Displaying publications 1 - 20 of 33 in total

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  1. Fulltext Differential proteome analysis of chikungunya virus infection on host cells
    Thio CL, Yusof R, Abdul-Rahman PS, Karsani SA
    PLoS One, 2013;8(4):e61444.
    PMID: 23593481 DOI: 10.1371/journal.pone.0061444
    Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach.
    Matched MeSH terms: Viral Proteins/metabolism*
  2. Identification of phosphoprotein:phosphoprotein and phosphoprotein:nucleocapsid protein interaction domains of the Newcastle disease virus
    Jahanshiri F, Eshaghi M, Yusoff K
    Arch Virol, 2005 Mar;150(3):611-8.
    PMID: 15592890
    The yeast two-hybrid system has been used to identify domains of the Newcastle disease virus (NDV) phosphoprotein (P) involved in self-association and interaction with the nucleocapsid protein (NP). Deletion analysis was used to map the domain(s) of the P protein involved in P:P and P:NP interactions. The C-terminal 45 amino acids (residues 247-291) were shown to play a major role in both of the interactions. Comparison of these findings with other reports suggests that paramyxoviruses are different with respect to interaction domain(s) between these two essential viral proteins involved in genome replication.
    Matched MeSH terms: Viral Proteins/metabolism*
  3. Fulltext Directed evolution of nucleotide-based libraries using lambda exonuclease
    Lim BN, Choong YS, Ismail A, Glökler J, Konthur Z, Lim TS
    Biotechniques, 2012 Dec;53(6):357-64.
    PMID: 23227986 DOI: 10.2144/000113964
    Directed evolution of nucleotide libraries using recombination or mutagenesis is an important technique for customizing catalytic or biophysical traits of proteins. Conventional directed evolution methods, however, suffer from cumbersome digestion and ligation steps. Here, we describe a simple method to increase nucleotide diversity using single-stranded DNA (ssDNA) as a starting template. An initial PCR amplification using phosphorylated primers with overlapping regions followed by treatment with lambda exonuclease generates ssDNA templates that can then be annealed via the overlap regions. Double-stranded DNA (dsDNA) is then generated through extension with Klenow fragment. To demonstrate the applicability of this methodology for directed evolution of nucleotide libraries, we generated both gene shuffled and regional mutagenesis synthetic antibody libraries with titers of 2×108 and 6×107, respectively. We conclude that our method is an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries.
    Matched MeSH terms: Viral Proteins/metabolism*
  4. Fulltext Inhibition of dengue NS2B-NS3 protease and viral replication in Vero cells by recombinant retrocyclin-1
    Rothan HA, Han HC, Ramasamy TS, Othman S, Rahman NA, Yusof R
    BMC Infect Dis, 2012;12:314.
    PMID: 23171075 DOI: 10.1186/1471-2334-12-314
    Global resurgence of dengue virus infections in many of the tropical and subtropical countries is a major concern. Therefore, there is an urgent need for the development of successful drugs that are both economical and offer a long-lasting protection. The viral NS2B-NS3 serine protease (NS2B-NS3pro) is a promising target for the development of drug-like inhibitors, which are not available at the moment. In this study, we report retrocyclin-1 (RC-1) production in E. coli as a recombinant peptide to test against dengue NS2B-NS3pro.
    Matched MeSH terms: Viral Proteins/metabolism*
  5. Fulltext Proteolytic cleavage analysis at the Murray Valley encephalitis virus NS1-2A junction
    Addis SN, Lee E, Bettadapura J, Lobigs M
    Virol J, 2015;12:144.
    PMID: 26377679 DOI: 10.1186/s12985-015-0375-4
    Our understanding of the proteolytic processing events at the NS1-2A junction in the flavivirus polyprotein has not markedly progressed since the early work conducted on dengue virus (DENV). This work identified an octapeptide sequence located immediately upstream of the cleavage site thought to be important in substrate recognition by an as yet unknown, endoplasmic reticulum-resident host protease. Of the eight amino acid recognition sequence, the highly conserved residues at positions P1, P3, P5, P7 and P8 (with respect to N-terminus of NS2A) are particularly sensitive to amino acid substitutions in terms of DENV NS1-NS2A cleavage efficiency; however, the role of the octapeptide in efficient NS1 and NS2A production of other flaviviruses has not been experimentally addressed.
    Matched MeSH terms: Viral Proteins/metabolism*
  6. Fulltext Diversity and Evolutionary Histories of Human Coronaviruses NL63 and 229E Associated with Acute Upper Respiratory Tract Symptoms in Kuala Lumpur, Malaysia
    Al-Khannaq MN, Ng KT, Oong XY, Pang YK, Takebe Y, Chook JB, et al.
    Am J Trop Med Hyg, 2016 05 04;94(5):1058-64.
    PMID: 26928836 DOI: 10.4269/ajtmh.15-0810
    The human alphacoronaviruses HCoV-NL63 and HCoV-229E are commonly associated with upper respiratory tract infections (URTI). Information on their molecular epidemiology and evolutionary dynamics in the tropical region of southeast Asia however is limited. Here, we analyzed the phylogenetic, temporal distribution, population history, and clinical manifestations among patients infected with HCoV-NL63 and HCoV-229E. Nasopharyngeal swabs were collected from 2,060 consenting adults presented with acute URTI symptoms in Kuala Lumpur, Malaysia, between 2012 and 2013. The presence of HCoV-NL63 and HCoV-229E was detected using multiplex polymerase chain reaction (PCR). The spike glycoprotein, nucleocapsid, and 1a genes were sequenced for phylogenetic reconstruction and Bayesian coalescent inference. A total of 68/2,060 (3.3%) subjects were positive for human alphacoronavirus; HCoV-NL63 and HCoV-229E were detected in 45 (2.2%) and 23 (1.1%) patients, respectively. A peak in the number of HCoV-NL63 infections was recorded between June and October 2012. Phylogenetic inference revealed that 62.8% of HCoV-NL63 infections belonged to genotype B, 37.2% was genotype C, while all HCoV-229E sequences were clustered within group 4. Molecular dating analysis indicated that the origin of HCoV-NL63 was dated to 1921, before it diverged into genotype A (1975), genotype B (1996), and genotype C (2003). The root of the HCoV-229E tree was dated to 1955, before it diverged into groups 1-4 between the 1970s and 1990s. The study described the seasonality, molecular diversity, and evolutionary dynamics of human alphacoronavirus infections in a tropical region.
    Matched MeSH terms: Viral Proteins/metabolism
  7. Regions on nucleocapsid protein of Newcastle disease virus that interact with its phosphoprotein
    Kho CL, Tan WS, Tey BT, Yusoff K
    Arch Virol, 2004 May;149(5):997-1005.
    PMID: 15098113 DOI: 10.1007/s00705-003-0273-8
    The nucleocapsid (NP) and phospho-(P) proteins of paramyxoviruses are involved in transcription and replication of the viral genome. An in vitro protein binding assay was used to investigate the regions on NP protein that interact with the P protein of Newcastle disease virus (NDV). Truncated NP mutants were first immobilised on a solid phase and then interacted with radio-labelled [(35)S]-P protein synthesised in rabbit reticulocyte. The interaction affinity was quantitated by measuring the radioactivity that was retained on the solid phase. Using this approach, a highly interactive region was identified to be resided at the first 25 amino acids of NP N-terminus. The interaction between these two proteins remained strong even with the removal of 114 amino acids from the C-terminal end of NP. However, it is possible that the 49 amino acids at the C-terminal end might have another contact region for P protein, which is not as critical as the N-terminal end. The interaction regions mapped in this study are significantly different from the other two paramyxoviruses: Sendai and measles viruses in which the C-termini of their NP proteins play an important role in binding to the P.
    Matched MeSH terms: Viral Proteins/metabolism*
  8. Fulltext In silico evidence of de novo interactions between ribosomal and Epstein - Barr virus proteins
    Sim EU, Talwar SP
    BMC Mol Cell Biol, 2019 08 15;20(1):34.
    PMID: 31416416 DOI: 10.1186/s12860-019-0219-y
    BACKGROUND: Association of Epstein-Barr virus (EBV) encoded latent gene products with host ribosomal proteins (RPs) has not been fully explored, despite their involvement in the aetiology of several human cancers. To gain an insight into their plausible interactions, we employed a computational approach that encompasses structural alignment, gene ontology analysis, pathway analysis, and molecular docking.

    RESULTS: In this study, the alignment analysis based on structural similarity allows the prediction of 48 potential interactions between 27 human RPs and the EBV proteins EBNA1, LMP1, LMP2A, and LMP2B. Gene ontology analysis of the putative protein-protein interactions (PPIs) reveals their probable involvement in RNA binding, ribosome biogenesis, metabolic and biosynthetic processes, and gene regulation. Pathway analysis shows their possible participation in viral infection strategies (viral translation), as well as oncogenesis (Wnt and EGFR signalling pathways). Finally, our molecular docking assay predicts the functional interactions of EBNA1 with four RPs individually: EBNA1-eS10, EBNA1-eS25, EBNA1-uL10 and EBNA1-uL11.

    CONCLUSION: These interactions have never been revealed previously via either experimental or in silico approach. We envisage that the calculated interactions between the ribosomal and EBV proteins herein would provide a hypothetical model for future experimental studies on the functional relationship between ribosomal proteins and EBV infection.

    Matched MeSH terms: Viral Proteins/metabolism*
  9. Fulltext EGCG as an anti-SARS-CoV-2 agent: Preventive versus therapeutic potential against original and mutant virus
    Tsvetkov V, Varizhuk A, Kozlovskaya L, Shtro A, Lebedeva O, Komissarov A, et al.
    Biochimie, 2021 Dec;191:27-32.
    PMID: 34389380 DOI: 10.1016/j.biochi.2021.08.003
    In the search for anti-SARS-CoV-2 drugs, much attention is given to safe and widely available native compounds. The green tea component epigallocatechin 3 gallate (EGCG) is particularly promising because it reportedly inhibits viral replication and viral entry in vitro. However, conclusive evidence for its predominant activity is needed. We tested EGCG effects on the native virus isolated from COVID-19 patients in two independent series of experiments using VERO cells and two different treatment schemes in each series. The results confirmed modest cytotoxicity of EGCG and its substantial antiviral activity. The preincubation scheme aimed at infection prevention has proven particularly beneficial. We complemented that finding with a detailed investigation of EGCG interactions with viral S-protein subunits, including S2, RBD, and the RBD mutant harboring the N501Y mutation. Molecular modeling experiments revealed N501Y-specific stacking interactions in the RBD-ACE2 complex and provided insight into EGCG interference with the complex formation. Together, these findings provide a molecular basis for the observed EGCG effects and reinforce its prospects in COVID-19 prevention therapy.
    Matched MeSH terms: Viral Proteins/metabolism
  10. Fulltext Protein-protein interactions between A. aegypti midgut and dengue virus 2: two-hybrid screens using the midgut cDNA library
    Tham HW, Balasubramaniam VR, Chew MF, Ahmad H, Hassan SS
    J Infect Dev Ctries, 2015 Dec 30;9(12):1338-49.
    PMID: 26719940 DOI: 10.3855/jidc.6422
    INTRODUCTION: Dengue virus (DENV) is principally transmitted by the Aedes aegypti mosquito. To date, mosquito population control remains the key strategy for reducing the continuing spread of DENV. The focus on the development of new vector control strategies through an understanding of the mosquito-virus relationship is essential, especially targeting the midgut, which is the first mosquito organ exposed to DENV infection.
    METHODOLOGY: A cDNA library derived from female adult A. aegypti mosquito midgut cells was established using the switching mechanism at the 5' end of the RNA transcript (SMART), in combination with a highly potent recombination machinery of Saccharomyces cerevisiae. Gal4-based yeast two-hybrid (Y2H) assays were performed against DENV-2 proteins (E, prM, M, and NS1). Mammalian two-hybrid (M2H) and double immunofluorescence assays (IFA) were conducted to validate the authenticity of the three selected interactions.
    RESULTS: The cDNA library was of good quality based on its transformation efficiency, cell density, titer, and the percentage of insert size. A total of 36 midgut proteins interacting with DENV-2 proteins were identified, some involved in nucleic acid transcription, oxidoreductase activity, peptidase activity, and ion binding. Positive outcomes were obtained from the three selected interactions validated using M2H and double IFA assays.
    CONCLUSIONS: The identified proteins have different biological activities that may aid in the virus replication pathway. Therefore, the midgut cDNA library is a valuable tool for identifying DENV-2 interacting proteins. The positive outcomes of the three selected proteins validated supported the quality of the cDNA library and the robustness of the Y2H mechanisms.
    Matched MeSH terms: Viral Proteins/metabolism*
  11. Nodamura virus B2 amino terminal domain sensitivity to small interfering RNA
    Ali PS, John J, Selvaraj M, Kek TL, Salleh MZ
    Microbiol. Immunol., 2015 May;59(5):299-304.
    PMID: 25753649 DOI: 10.1111/1348-0421.12253
    Nodamura virus (NoV) B2, a suppressor of RNA interference, binds double stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) corresponding to Dicer substrates and products. Here, we report that the amino terminal domain of NoV B2 (NoV B2 79) specifically binds siRNAs but not dsRNAs. NoV B2 79 oligomerizes on binding to 27 nucleotide siRNA. Mutation of the residues phenylalanine49 and alanine60 to cysteine and methionine, respectively enhances the RNA binding affinity of NoV B2 79. Circular dichroism spectra demonstrated that the wild type and mutant NoV B2 79 have similar secondary structure conformations.
    Matched MeSH terms: Viral Proteins/metabolism*
  12. Induction of protein expression within Escherichia coli vector for entry into mammalian cells
    Chen Q, Lee CW, Sim EU, Narayanan K
    Hum Gene Ther Methods, 2014 Feb;25(1):40-7.
    PMID: 24134118 DOI: 10.1089/hgtb.2012.188
    Direct protein delivery into the cytosol of mammalian cells by invasive Escherichia coli (E. coli) bacterial vector will bypass the need to achieve nuclear entry and transcription of DNA, a major hurdle that is known to seriously limit gene transfer. The bacterial vector is induced to express the protein during its growth phase, before presentation for entry into mammalian cells and release of its content into the cellular environment. For this class of vector, crossing the plasma membrane becomes the primary step that determines the success of protein delivery. Yet, how the mechanics of protein expression within the vector affect its entry into the host is poorly understood. We found the vector's effectiveness to enter HeLa cells diminished together with its viability when phage N15 protelomerase (TelN) expression was induced continuously in the invasive E. coli despite producing an abundant amount of functional protein. By comparison, shorter induction, even as little as 3 hr, produced sufficient amounts of functional TelN and showed more effective invasion of HeLa cells, comparable to that of uninduced invasive E. coli. These results demonstrate that brief induction of protein expression during vector growth is essential for optimal entry into mammalian cells, an important step for achieving bacteria-mediated protein delivery.
    Matched MeSH terms: Viral Proteins/metabolism
  13. Comparative evaluation of three purification methods for the nucleocapsid protein of Newcastle disease virus from Escherichia coli homogenates
    Tan YP, Ling TC, Yusoff K, Tan WS, Tey BT
    J Microbiol, 2005 Jun;43(3):295-300.
    PMID: 15995649
    In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.
    Matched MeSH terms: Viral Proteins/metabolism
  14. Fulltext Observation of risk factors, clinical manifestations and genetic characterization of recent Newcastle Disease Virus outbreak in West Malaysia
    Jaganathan S, Ooi PT, Phang LY, Allaudin ZN, Yip LS, Choo PY, et al.
    BMC Vet Res, 2015;11:219.
    PMID: 26293577 DOI: 10.1186/s12917-015-0537-z
    Newcastle disease virus remains a constant threat in commercial poultry farms despite intensive vaccination programs. Outbreaks attributed to ND can escalate and spread across farms and states contributing to major economic loss in poultry farms.
    Matched MeSH terms: Viral Proteins/metabolism
  15. A Self-Replicating Linear DNA
    Liew PS, Tan TH, Wong YC, Sim EUH, Lee CW, Narayanan K
    ACS Synth Biol, 2020 04 17;9(4):804-813.
    PMID: 32196315 DOI: 10.1021/acssynbio.9b00478
    TelN and tos are a unique DNA linearization unit isolated from bacteriophage N15. While being transferable, the TelN cleaving-rejoining activities remained stable to function on tos in both bacterial and mammalian environments. However, TelN contribution in linear plasmid replication in mammalian cells remains unknown. Herein, we investigated the association of TelN in linear tos-containing DNA (tos-DNA) replication in mammalian cells. Additionally, the mammalian origin of replication (ori) that is well-known to initiate the replication event of plasmid vectors was also studied. In doing so, we identified that both TelN and mammalian initiation sites were essential for the replication of linear tos-DNA, determined by using methylation sensitive DpnI/MboI digestion and polymerase chain reaction (PCR) amplification approaches. Furthermore, we engineered the linear tos-DNA to be able to retain in mammalian cells using S/MAR technology. The resulting S/MAR containing tos-DNA was robust for at least 15 days, with (1) continuous tos-DNA replication, (2) correct splicing of gene transcripts, and (3) stable exogenous gene expression that was statistically comparable to the endogenous gene expression level. Understanding the activities of TelN and tos in mammalian cells can potentially provide insights for adapting this simple DNA linearization unit in developing novel genetic engineering tools, especially to the eukaryotic telomere/telomerase study.
    Matched MeSH terms: Viral Proteins/metabolism
  16. P-TEFb goes viral
    Zaborowska J, Isa NF, Murphy S
    Bioessays, 2016 07;38 Suppl 1:S75-85.
    PMID: 27417125 DOI: 10.1002/bies.201670912
    Positive transcription elongation factor b (P-TEFb), which comprises cyclin-dependent kinase 9 (CDK9) kinase and cyclin T subunits, is an essential kinase complex in human cells. Phosphorylation of the negative elongation factors by P-TEFb is required for productive elongation of transcription of protein-coding genes by RNA polymerase II (pol II). In addition, P-TEFb-mediated phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of pol II mediates the recruitment of transcription and RNA processing factors during the transcription cycle. CDK9 also phosphorylates p53, a tumor suppressor that plays a central role in cellular responses to a range of stress factors. Many viral factors affect transcription by recruiting or modulating the activity of CDK9. In this review, we will focus on how the function of CDK9 is regulated by viral gene products. The central role of CDK9 in viral life cycles suggests that drugs targeting the interaction between viral products and P-TEFb could be effective anti-viral agents.
    Matched MeSH terms: Viral Proteins/metabolism*
  17. Fulltext Molecular characterization of Malaysian fowl adenovirus (FAdV) serotype 8b species E and pathogenicity of the virus in specific-pathogen-free chicken
    Sabarudin NS, Tan SW, Phang YF, Omar AR
    J Vet Sci, 2021 Jul;22(4):e42.
    PMID: 34313038 DOI: 10.4142/jvs.2021.22.e42
    BACKGROUND: Inclusion body hepatitis (IBH) is an economically important viral disease primarily affecting broiler and breeder chickens. All 12 serotypes of fowl adenovirus (FAdV) can cause IBH.

    OBJECTIVES: To characterize FAdV isolates based on phylogenetic analysis, and to study the pathogenicity of FAdV-8b in specific-pathogen-free (SPF) chickens following virus inoculation via oral and intramuscular (IM) routes.

    METHODS: Suspected organ samples were subjected to virus isolation and polymerase chain reaction (PCR) for FAdV detection. Hexon gene sequencing and phylogenetic analysis were performed on FAdV-positive samples for serotype identification. One FAdV-8b isolate, UPM/FAdV/420/2017, was selected for fiber gene characterization and pathogenicity study and was inoculated in SPF chickens via oral and IM routes.

    RESULTS: The hexon gene phylogenetic analysis revealed that all isolates belonged to FAdV-8b. The fiber gene-based phylogenetic analysis of isolate UPM/FAdV/420/2017 supported the grouping of that isolate into FAdV species E. Pathogenicity study revealed that, chickens infected with UPM/FAdV/420/2017 via the IM route had higher clinical score values, higher percent mortality, higher degree of the liver lesions, higher antibody response (p < 0.05), and higher virus shedding amounts (p < 0.05) than those infected via the oral route. The highest virus copy numbers were detected in liver and gizzard.

    CONCLUSIONS: FAdV-8b is the dominant FAdV serotype in Malaysia, and pathogenicity study of the FAdV-8b isolate UPM/FAdV/420/2017 indicated its ability to induce IBH in young SPF chickens when infected via oral or IM routes.

    Matched MeSH terms: Viral Proteins/metabolism
  18. VP1 residues around the five-fold axis of enterovirus A71 mediate heparan sulfate interaction
    Tan CW, Sam IC, Lee VS, Wong HV, Chan YF
    Virology, 2017 01 15;501:79-87.
    PMID: 27875780 DOI: 10.1016/j.virol.2016.11.009
    Enterovirus A71 (EV-A71) is a neurotropic enterovirus that uses heparan sulfate as an attachment receptor. The molecular determinants of EV-A71-heparan sulfate interaction are unknown. With In silico heparin docking and mutagenesis of all possible lysine residues in VP1, we identified that K162, K242 and K244 are responsible for heparin interaction and inhibition. EV-A71 mutants with K242A and K244A rapidly acquired compensatory mutations, T100K or E98A, and Q145R-T237N respectively, which restored the heparin-binding phenotype. Both VP1-98 and VP1-145 modulates heparin binding. Heparin-binding phenotype was completely abolished with VP1-E98-E145, but was restored by an E98K or E145Q substitution. During cell culture adaptation, EV-A71 rapidly acquired K98 or Q/G145 to restore the heparin-binding phenotype. Together with next-generation sequencing analysis, our results implied that EV-A71 has high genetic plasticity by modulating positively-charged residues at the five-fold axis during in vitro heparin adaptation. Our finding has impact on EV-A71 vaccine production, evolutionary studies and pathogenesis.
    Matched MeSH terms: Viral Proteins/metabolism*
  19. Fulltext Binding of Duck Tembusu Virus Nonstructural Protein 2A to Duck STING Disrupts Induction of Its Signal Transduction Cascade To Inhibit Beta Interferon Induction
    Zhang W, Jiang B, Zeng M, Duan Y, Wu Z, Wu Y, et al.
    J Virol, 2020 04 16;94(9).
    PMID: 32075929 DOI: 10.1128/JVI.01850-19
    Duck Tembusu virus (DTMUV), which is similar to other mosquito-borne flaviviruses that replicate well in most mammalian cells, is an emerging pathogenic flavivirus that has caused epidemics in egg-laying and breeding waterfowl. Immune organ defects and neurological dysfunction are the main clinical symptoms of DTMUV infection. Preinfection with DTMUV makes the virus impervious to later interferon (IFN) treatment, revealing that DTMUV has evolved some strategies to defend against host IFN-dependent antiviral responses. Immune inhibition was further confirmed by screening for DTMUV-encoded proteins, which suggested that NS2A significantly inhibited IFN-β and IFN-stimulated response element (ISRE) promoter activity in a dose-dependent manner and facilitated reinfection with duck plague virus (DPV). DTMUV NS2A was able to inhibit duck retinoic acid-inducible gene-I (RIG-I)-, and melanoma differentiation-associated gene 5 (MDA5)-, mitochondrial-localized adaptor molecules (MAVS)-, stimulator of interferon genes (STING)-, and TANK-binding kinase 1 (TBK1)-induced IFN-β transcription, but not duck TBK1- and interferon regulatory factor 7 (IRF7)-mediated effective phases of IFN response. Furthermore, we found that NS2A competed with duTBK1 in binding to duck STING (duSTING), impaired duSTING-duSTING binding, and reduced duTBK1 phosphorylation, leading to the subsequent inhibition of IFN production. Importantly, we first identified that the W164A, Y167A, and S361A mutations in duSTING significantly impaired the NS2A-duSTING interaction, which is important for NS2A-induced IFN-β inhibition. Hence, our data demonstrated that DTMUV NS2A disrupts duSTING-dependent antiviral cellular defenses by binding with duSTING, which provides a novel mechanism by which DTMUV subverts host innate immune responses. The potential interaction sites between NS2A and duSTING may be the targets of future novel antiviral therapies and vaccine development.IMPORTANCE Flavivirus infections are transmitted through mosquitos or ticks and lead to significant morbidity and mortality worldwide with a spectrum of manifestations. Infection with an emerging flavivirus, DTMUV, manifests with clinical symptoms that include lesions of the immune organs and neurological dysfunction, leading to heavy egg drop and causing serious harm to the duck industry in China, Thailand, Malaysia, and other Southeast Asian countries. Mosquito cells, bird cells, and mammalian cell lines are all susceptible to DTMUV infection. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and may pose a threat to mammalian health. However, the pathogenesis of DTMUV is largely unclear. Our results show that NS2A strongly blocks the STING-induced signal transduction cascade by binding with STING, which subsequently blocks STING-STING binding and TBK1 phosphorylation. More importantly, the W164, Y167, or S361 residues in duSTING were identified as important interaction sites between STING and NS2A that are vital for NS2A-induced IFN production and effective phases of IFN response. Uncovering the mechanism by which DTMUV NS2A inhibits IFN in the cells of its natural hosts, ducks, will help us understand the role of NS2A in DTMUV pathogenicity.
    Matched MeSH terms: Viral Proteins/metabolism*
  20. Influenza A virus neuraminidase protein interacts with Hsp90, to stabilize itself and enhance cell survival
    Kumar P, Gaur P, Kumari R, Lal SK
    J Cell Biochem, 2019 04;120(4):6449-6458.
    PMID: 30335904 DOI: 10.1002/jcb.27935
    Neuraminidase protein (NA) of influenza A virus (IAV) is popularly known for its sialidase function to assist in the release of progeny virus. However, involvement of NA in other stages of the IAV life cycle also indicates its multifunctional nature and necessity to interact with other host proteins. Here, we report a host protein-heat shock protein 90 (Hsp90), as a novel interacting partner of IAV NA. A classical yeast two-hybrid screen was conducted to identify a new host interacting partner for NA and the interaction was further validated by coimmunoprecipitation from cells, transiently expressing both proteins and also from IAV-infected cells. Confocal imaging showed that both proteins colocalized in the cytoplasm in transfected host cells. Interestingly, increased levels of NA in the presence of Hsp90 was observed, which tends to decrease if adenosine triphosphatase activity of Hsp90 is inhibited using 17-N-allylamino-17-demethoxygeldanamycin (17AAG). This establishes viral NA as a client protein of host chaperone Hsp90 contributing toward NA's stability via the NA-Hsp90 interaction. This is the first report showing the interaction of NA with Hsp90 and its role in stabilizing viral NA thus preventing it from degradation. Enhanced cell survival in the presence of this interaction was also observed, thus suggesting the requirement of stable viral NA, post-IAV infection, for efficient virus production in infected mammalian cells.
    Matched MeSH terms: Viral Proteins/metabolism*
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