotoxins from Conus geographus and a proline-rich peptide library mimicking lebocin 1 and 2 from Bombyx mori by using influenza virus RNA polymerase (PB1, PB2 and PA) and nucleoprotein (NP) as baits. Results: Among the 22 peptides selected from the libraries, we found that the NP-binding proline-rich peptide, PPWCCCSPMKRASPPPAQSDLPATPKCPP, inhibited influenza replicon activity to mean ±SD 40.7% ±15.8% when expressed as a GFP fusion peptide in replicon cells. Moreover, when the GFP fusion peptide was transduced into cells by an HIV-TAT protein transduction domain sequence, the replication of influenza virus A/WSN/33 (WSN) at a multiplicity of infection of 0.01 was inhibited to 20% and 69% at 12 and 24 h post-infection, respectively. In addition, the TAT-GFP fusion peptide was able to slightly protect Balb/c mice from WSN infection when administrated prior to the infection. Conclusions: These results suggest the potential of this peptide as the seed of an anti-influenza drug and reveal the usefulness of the constrained peptide strategy for generating inhibitors of influenza infection. The results also suggest that influenza NP, which is conserved among the influenza A viruses, is a good target for influenza inhibition, despite being the most abundant protein in infected cells.

Influenza Avirus is one of the major pathogens that pose a large threat to human health worldwide and has caused pandemics. Influenza A virus is the Orthomyxoviridae prototype, and has 8 segmented negative-sense single-stranded RNA (vRNA) as its genome. Influenza virus RNA polymerase (RdRp) consists of three subunits PB2, PB1 and PA, and catalyzes both transcription and replication. Recently, intensive biochemical and structural analysis of its RdRp has been performed. In this paper, we review the details from the biochemical analysis of the purified influenza virus RdRp and the classical ribonucleoprotein complex, as well as piece together their structures to form an overall picture.

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.

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.

A natural variation is observed at position 4 of the 3′-end of influenza A virus genomes, where U (U4) or C (C4) is present. The replicon activity of C4 was 28% of U4. We compared the transcription and replication activity of U4 [v84(U4)], C4 [v84(C4)] and the complimentary RNA (c84) using the purified influenza virus RNA polymerase in vitro. ApG-primed replication activities of v84(C4) and c84 were 23.8% and 7.8% of v84(U4). Globin mRNA-primed transcription activities of v84(C4) and c84 were 36.9% and 6.81% of v84(U4). De novo replication activities of v84(C4) and c84 were 21.3 and 10.2% of v84(U4). This difference came from their polymerase binding activity. When all the eight genome segments of WSN strain were changed to U4, the virus titer was 760 times higher than the wild type. However, its pathogenicity in mice was lower than the wild type.

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.

International audience; 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.

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.

Serine at position 624 of the PA subunit of the influenza A virus RNA polymerase is the active site of a serine protease. To examine the role of this protease activity in the viral infection cycle, we compared the growth and pathogenesis of influenza virus A/WSN/33 and the virus encoding a PA with an S624A mutation (S624A virus). The growth and pathogenesis of the S624A virus was less than that of WSN in cells, indicating that the protease activity of PA is not essential for viral growth and pathogenesis, but is required for maximal viral growth.

Zanamivir has been evaluated for treatment and prophylaxis of influenza A and B viruses. However, it is not recommended for children younger than 6 years old because the use of the Diskhaler is required. We developed a new method of administering zanamivir with a straw in order to expand the application of zanamivir to those who are not yet able to use the Diskhaler. Of 142 patients aged 2 months to 5 years old who received zanamivir in this study, 126 were relieved from fever (

Gene expression patterns between encephalitis/encephalopathy patients and respiratory infection patients by the influenza viral infection were compared by DNA microarray analysis. Expression of six genes (interferon (α, β, ω) receptor, ephrin-B2, RNA binding motif protein 5 (RBM5), transcription factor EC (TFEC), Calsenilin/DREAM/KChIP3, and MEGF6 protein) was elevated and that of three genes (non-metastatic protein 23 (Nm23/Awd), neuropeptide Y/peptide YY receptor, and hippocalcin-like protein 4 (HPCAL4)) was decreased in encephalitis/encephalopathy patients.

OBJECTIVE— To describe the clinical and immunologic characteristics of fulminant type 1 diabetes, a novel subtype of type 1 diabetes, we conducted a nationwide survey. RESEARCH DESIGN AND METHODS—History and laboratory data, including islet-related autoantibodies, were examined in 222 patients with fulminant and nonfulminant type 1 diabetes in our hospitals in addition to another 118 patients with fulminant type 1 diabetes located outside our hospitals in Japan. RESULTS—In our hospitals, of the 222 patients studied, 43 (19.4%) were diagnosed with fulminant type 1 diabetes, 137 (61.7%) were classified as having autoimmune type 1 diabetes, and 42 were type 1 diabetic subjects who were not fulminant and did not have anti-islet antibodies. An additional 118 fulminant patients outside our hospitals were enrolled, making a total of 161 fulminant type 1 diabetic subjects (83 male and 78 female subjects; 14 children/adolescents and 147 adults) identified from all over Japan. (In 2000, the average incidence was three cases per month.) Flu-like symptoms and pregnancy were more frequently observed in the fulminant than in the autoimmune group (P < 0.001). In the fulminant patients, 4.8% were positive for anti-GAD antibodies and none were positive for anti–islet antigen 2 antibodies. CONCLUSIONS—Fulminant type 1 diabetes is a distinct subtype and accounts for ∼20% of the ketosis-onset type 1 diabetes cases in Japan. Flu-like symptoms are characteristic of disease onset. Metabolic derangement is more severe in this subtype than in autoimmune type 1 diabetes.

The biochemical properties of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) truncated with C-terminal 21 amino acids and expressed in insect cells were analyzed. The enzyme carried copy-back and de novo RNA synthesis activity but not terminal nucleotidyl transferase activity. k(pol) and K(m) for de novo RNA synthesis were calculated as 10.0 pmol/microg/h and 2.5 microM under 0.5 mM GTP and 2.0 pmol/microg/h and 3.5 microM under 50 microM GTP, respectively. Those for copy-back RNA synthesis were similar under both conditions (k(pol), 1.8 pmol/microg/h; K(m), 3.0 microM). De novo RNA synthesis was activated by 0.5 mM GTP. However, the ratio of GTP to three other NTPs was important for activation. Our HCV RdRp showed high activity for the complementary sequence of the HCV internal ribosomal entry site and a synergistic effect of Mg(2+) to Mn(2+).

Background: Influenza virus RNA polymerase consists of three subunits, PB1, PB2 and PA, and catalyzes both transcription and replication of the RNA genome. PB1 is a catalytic subunit of RNA polymerization and a core of subunit assembly. Previously, we mapped the PB1–PB2 binding sites (1–259 of PB2 and 501–757 of PB1), and the PB1–PA binding sites (1–140 of PB1 and 201–716 of PA). In order to determine the fine map of the subunit binding sites, we continued the same line of experiments. Methods: Serial deletion mutants of each subunit were expressed with N-terminal HA-tag, and co-immunoprecipitated with the non-tagged wild-type subunits by anti-HA or subunit-specific antibodies. Results: PB1N599 and PB1C10 were co-immunoprecipitated with PB2wt, but PB1N625 and PB1N700 were weakly co-immunoprecipitated with PB2wt. PB1C25 or PB1C40 were not co-immunoprecipitated with PB2wt. PB2N50 and PB2N75 were co-immunoprecipitated with PB1wt. However, PB2N104 or PB2N150 were not co-immunoprecipitated with PB1wt. PB1C535 and PB1C617 were co-immunoprecipitated with PAwt, but PB1N25 was not. PAN300, PAN400, PAN500 and PA501–692 were co-immunoprecipitated with PB1wt, but PAC49 was not. Conclusions: PB1–PB2 binding sites are mapped in PB1(600–747) and PB2(76–104), and PB1–PA binding sites are in PB1(1–25) and PA(668–692).

Influenza virus RNA polymerase is a multifunctional enzyme that catalyzes both transcription and replication of the RNA genome. The function of the influenza virus RNA polymerase PA subunit in viral replication is poorly understood although the enzyme is known to be required for cRNA→vRNA synthesis. Protease related activity of PA has been discussed ever since protease-inducing activity was demonstrated in transfection experiments. PA protein was highly purified from baculovirus-infected insect cells and a novel chymotrypsin-type serine protease activity with serine624 as an active site was identified in the protein. These results constitute the first demonstration of protease activity in RNA polymerase complexes.

Rapid progressive encephalopathy showing a high fever, consciousness loss and recurrent convulsions has been occasionally reported in childhood during influenza epidemics in Japan since 1995. A clinicopathological study of a 2-year-old female diagnosed with hemorrhagic shock and encephalopathy syndrome associated with influenza A virus (A/Nagasaki/76/98; H3N2) infection showed that the virus antigens are present in CD8-positive T lymphocytes from the lung and spleen, suggesting the possible route of the virus spread. The virus antigens are confined to a very limited part of the brain, especially Purkinje cells in the cerebellum and many neurons in the pons, without inducing an overt immunological reaction of the host. RT-PCR for detecting the hemagglutinin gene demonstrated definite positive bands in all frozen tissues and cerebrospinal fluids taken at autopsy but not in samples on admission. The pathological change induced by the direct viral invasion cannot be sufficient for the rapid and severe clinical course of the disease within 24–48 h after the initial respiratory symptoms. The rapid production of several inflammatory cytokines together with the breakdown of the blood–brain barrier, which will induce severe brain edema, may be the major pathological processes. Any therapeutic strategy to control this multi-step progression could be effective.

An epidemic of aseptic meningitis caused by human echovirus 9 (E-9) occurred in the summer of 1997 in northern Kyushu, Japan. Sequences of genome position 2504-3358, which encoded a part of VP1, of the nine isolated viruses were determined. An RGD motif and B-C loop were found in all. They were almost identical and closely related to the virulent strain Barty.

Background Influenza virus RNA polymerase is a multifunctional enzyme that catalyses both transcription and replication of the RNA genome. The function of the influenza virus RNA polymerase PA subunit in viral replication is poorly understood, although the enzyme is known to be required for cRNA vRNA synthesis. The protease related activity of PA has been discussed ever since protease-inducing activity was demonstrated in transfection experiments. Results PA protein was highly purified from insect cells infected with the recombinant baculovirus carrying PA cDNA, and a novel chymotrypsin-type serine protease activity was identified with the synthetic peptide, Suc-LLVY-MCA, in the PA protein. [3H]DFP was crosslinked with PA and a mutational analysis revealed that serine624 was as an active site for the protease activity. Conclusions These results constitute the demonstration of protease activity in PA subunit of the influenza virus RNA polymerase complexes.

Influenza A viruses (H3N2) isolated in 1998 in Nagasaki, Japan, carried a mutation (384R --> G) in one of the anchor amino acids of the HLA-B27-restricted cytotoxic T lymphocyte (CTL) epitope of NP (383-391). Phylogenetic analysis revealed that these viruses have been isolated only in Japan to date and belong to the unique lineages.

Influenza virus replication has been effectively inhibited by antisense phosphothioate oligonucleotides targeting the AUG initiation codon of PB2 mRNA. We designed RNA-cleaving DNA enzymes from 10-23 catalytic motif to target PB2-AUG initiation codon and measured their RNA-cleaving activity in vitro. Although the RNA-cleaving activity was not optimal under physiological conditions, DNA enzymes inhibited viral replication in cultured cells more effectively than antisense phosphothioate oligonucleotides. Our data indicated that DNA enzymes could be useful for the control of viral infection.

To obtain reagents to functionally map the PA protein, we produced monoclonal antibodies specific to this protein. Twenty-two monoclonal antibodies reacting with PA protein in ELISA were divided into 10 groups on the basis of competitive binding patterns to this protein. Of these, seventeen monoclonal antibodies bound to PA polypeptide spanning amino acids 101-400 and three bound to that of amino acids 518-600, while the other two did not react with any PA polypeptides tested with the exception of full-length PA. Among these monoclonal antibodies, only five reacted with PA in A/PR/8/34 virus-infected cells in indirect immunofluorescence assay. Thus, we obtained monoclonal antibodies that recognize at least 10 distinct regions of the PA molecule. These monoclonal antibodies should be useful in dissecting functions of the PA protein.

Background: Rapid progressive encephalopathy with a high fever, consciousness loss and recurrent convulsions has been occasionally reported in children during influenza pandemics in Japan since 1995. We examined a 2-year old girl with hemorrhagic shock and encephalopathy syndrome associated with acute influenza A virus infection (A/Nagasaki/76/98; H3N2), to answer several questions for which no histologic or virologic data exist. Methods: A clinicopathologic study using immunohistochemical staining and viral genome detection by reverse transcriptase polymerase chain reaction (RT-PCR) was performed with this autopsied case. Results: The virus antigen was positive in CD8+ T lymphocytes from the lung and spleen. The virus infected a very limited part of the brain, especially Purkinje cells in the cerebellum and many neurons in the pons, without inducing an overt immunologic reaction from the host. The RT-PCR used for detecting the hemagglutinin gene demonstrated positive bands in all frozen tissues and cerebrospinal fluid taken at autopsy and not in samples obtained on admission. Conclusions: The pathologic change induced by the direct viral invasion cannot be responsible for all of the symptoms, especially for the rapid and severe clinical course of the disease within 24–48 h after the initial respiratory symptoms. Together with the rapid production of several inflammatory cytokines, the breakdown of the blood–brain barrier may induce severe brain edema and can be a major pathologic change for the disease. Any therapeutic strategy to control this multistep progression of the disease could be effective.

There is controversy over the prevalence of Borna disease virus (BDV) antibodies and its RNA in the peripheral blood mononuclear cells (PBMCs) of psychiatric patients, and the contribution of BDV to human psychiatric disorders. We examined 299 plasma and 229 PBMC samples. No plasma samples were positive for BDV-p40, p24 or gp18 antibodies by western blot analysis. The prevalence of BDV RNA in the psychiatric (schizophrenic) patients (1.8%) was not significantly different from that in the healthy volunteers (0.6%). The nucleotide sequences of BDV p40 and p24 were highly conserved with those of BDV He/80. Our results suggested that there is a lack of association between BDV infection and psychiatric disorders among the patients in Northern Kyushu, Japan.

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome, including the unique cap-I-dependent RNase activity. To map the important domains for RNA polymerization, cap-I-dependent RNase, and cap-I-binding activity, we generated site-specific antibodies against overlapping 150-amino-acid peptides that cover each entire subunit. Monospecific antibodies against each subunit inhibited RNA synthesis in vitro. Those against PB1 and PB2 inhibited the cap-I-dependent RNase activity, but those against PB2 alone slightly inhibited the cap-I-binding activity. Antibodies against the N-terminal amino acids 1-159 of PB2 that overlap the PB1-binding site on PB2 and the C-terminal amino acids 501-617 of PA that overlap the putative nucleotide-binding site and PB1-binding site on PA inhibited RNA polymerizing activity as well as monospecific antibodies. Those against the N-terminal (amino acids 1-159); the central region (amino acids 305-559) of PB2, where a part of the cap-binding domain predicted previously is localized; the N-terminal (amino acids 1-222) of PB1; and amino acids 301-517 and 601-716 of PA inhibited the cap-I-dependent RNase activity. The cap-binding domain on PB2 could be mapped in amino acids 402-559, where one of the cap-binding domains mapped previously overlapped.

The nucleotide sequences of the genome RNA encoding the RNA polymerase and the 3' non-coding region (NCR) of bovine enterovirus (BEV) serotype I Japanese isolate, MZ468, were determined. The genetic distance between the two BEV serotype I strains, MZ468 and VG-5-27, was calculated by pairwise comparison of nucleotide sequences. The synonymous substitution rate was high (1.40 x 10(-2)/site/year), and of the same order as those of influenza virus HA, HIV-1 gag and env, and enterovirus 70 VP1 genes.

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. By transfection of various combinations of cDNA encoding wild-type and serial deletion mutants of each P protein subunit and co-immunoprecipitation with subunit-specific antibodies, the subunit-subunit contact sites on all three of the P proteins were determined. Results indicate that binary complexes are formed between PB1-PB2 and PB1-PA but not between PB2-PA. Therefore, we concluded that PB1 is the core subunit for assembly of the virus RNA polymerase. The C-terminal 158 amino acids of PB1 bound to the N-terminal 249 amino acids of PB2, while the N-terminal 140 amino acids of PB1 bound to the C-terminal two-thirds of PA. PB2-PA binding was not detected when they were expressed in the absence of the PB1 subunit.

Human papillomavirus (HPV) types 16 and 6b E7 proteins and their chimeric or mutant proteins were analyzed for oligonucleotide-binding activity by surface plasmon resonance-based biomolecular interaction analysis. The results indicated that type 16 E7 protein has stronger nucleic acid-binding activity than that of type 6b E7 protein. In addition, the results also indicated that the zinc finger-like motif in the C-terminal region of the type 16 E7 protein plays an important role in this activity.

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.

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.

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., Author Summary One of the key components for hepatitis C virus (HCV) propagation is lipids, some of which comprise membranous replication complexes for HCV replication. Research on cofactors that are involved in the formation of the membranous replication complex has advanced steadily; on the other hand, the lipids constituting the membranous replication complex remain to be elucidated. Here, we report that HCV modulates sphingolipid metabolism by promoting sphingolipid biosynthesis, to enhance viral replication. Specifically a specific molecular species of sphingomyelin (SM), a type of sphingolipid interacts with HCV nonstructural 5B polymerase, enhancing HCV replication. This work highlights the relationship between specific molecular species of SMs and HCV replication, giving new insight into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Influenza virus nucleoprotein (NP) is associated with the genome RNA, forming ribonucleoprotein cores. To identify the amino acid sequence involved in RNA binding, we performed Northwestern blot analysis with a set of N- and C-terminal deletion mutants of NP produced in Escherichia coli. The RNA binding region has been mapped between amino acid residues 91 and 188, a stretch of residues that contains a sequence that is not only highly conserved among NPs from A-, B-, and C-type influenza viruses but also similar to the RNA binding domain of a plant virus movement protein.

Nonlinear forced oscillation of a rotating circular disk excited at a fixed point in space is considered. For theoretical analysis, modal equations are derived from the governing partial differential equations. Then, based on these modal equations, the characteristics of oscillation induced by harmonic excitation are investigated. Numerical calculation is conducted for a typical case in which the excitation frequency approches the primary resonance points of the forward and backward traveling wave modes. It is shown that near the primary resonance point of the backward traveling mode, forward traveling mode oscillation occurs in addition to backward traveling mode oscillation, and conversely, near the primary resonance point of the forward traveling mode, backward traveling mode oscillation occurs in addition to forward traveling mode oscillation. An experimental analysis is also conducted, which confirms the validity of the theoretical analysis.