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Bobo Wing-Yee Mok,
Wenjun Song, Pui Wang,
Hung Tai,
Yixin Chen,
Min Zheng,
Xi Wen,
Siu-Ying Lau,
Wai Lan Wu,
Ken Matsumoto,
Kwok-Yung Yuen,
Honglin Chen
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ABSTRACT: The non-structural protein (NS1) of influenza A virus exhibits multiple functions in the virus life cycle. Proteomics screening for cellular proteins which interact with NS1 identified the cellular protein, RAP55, which is one of the components of cellular processing bodies (P-bodies) and stress granules. To verify whether NS1 interacts with cellular P-bodies, interactions between NS1 and RAP55 and other P-body associated proteins (Ago1, Ago2 and DCP1a) were confirmed using co-immunoprecipitation and cellular co-localization assays. Over-expression of RAP55 induces RAP55-associated stress granule formation and suppresses virus replication. Knockdown of RAP55 with siRNA or expression of a dominant negative mutant RAP55 with defective interaction with P-bodies blocks NS1 co-localization to P-bodies in cells. Expression of NS1 inhibits RAP55 expression and formation of RAP55-associated P-bodies/stress granules. Viral nucleoprotein (NP) was found to be targeted to stress granules in the absence of NS1 but localized to P-bodies when NS1 was co-expressed. Restriction of virus replication via P-bodies occurs in the early phases of infection, as the numbers of RAP55-associated P-bodies in cells diminish over the course of virus infection. NS1 interaction with RAP55-associated P-bodies/stress granules is RNA binding associated and mediated via a PKR-interacting viral element. Mutations introduced into either RNA binding sites, namely R38/K41, or PKR interaction sites, namely I123/M124/K126/N127, cause NS1 proteins to lose the ability to interact with RAP55 and to inhibit stress granules. These results reveal an interplay between virus and host during virus replication in which NP is targeted to P-bodies/stress granules, while NS1 counteracts this host restriction mechanism.
Journal of Virology 09/2012; · 5.40 Impact Factor
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Wai Lan Wu,
Siu-Ying Lau,
Yixin Chen,
Genyan Wang,
Bobo Wing-Yee Mok,
Xi Wen, Pui Wang,
Wenjun Song,
Tianwei Lin,
Kwok-Hung Chan,
Kwok-Yung Yuen,
Honglin Chen
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ABSTRACT: A naturally-occurring H275Y oseltamivir resistant variant of influenza A (H1N1) virus emerged in 2007, subsequently becoming prevalent worldwide, via an undetermined mechanism. To understand the antigenic properties of the H275Y variant, oseltamivir resistant and susceptible strains of H1N1 viruses were analyzed by hemagglutination inhibition (HI) and microneutralization assays. HI analysis with H1-positive sera obtained from seasonal flu vaccine immunized and non-immunized individuals, and H1-specific monoclonal antibodies, revealed that resistant strains exhibited a reduced reactivity to these antisera and antibodies in the HI assay, as compared to susceptible strains. Neutralization assay testing demonstrated that oseltamivir resistant H1N1 strains are also less susceptible to antibody inhibition during infection. Mice inoculated with a resistant clinical isolate exhibit 4-fold lower virus-specific antibody titers than mice infected with a susceptible strain under the same conditions. Resistant and sensitive variants of 2009 pandemic H1N1 virus did not exhibit such differences. While HA1 and NA phylogenetic trees show that both oseltamivir resistant and susceptible strains belong to clade 2B, NA D354G and HA A189T substitutions were found exclusively, and universally, in oseltamivir resistant variants. Our results suggest that the reduced susceptibility to antibody inhibition and lesser in vivo immunogenicity of the oseltamivir resistant 2008-2009 H1N1 influenza A virus is conferred by coupled NA and HA mutations, and may contribute to the prevalence of this H1N1 variant.
Antiviral research 11/2011; 93(1):144-53. · 3.61 Impact Factor
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ABSTRACT: Metabolomics is the downstream of systems biology and has drawn significant interest for studying the metabolic networks from cells to organisms. To profile the metabolites in two different cell lines (A549 and AGS) infected with influenza A virus, gas chromatography coupled with mass spectrometry (GC/MS) was employed. Some differentiating metabolites in the cell lines were tentatively identified using reference library, interpreted and visualized by applying principal components analysis (PCA) and cluster heat map. Consequently, metabolic flux profiling allowed the differentiation of fatty acid biosynthesis and cholesterol metabolism during viral replication in the cell lines. The change in fatty acid turnover was also observed. Metabolomics investigation also revealed the different responses between A549 and AGS cell lines to the virus infection. From the pattern recognition results, AGS cell line might be more susceptible to influenza A virus. Regarding the fact that AGS is a poorly differentiated gastric adenocarcinoma cell line whereas A549 is a relatively differentiated lung tumor one, it is speculated that viral replication might be associated with the cell differentiations.
Talanta 11/2010; 83(1):262-8. · 3.79 Impact Factor
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Richard Y Kao,
Dan Yang,
Lai-Shan Lau,
Wayne H W Tsui,
Lihong Hu,
Jun Dai,
Mei-Po Chan,
Che-Man Chan, Pui Wang,
Bo-Jian Zheng,
Jian Sun,
Jian-Dong Huang,
Jason Madar,
Guanhua Chen,
Honglin Chen,
Yi Guan,
Kwok-Yung Yuen
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ABSTRACT: Influenza A remains a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. Antiviral resistance to available drugs such as adamantanes or neuraminidase inhibitors has appeared rapidly, creating a need for new antiviral targets and new drugs for influenza virus infections. Using forward chemical genetics, we have identified influenza A nucleoprotein (NP) as a druggable target and found a small-molecule compound, nucleozin, that triggers the aggregation of NP and inhibits its nuclear accumulation. Nucleozin impeded influenza A virus replication in vitro with a nanomolar median effective concentration (EC(50)) and protected mice challenged with lethal doses of avian influenza A H5N1. Our results demonstrate that viral NP is a valid target for the development of small-molecule therapies.
Nature Biotechnology 06/2010; 28(6):600-5. · 29.50 Impact Factor
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Honglin Chen,
Xi Wen,
Kelvin K W To, Pui Wang,
Herman Tse,
Jasper F W Chan,
Hoi-Wah Tsoi,
Kitty S C Fung,
Cindy W S Tse,
Rodney A Lee,
Kwok-Hung Chan,
Kwok-Yung Yuen
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ABSTRACT: The D225G (aspartic acid to glycine) substitution in the hemagglutinin of H1N1 influenza virus may alter its receptor-binding specificity. Direct analysis of polymorphisms in 126 amino acids spanning the receptor-binding site in the hemagglutinin of pandemic H1N1 2009 virus from 117 clinical specimens in Hong Kong found the D225G substitution for 7 (12.5%) of 57 patients with severe disease and for 0 (0%) of 60 patients with mild disease. D225G quasispecies were identified mainly in endotracheal aspirate samples and were identified less frequently in nasopharyngeal aspirate samples from patients with severe disease. Continuous monitoring of the prevalence and tissue tropism of this variant during its circulation among humans is important.
The Journal of Infectious Diseases 04/2010; 201(10):1517-21. · 6.41 Impact Factor
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Jinxia Zhang,
Zengfeng Zhang,
Xiaohui Fan,
Yuansheng Liu,
Jia Wang,
Zuoyi Zheng,
Rirong Chen, Pui Wang,
Wenjun Song,
Honglin Chen,
Yi Guan
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ABSTRACT: The replication activity of 2009 pandemic H1N1 influenza virus in human lung cells was evaluated in this study. Twenty-two surgically removed human lung tissue samples were infected ex vivo with pandemic H1N1 influenza virus (A/California/04/2009), seasonal human H1N1 influenza virus (A/Shantou/92/09), or a highly pathogenic H5N1 influenza virus (A/Vietnam/1194/04). Examination of nucleoprotein expression and viral RNA replication in the infected human lung tissue samples showed that whereas the replication of pandemic H1N1 influenza virus varied between tissue samples, overall this virus replicated more efficiently than seasonal H1N1 influenza virus but less efficiently than H5N1 influenza virus. Double-immunostaining for viral antigens and cellular markers indicated that pandemic H1N1 influenza virus replicates in type 2 alveolar epithelial cells.
The Journal of Infectious Diseases 04/2010; 201(10):1522-6. · 6.41 Impact Factor
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ABSTRACT: Unusual truncated forms of nucleocapsid protein (NP) were identified in the lysate of MDCK cells infected by Avian influenza virus (H9N2) using MS-based proteomics approach. Moreover, O-sulfonation that was considered as an unusual modification was identified in one of the tryptic peptides from the truncated NP. The findings might have implications on better understanding on the role of nucleoprotein in Avian influenza virus-host interaction.
Proteomics 03/2010; 10(9):1875-9. · 4.43 Impact Factor
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Huachen Zhu,
Jia Wang, Pui Wang,
Wenjun Song,
Zuoyi Zheng,
Rirong Chen,
Kunyuan Guo,
Taixing Zhang,
Joseph S M Peiris,
Honglin Chen,
Yi Guan
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ABSTRACT: A lysine at the 627 position (627K) of PB2 protein of influenza virus has been recognized as a determinant for host adaptation and a virulent element for some influenza viruses. While seasonal influenza viruses exclusively contained 627K, the pandemic (H1N1) 2009 possessed a glutamic acid (627E), even after circulation in humans for more than 6months. To explore the potential role of E627K substitution in PB2 in the pandemic (H1N1) 2009 virus, we compared pathogenicity and growth properties between a recombinant virus containing 627K PB2 gene and the parental A/California/4/2009 strain containing 627E. Our results showed that substitution of 627K in PB2 gene does not confer higher virulence and growth rate for the pandemic (H1N1) 2009 virus in mice and cell culture respectively, suggesting 627K is not required for human adaptation of the pandemic (H1N1) 2009 virus.
Virology 03/2010; 401(1):1-5. · 3.35 Impact Factor
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ABSTRACT: Nucleoprotein (NP), the structural component of ribonucleoprotein complex of avian influenza virus, performs multiple essential functions in the regulation of viral RNA synthesis and in the control of nuclear traffic of viral proteins. Mutations have often been found in NP, some of which are relevant to viral survival strategies. In this study, we used nanospray-MS/MS to analyze tryptic digestion of nucleoprotein of avian influenza virus (H5N1) and to identify three mutated peptides. The MS/MS analyses allowed the confident determination of the three mutated amino acid residues F313Y, I194V and V408I/L in the mutated peptides of LLQNSQVYSLIRPNENPAHK, GVGTMVMELVR and ASAGQI/LSVQPTFSVQR, respectively.
Talanta 07/2009; 78(4-5):1492-6. · 3.79 Impact Factor
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Pui Wang,
Wenjun Song,
Bobo Wing-Yee Mok,
Pengxi Zhao,
Kun Qin,
Alexander Lai,
Gavin J D Smith,
Jinxia Zhang,
Tianwei Lin,
Yi Guan,
Honglin Chen
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ABSTRACT: Interactions between host factors and the viral replication complex play important roles in host adaptation and regulation of influenza virus replication. A cellular protein, nuclear factor 90 (NF90), was copurified with H5N1 viral nucleoprotein (NP) from human cells in which NP was transiently expressed and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. In vitro coimmunoprecipitation of NF90 and NP coexpressed in HEK 293T cells or individually expressed in bacterial and HEK 293T cells, respectively, confirmed a direct interaction between NF90 and NP, independent of other subunits of the ribonucleoprotein complex. This interaction was prevented by a mutation, F412A, in the C-terminal region of the NP, indicating that the C-terminal of NP is required for NF90 binding. RNase V treatment did not prevent coprecipitation of NP and NF90, which demonstrates that the interaction is RNA binding independent. After small interfering RNA knockdown of NF90 expression in A549 and HeLa cells, viral polymerase complex activity and virus replication were significantly increased, suggesting that NF90 negatively affects viral replication. Both NP and NF90 colocalized in the nucleus of virus-infected cells during the early phase of infection, suggesting that the interaction between NF90 and NP is an early event in virus replication. Quantitative reverse transcription-PCR showed that NF90 downregulates both viral genome replication and mRNA transcription in infected cells. These results suggest that NF90 inhibits influenza virus replication during the early phase of infection through direct interaction with viral NP.
Journal of Virology 07/2009; 83(16):7850-61. · 5.40 Impact Factor
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ABSTRACT: Matrix protein 1 (M1), the major structural protein of the avian influenza virus, plays a critical role in regulation of viral RNA transcription via interaction with RNA and transportation of RNP cores. Mutations in M1 have been frequently observed in the highly virulent avian influenza H5N1 virus, which might be crucial to the pathogenic function. Here we report the characterization of mutated peptides in M1 purified from highly pathogenic avian influenza virus H5N1 by nanoelectrospray MS and MS/MS analyses on a quadrupole-time-of-flight mass spectrometer (Q-TOFMS). The specificity of tandem mass spectrometry allowed the identification of six amino acid (AA) substitutions in M1, including R95K, A166V, I168T, N207S, N224S, and R230K. Two commonly observed modifications such as oxidation and deamidation were accurately assigned in the protein. Bioinformatics analysis suggested some relationship between the amino acid substitution and structural property of M1 protein. Discussions on de novo sequencing of MS/MS spectra, especially in dealing with the AA substitutions, were provided.
Journal of the American Society for Mass Spectrometry 11/2008; 20(2):312-20. · 4.00 Impact Factor
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ABSTRACT: We present the first proteomic analysis on the cellular responses to avian influenza virus (H9N2) infection in a human cell line in different time courses in order to search for target proteins for viral pathogenesis/adaptation studies. By using 2-DE coupled with MALDI-TOF MS and nano-ESI-MS/MS, we identified a set of differentially expressed cellular proteins, including cytoplasmic actin, cytokeratin, prohibitin, enoyl-CoA hydratase, peptide-prolyl cis-trans isomerase A (PPIase A), chloride intracellular channel protein 1, pyruvate dehydrogenase E1 component subunit beta, adenine phosphoribosyltransferase, guanine nucleotide-binding protein subunit beta, nucleoside diphosphate kinase A, elongation factor 1-beta and splicing factor, arginine/serine rich 1. The most significant changes in different time courses were found in cytoplasmic actin and cytokeratin, both of which constituted the major components of cytoskeleton network in the cells. The obtained data suggested a possible role of the cytoskeleton during avian influenza virus infection of mammalian cells, which might help for better understanding of the dynamics of avian influenza virus and host interaction in mammalian cell setting.
Proteomics 06/2008; 8(9):1851-8. · 4.43 Impact Factor
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Wai Lan Wu,
Yixin Chen, Pui Wang,
Wenjun Song,
Siu-Ying Lau,
Jane M Rayner,
Gavin J D Smith,
Robert G Webster,
J S Malik Peiris,
Tianwei Lin,
Ningshao Xia,
Yi Guan,
Honglin Chen
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ABSTRACT: Antigenic profiles of post-2002 H5N1 viruses representing major genetic clades and various geographic sources were investigated using a panel of 17 monoclonal antibodies raised from five H5N1 strains. Four antigenic groups from seven clades of H5N1 virus were distinguished and characterized based on their cross-reactivity to the monoclonal antibodies in hemagglutination inhibition and cell-based neutralization assays. Genetic polymorphisms associated with the variation of antigenicity of H5N1 strains were identified and further verified in antigenic analysis with recombinant H5N1 viruses carrying specific mutations in the hemagglutinin protein. Modification of some of these genetic variations produced marked improvement to the immunogenicity and cross-reactivity of H5N1 strains in assays utilizing monoclonal antibodies and ferret antisera raised against clade 1 and 2 H5N1 viruses, suggesting that these sites represent antigenically significant amino acids. These results provide a comprehensive antigenic profile for H5N1 virus strains circulating in recent years and will facilitate the recognition of emerging antigenic variants of H5N1 virus and aid in the selection of vaccine strains.
Journal of Virology 03/2008; 82(4):1798-807. · 5.40 Impact Factor
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Chung-Lam Cheung,
Jane M Rayner,
Gavin J D Smith, Pui Wang,
T S P Naipospos,
Jinxia Zhang,
Kwok-Yung Yuen,
Robert G Webster,
J S Malik Peiris,
Yi Guan,
Honglin Chen
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ABSTRACT: We examined the distribution of genetic mutations associated with resistance to the M2 ion channel-blocking adamantane derivatives, amantadine and rimantadine, among H5N1 viruses isolated in Vietnam, Thailand, Cambodia, Indonesia, Hong Kong, and China. More than 95% of the viruses isolated in Vietnam and Thailand contained resistance mutations, but resistant mutants were less commonly isolated in Indonesia (6.3% of isolates) and China (8.9% of isolates), where human infection was recently reported. The dual mutation motif Leu26Ile-Ser31Asn (leucine-->isoleucine at aa 26 and serine-->asparagine at aa 31) was found almost exclusively in all resistant isolates from Vietnam, Thailand, and Cambodia, suggesting the biological selection of these mutations.
The Journal of Infectious Diseases 07/2006; 193(12):1626-9. · 6.41 Impact Factor
