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N-linked glycosylation of dengue virus NS1 protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement.

ABSTRACT: Dengue virus (DENV) NS1 is a versatile non-structural glycoprotein that is secreted as a hexamer, binds to the cell surface of infected and uninfected cells, and has immune evasive functions. DENV NS1 displays two conserved N-linked glycans at N130 and N207. In this study, we examined the role of these two N-linked glycans on NS1 secretion, stability, and function. Because some groups have reported reduced yields of infectious DENV when N130 and N207 are changed, we analyzed glycosylation-deficient NS1 phenotypes using a transgenic expression system. We show that the N-linked glycan at position 130 is required for stabilization of the secreted hexamer whereas the N-linked glycan at residue 207 facilitates secretion and extracellular protein stability. Moreover, NS1 mutants lacking an N-linked glycan at N130 did not interact efficiently with complement components C1s and C4. In summary, our results elucidate the contribution of N-linked glycosylation to the function of DENV NS1.

SUBMITTER: Somnuke P

PROVIDER: S-EPMC3089955 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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N-linked glycosylation of dengue virus NS1 protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement.

Somnuke Pawit P Hauhart Richard E RE Atkinson John P JP Diamond Michael S MS Avirutnan Panisadee P

Virology 20110322 2

Dengue virus (DENV) NS1 is a versatile non-structural glycoprotein that is secreted as a hexamer, binds to the cell surface of infected and uninfected cells, and has immune evasive functions. DENV NS1 displays two conserved N-linked glycans at N130 and N207. In this study, we examined the role of these two N-linked glycans on NS1 secretion, stability, and function. Because some groups have reported reduced yields of infectious DENV when N130 and N207 are changed, we analyzed glycosylation-defici ...[more]

PMID: 21429549

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Project description:Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV.Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the intracellular and the extracellular milieus. Despite the fact that NS1 has been commonly associated with DENV pathogenesis, it plays a pivotal but unknown role in the replication process. In an effort to understand the role of intracellular NS1, we demonstrate that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with NS1. Our results indicate that NS1 increases the glycolytic activity of GAPDH in vitro. Interestingly, the GAPDH activity was increased during DENV infection, and NS1 expression alone was sufficient to enhance intracellular GAPDH activity in BHK-21 cells. Overall, our findings suggest that NS1 is an important modulator of cellular energy metabolism by increasing glycolytic flux.

Project description:BACKGROUND: Early diagnosis of dengue infection is crucial for better management of the disease. Diagnostic tests based on the detection of dengue virus (DENV) Non Structural Protein 1 (NS1) antigen are commercially available with different sensitivities and specificities observed in various settings. Dengue is endemic in Indonesia and clinicians are increasingly using the NS1 detection for dengue confirmation. This study described the performance of Panbio Dengue Early NS1 and IgM Capture ELISA assays for dengue detection during our surveillance in eight cities in Indonesia as well as the genetic diversity of DENV NS1 genes and its relationship with the NS1 detection. METHODS: The NS1 and IgM/IgG ELISA assays were used for screening and confirmation of dengue infection during surveillance in 2010-2012. Collected serum samples (n?=?440) were subjected to RT-PCR and virus isolation, in which 188 samples were confirmed for dengue infection. The positivity of the ELISA assays were correlated with the RT-PCR results to obtain the sensitivity of the assays. The NS1 genes of 48 Indonesian virus isolates were sequenced and their genetic characteristics were studied. RESULTS: Using molecular data as gold standard, the sensitivity of NS1 ELISA assay for samples from Indonesia was 56.4% while IgM ELISA was 73.7%. When both NS1 and IgM results were combined, the sensitivity increased to 89.4%. The NS1 sensitivity varied when correlated with city/geographical origins and DENV serotype, in which the lowest sensitivity was observed for DENV-4 (19.0%). NS1 sensitivity was higher in primary (67.6%) compared to secondary infection (48.2%). The specificity of NS1 assay for non-dengue samples were 100%. The NS1 gene sequence analysis of 48 isolates revealed the presence of polymorphisms of the NS1 genes which apparently did not influence the NS1 sensitivity. CONCLUSIONS: We observed a relatively low sensitivity of NS1 ELISA for dengue detection on RT-PCR-positive dengue samples. The detection rate increased significantly when NS1 data was combined with IgM. In our study, the low sensitivity of NS1 antigen detection did not relate to NS1 genetic diversity. Rather, the performance of the NS1 antigen test was affected by the infection status of patients and geographical origin of samples.

Dataset Information

N-linked glycosylation of dengue virus NS1 protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement.

Publications

N-linked glycosylation of dengue virus NS1 protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement.

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