A small fragmented P protein of respiratory syncytial virus inhibits virus infection by targeting P protein.

Peptide-based inhibitors hold promising potential in the development of antiviral therapy. Here, we investigated the antiviral potential of fragmented viral proteins derived from ribonucleoprotein (RNP) components of the human respiratory syncytial virus (HRSV). Based on a mimicking approach that targets the functional domains of viral proteins, we designed various fragments of nucleoprotein (N), matrix protein M2-1 and phosphoprotein (P) and tested the antiviral activity in an RSV mini-genome system. We found that the fragment comprising residues 130-180 and 212-241 in the C-terminal region of P (81 amino acid length), denoted as P Fr, significantly inhibited the polymerase activity through competitive binding to the full-length P. Further deletion analysis of P Fr suggested that three functional domains in P Fr (oligomerization, L-binding and nucleocapsid binding) are required for maximum inhibitory activity. More importantly, a purified recombinant P Fr displayed significant antiviral activity at low nanomolar range in RSV-infected HEp-2 cells. These results highlight P as an important target for the development of antiviral compounds against RSV and other paramyxoviruses.

Basic amino acids in the N-terminal half of the PB2 subunit of influenza virus RNA polymerase are involved in both transcription and replication.

The PB2 subunit of influenza virus RNA polymerase is known to be involved in the initiation of transcription of the virus genome via cap binding. However, other specific roles of PB2 for viral RNA synthesis are not well understood. Here, we demonstrate that basic residues, 124R, 142R, 143R, 268R and 331K/332R, in the N-terminal half of PB2 are important for the polymerase activity. Notably, R124A mutation remarkably reduced the synthesis of mRNA, cRNA and vRNA in vivo, which was in good agreement with the data obtained in vitro. Cross-linking studies suggested that a reduction of the polymerase activity in the R124A mutant was due to a significant decrease in binding to the viral RNA promoter. In the three-dimensional structure of the polymerase, 124R is visible through the NTP tunnel and is located close to the polymerase active site. We propose that 124R plays a key role in promoter binding during RNA synthesis.

Dengue Hemorrhagic Fever in a Japanese Traveler with Pre-existing Japanese Encephalitis Virus Antibody.

An adult Japanese man who had just returned from Thailand developed dengue hemorrhagic fever (DHF). A primary infection of dengue virus (DENV) was confirmed, specifically DENV serotype 2 (DENV-2), on the basis of the detection of the virus genome, a significant increase in the neutralizing antibody and the isolation of DENV-2. DHF is often observed following a secondary infection from another serotype of dengue virus, particularly in children, but this case was a primary infection of DENV. Japan is a non-endemic country for dengue disease. In fact, only Japanese encephalitis (JE) is known to be a member of the endemic flavivirus family. In this study, IgG antibody against Japanese encephalitis virus (JEV) was detected. JEV belongs to the family of dengue virus and prevails in Japan, particularly Kyushu. Among many risk factors for the occurrence of DHF, a plausible candidate could be a cross-reactive antibody-dependent enhancement (ADE) mechanism caused by JEV antibody. This indicates that most Japanese travelers who living in dengue non-endemic areas, particularly Kyushu, should be aware of the occurrence of DHF.

The N-terminal fragment of PA subunit of the influenza A virus effectively inhibits ribonucleoprotein (RNP) activity via suppression of its RNP expression.

The influenza RNP, which is formed from PB1, PB2, PA, NP subunits, and vRNA, is autonomously replicated and transcribed in the infected cell. The simplest method to inhibit RNP activity is to impair the formation of the RNP. Thereupon we confirmed whether the peptides/fragments mimicking one of RNP components can interfere with their formation. During the process of this inhibitory study we found interesting suppression of protein expression of the RNP components by the N-terminal fragment of PA subunit. Especially, we found two residues (D108 and K134) on the fragment that were critical for the suppression. Furthermore, we determined the combination of three amino acids (P28, M86 and E100) on the fragment that are important for the strong suppression, and identified the minimum essential region (residues from 1 to 188) of the PA subunit that allowed its suppression. Our findings indicate that the N-terminal fragment of PA subunit may become one of candidates for an effective inhibitor of influenza RNP activity.

The N-terminal fragment of a PB2 subunit from the influenza A virus (A/Hong Kong/156/1997 H5N1) effectively inhibits RNP activity and viral replication.

BACKGROUND: Influenza A virus has a RNA-dependent RNA polymerase (RdRp) that is composed of three subunits (PB1, PB2 and PA subunit), which assemble with nucleoproteins (NP) and a viral RNA (vRNA) to form a RNP complex in the host nucleus. Recently, we demonstrated that the combination of influenza ribonucleoprotein (RNP) components is important for both its assembly and activity. Therefore, we questioned whether the inhibition of the RNP combination via an incompatible component in the RNP complex could become a methodology for an anti-influenza drug. METHODOLOGY/PRINCIPAL FINDINGS: We found that a H5N1 PB2 subunit efficiently inhibits H1N1 RNP assembly and activity. Moreover, we determined the domains and important amino acids on the N-terminus of the PB2 subunit that are required for a strong inhibitory effect. The NP binding site of the PB2 subunit is important for the inhibition of RNP activity by another strain. A plaque assay also confirmed that a fragment of the PB2 subunit could inhibit viral replication. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the N-terminal fragment of a PB2 subunit becomes an inhibitor that targets influenza RNP activity that is different from that targeted by current drugs such as M2 and NA inhibitors.

Co-incorporation of the PB2 and PA polymerase subunits from human H3N2 influenza virus is a critical determinant of the replication of reassortant ribonucleoprotein complexes.

The influenza virus RNA polymerase, composed of the PB1, PB2 and PA subunits, has a potential role in influencing genetic reassortment. Recent studies on the reassortment of human H3N2 strains suggest that the co-incorporation of PB2 and PA from the same H3N2 strain appears to be important for efficient virus replication; however, the underlying mechanism remains unclear. Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes and demonstrated that the RNP activity was severely impaired when the PA subunit of H3N2 strain A/NT/60/1968 (NT PA) was introduced into H1N1 or H5N1 polymerase. The NT PA did not affect the correct assembly of the polymerase trimeric complex, but it significantly reduced replication-initiation activity when provided with a vRNA promoter and severely impaired the accumulation of RNP, which led to the loss of RNP activity. Mutational analysis demonstrated that PA residues 184N and 383N were the major determinants of the inhibitory effect of NT PA and 184N/383N sequences were unique to human H3N2 strains. Significantly, NT PB2 specifically relieved the inhibitory effect of NT PA, and the PB2 residue 627K played a key role. Our results suggest that PB2 from the same H3N2 strain might be required for overcoming the inhibitory effect of H3N2 PA in the genetic reassortment of influenza virus.

[Review of a community-based laboratory diagnosis of pandemic influenza A (H1N1) 2009].

We performed a community-based laboratory diagnosis of pandemic influenza A (H1N1) 2009 with the RT-PCR technique using originally constructed primers. Of 30 patients who were suspected to be infected with the influenza virus from May 2009 until January 2010, the A (H1N1) 2009 virus was detected in 13 patients (43.3%). Three cases were immunologically confirmed to be infected with the A (H1N1) 2009 virus, because significant increases in the HI titer were observed in the convalescent sera. We also measured the antibody titers to the A (H1N1) 2009 virus in 13 healthy individuals with the HI assay using originally isolated virus. In most cases, the HI antibody titers were less than 10, except two cases with titers of 40 and 20. Our inspection system organized in the early phase of the A (H1N1) 2009 pandemic contributed to disease control in an outpatient clinic and a hospital in a small city. The process which we used to construct the system would be a good reference for a treatment protocol in the case of a future literal pandemic.

Intrahost emergent dynamics of oseltamivir-resistant virus of pandemic influenza A (H1N1) 2009 in a fatally immunocompromised patient.

The oseltamivir-resistant pandemic influenza virus A (2009 H1N1) with H275Y mutation in neuraminidase (NA) has been sporadically reported, and its wide spread remains a potential threat. Here we detected the uneven distribution of H275Y mutant virus in a patient who received a 21-day long-term administration of oseltamivir. Intrahost variation of the virus showed that the H275Y mutant virus was the predominant population in both nasopharynx and right lung, whereas the oseltamivir-sensitive virus comprised half the population in the left lung. By constructing minimum spanning trees, it is proposed that the H275Y mutant might be generated primarily in the nasopharynx, then spread to the right and left lungs.

The RNA polymerase PB2 subunit of influenza A/HongKong/156/1997 (H5N1) restricts the replication of reassortant ribonucleoprotein complexes [corrected].

BACKGROUND: Genetic reassortment plays a critical role in the generation of pandemic strains of influenza virus. The influenza virus RNA polymerase, composed of PB1, PB2 and PA subunits, has been suggested to influence the efficiency of genetic reassortment. However, the role of the RNA polymerase in the genetic reassortment is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes, and demonstrated that the PB2 subunit of A/HongKong/156/1997 (H5N1) [HK PB2] dramatically reduced the synthesis of mRNA, cRNA and vRNA when introduced into the polymerase of other influenza strains of H1N1 or H3N2. The HK PB2 had no significant effect on the assembly of the polymerase trimeric complex, or on promoter binding activity or replication initiation activity in vitro. However, the HK PB2 was found to remarkably impair the accumulation of RNP. This impaired accumulation and activity of RNP was fully restored when four amino acids at position 108, 508, 524 and 627 of the HK PB2 were mutated. CONCLUSIONS/SIGNIFICANCE: Overall, we suggest that the PB2 subunit of influenza polymerase might play an important role for the replication of reassortant ribonucleoprotein complexes.

Clinical study concerning the relationship between community-acquired pneumonia and viral infection in northern Thailand.

OBJECTIVE: The etiological agents associated with community-acquired pneumonia (CAP) in Thailand have been studied extensively in bacterial pathogens, but not in viral pathogens. To clarify the association of viral pathogens with CAP, we conducted a comprehensive study of viral and bacterial pathogens in patients with CAP. METHODS: We enrolled 119 hospitalized patients with CAP in Nakornping Hospital, Chiang Mai, Thailand between 2006 and 2008. The severity of pneumonia was classified and the risk factors for death were estimated. Bacterial and fungal pathogens were determined from specimens taken from blood and sputum, and viral pathogens were identified from nasopharyngeal specimens by RT-PCR using primers specific for 7 respiratory viruses. RESULTS: Overall, 29 patients were HIV-infected and 90 patients were non-HIV-infected. The microbial pathogens most commonly isolated among HIV-infected patients were: 4 Klebsiella pneumoniae, 4 Mycobacterium tuberculosis and 3 Haemophilus influenzae. Among non-HIV infected patients, predominant microbial pathogens were: 6 Pseudomonas aeruginosa, 5 Haemophilus influenzae and 4 Klebsiella pneumoniae. As for viral pathogens for CAP, influenza virus was identified from 2 HIV-infected patients and 5 non-HIV infected patients. In addition, human rhinovirus (HRV) and respiratory syncytial virus (RSV) were identified from 2 patients each among non-HIV-infected patients. CONCLUSION: Our study demonstrates that the most common viral agent was influenza virus (5%), followed by HRV (2%) and RSV (2%) among CAP patients in northern Thailand. The underlying chronic obstructive pulmonary disease (COPD) seems to be correlated with the severity of illness.

Artificial hybrids of influenza A virus RNA polymerase reveal PA subunit modulates its thermal sensitivity.

BACKGROUND: Influenza A virus can infect a variety of different hosts and therefore has to adapt to different host temperatures for its efficient viral replication. Influenza virus codes for an RNA polymerase of 3 subunits: PB1, PB2 and PA. It is well known that the PB2 subunit is involved in temperature sensitivity, such as cold adaptation. On the other hand the role of the PA subunit in thermal sensitivity is still poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: To test which polymerase subunit(s) were involved in thermal stress we reconstituted artificial hybrids of influenza RNA polymerase in ribonucleoprotein (RNP) complexes and measured steady-state levels of mRNA, cRNA and vRNA at different temperatures. The PA subunit was involved in modulating RNP activity under thermal stress. Residue 114 of the PA subunit was an important determinant of this activity. CONCLUSIONS/SIGNIFICANCE: These findings suggested that influenza A virus may acquire an RNA polymerase adapted to different body temperatures of the host by reassortment of the RNA polymerase genes.

A mutational shift from domain III to II in the internal ribosome entry site of hepatitis C virus after interferon-ribavirin therapy.

We focused on the relationship between variation in the IRES of hepatitis C virus (HCV) genotype 1b and clinical outcome, since the internal ribosome entry site (IRES) has a comparatively low heterogeneity and it might be easy to find unique substitutions. Patients infected with HCV were selected using strict criteria, and unique mutations in the IRES were extracted by the subtraction of common mutations. We found that most mutations accumulated in domain III (dIII) of IRES in sustained virological responders (SVRs) and non-SVRs before therapy. However, these mutations were exclusively observed in domain II (dII) in non-SVR at 2 weeks after the start of therapy.

Differential role of the influenza A virus polymerase PA subunit for vRNA and cRNA promoter binding.

The RNA polymerase of influenza A virus is a heterotrimeric complex of PB1, PB2 and PA subunits that is required for transcription and replication of the viral genome. Here, we demonstrate a differential requirement of the PA subunit for binding to the vRNA and cRNA promoters--specifically, PA is more important for binding to the cRNA than the vRNA promoter. Furthermore, five point mutations were identified in the L163-I178 region of PA, which resulted in an inhibition of polymerase activity when provided with a cRNA compared to vRNA promoter. Cross-linking studies suggested that this inhibition was due to a reduction in promoter binding of the mutant polymerases to the cRNA promoter. We conclude that the L163-I178 region of PA is directly or indirectly involved in cRNA promoter binding and suggest a novel function for PA in modulating promoter binding.

Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding.

The RNA-dependent RNA polymerase of influenza virus is a heterotrimer formed by the PB1, PB2, and PA subunits. Although PA is known to be required for polymerase activity, its precise role is still unclear. Here, we investigated the function of the N-terminal region of PA. Protease digestion of purified recombinant influenza virus A/PR/8/34 PA initially suggested that its N-terminal region is folded into a 25-kDa domain. We then systematically introduced point mutations into evolutionarily conserved amino acids in the N-terminal region of influenza virus A/WSN/33. Most alanine-scanning mutations between residues L109 and F117 caused PA degradation, mediated by a proteasome-ubiquitin pathway, and as a consequence interfered with polymerase activity. Three further PA mutations, K102A, D108A, and K134A, were investigated in detail. Mutation K102A caused a general decrease both in transcription and replication in vivo, whereas mutations D108A and K134A selectively inhibited transcription. Both the D108A and K134A mutations completely inhibited endonuclease activity in vitro, explaining their selective defect in transcription. K102A, on the other hand, resulted in a significant decrease in both cap binding and viral RNA promoter-binding activity and consequently inhibited both transcription and replication. These results suggest that the N-terminal region of PA is involved in multiple functions of the polymerase, including protein stability, endonuclease activity, cap binding, and promoter binding.

Inhibition of the protease activity of influenza virus RNA polymerase PA subunit by viral matrix protein.

Influenza virus PA is a subunit of RNA-dependent RNA polymerase. We demonstrated that PA has a unique chymotrypsin-like serine protease activity with Ser624 as an active site. To obtain further insight into the role of the protease activity of PA in viral proliferation, we examined the interaction between PA and matrix protein (M1). Both M1 purified from virion and hexa-histidine-tagged M1 expressed in Escherichia coli bound to PA. Hexa-histidine-tagged M1 pulled down PA. The interaction of PA with M1 was sensitive to ionic strength, suggesting that the interaction is formed by electrostatic force. Using Suc-Leu-Leu-Val-Tyr-MCA, a specific substrate for PA protease, M1 was demonstrated to inhibit the amidolytic activity of PA, whereas M1 did not inhibit that of chymotrypsin or trypsin at all. These results suggest that M1 binds to and inhibits the amidolytic activity of PA.

Establishment and characterization of a continuous cell line from pupal ovaries of Japanese oak silkworm Antheraea yamamai Guerin-Meneville.

Pupal ovaries of the wild oak silkworm Antheraea yamamai Guerin-Meneville were cultured in MGM-448 (Modified Grace Medium-448) medium containing 10% fetal bovine serum. After the primary culture was set up in 1988, a continuous cell line was obtained in 1991, designated as NISES-Anya-0611 (Anya-0611). The population doubling time was 54 hrs. and 19 min. at 96 passages and 88 hrs. and 29 min. at 387 passages. Spindle-shaped and spherical cells coexisted in the cell group. The cell line karyotype line was typical of lepidopteran cell lines, consisting of numerous small chromosomes. The cell line was distinguished from other lepidopteran cell lines by comparing malic enzyme, phosphoglucose isomerase, phosphoglucose mutase, and isocitric dehydrogenase isozyme patterns. The cell line was highly infected to the Antheraea yamamai nuclear polyhedrosis virus (Anya NPV). The luciferase gene of recombinant Bm NPV (BmNPVP6ETL) was able to express in the cell line, too, so that luciferase recombinant products were able to be detected in the cell body and in supernatant. The Anya NPV clone group was isolated on the cell seat using plaque purification.

Promoter/origin structure of the complementary strand of hepatitis C virus genome.

Hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp). Sequences in the 3' termini of both the plus and minus strand of HCV genomic RNA harbor the activity of a replication origin and a transcription promoter. There are unique stem-loop structures in both termini of the viral RNA. We found that the complementary strand of the internal ribosome-binding site (IRES) showed strong template activity in vitro. The complementary strand RNA of the HCV genome works as a template for mRNA and viral genomic RNA. We analyzed the promoter/origin structure of the complementary sequence of IRES and found that the first and second stem-loops worked as negative and positive elements in RNA synthesis, respectively. The complementary strand of the second stem-loop of IRES was an important element also for binding to HCV RdRp.

Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis.

Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER). At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown. Each RTN gene produces 1-3 proteins by different promoters and alternative splicing. In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C. mRNA of nRTN-C is expressed in germ cells and embryos. However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch. By yeast two-hybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated. These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis.

Molecular cloning and characterization of the mouse reticulon 3 cDNA.

cDNA of mouse reticulon 3 (mRTN3) was cloned. The cloned cDNA is 1745 bp long and contains an open reading frame of 237 amino acids for a 30 kDa protein. The gene was mapped at band B of chromosome 19 by FISH. Two altematively spliced transcripts, 3.4 and 2.3 kb, of mRTN3 were found by Northem blot analysis. Both transcripts were expressed in many tissues and embryos and the highest expression of the 3.4 kb-transcript was observed in the brain, especially in neurons. The expression of 30 kDa-mRTN3 protein was also greatest in the brain. Both N and C-termini of mRTN3 faced the cytosol, indicating that they may recruit other proteins to the endoplasmic reticulum.

Kinetic analysis of C-terminally truncated RNA-dependent RNA polymerase of hepatitis C virus.

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+).