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Annexin II as a Dengue Virus Serotype 2 Interacting Protein Mediating Virus Interaction on Vero Cells.

ABSTRACT: Recent evidence has demonstrated that dengue virus requires active filopodia formation for a successful infection. However, the cellular factor involved in the interaction has not been fully elucidated. We used a combination of virus overlay protein binding assay and LC-MS/MS, and identified annexin II as a dengue virus serotype 2 (DENV2) interacting protein on Vero cells, upon filopodia induction. Flow cytometry analysis showed annexin II on the Vero cells surface increased when DENV2 was added. The amount of annexin II in the plasma membrane fraction was reduced as the infection progressed. Antibody-mediated inhibition of infection and siRNA-mediated knockdown of annexin II expression significantly reduced DENV2 infection and production levels. Collectively, we demonstrated that annexin II is one of the host factor involved in DENV2 binding on Vero cells.

SUBMITTER: Aliyu IA

PROVIDER: S-EPMC6520844 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Annexin II as a Dengue Virus Serotype 2 Interacting Protein Mediating Virus Interaction on Vero Cells.

Aliyu Isah Abubakar IA Ling King-Hwa KH Md Hashim Nur Fariesha NF Lam Jia-Yong JY Chee Hui-Yee HY

Viruses 20190409 4

Recent evidence has demonstrated that dengue virus requires active filopodia formation for a successful infection. However, the cellular factor involved in the interaction has not been fully elucidated. We used a combination of virus overlay protein binding assay and LC-MS/MS, and identified annexin II as a dengue virus serotype 2 (DENV2) interacting protein on Vero cells, upon filopodia induction. Flow cytometry analysis showed annexin II on the Vero cells surface increased when DENV2 was added ...[more]

PMID: 30970587

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Project description:UnlabelledFlavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimated Kd (dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix α1, comprised of residues 17 to 32, and helix α2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach.ImportanceFlavivirus NS4A and NS4B proteins are essential components of the ER membrane-associated replication complex. The current study systematically characterizes the interaction between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts with NS4B in virus-infected cells, in cells transiently expressing NS4A and NS4B proteins, or in vitro with recombinant NS4A and NS4B proteins. We mapped the minimal regions required for the NS4A-NS4B interaction to be amino acids 40 to 76 of NS4A and amino acids 84 to 146 of NS4B. NMR analysis revealed the secondary structure of amino acids 17 to 80 of NS4A and the NS4A amino acids that may participate in the NS4A-NS4B interaction. Functional analysis showed a correlation between viral replication and NS4A-NS4B interaction, demonstrating the biological importance of the NS4A-NS4B interaction. The study has advanced our knowledge of the molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that inhibitors of the NS4A-NS4B interaction could be pursued for flavivirus antiviral development.

Project description:BackgroundA majority infections caused by dengue virus (DENV) are asymptomatic, but a higher incidence of severe illness, such as dengue hemorrhagic fever, is associated with secondary infections, suggesting that pre-existing immunity plays a central role in dengue pathogenesis. Primary infections are typically associated with a largely serotype-specific antibody response, while secondary infections show a shift to a broadly cross-reactive antibody response.Methods/principal findingsWe hypothesized that the basis for the shift in serotype-specificity between primary and secondary infections can be found in a change in the antibody fine-specificity. To investigate the link between epitope- and serotype-specificity, we assembled the Dengue Virus Antibody Database, an online repository containing over 400 DENV-specific mAbs, each annotated with information on 1) its origin, including the immunogen, host immune history, and selection methods, 2) binding/neutralization data against all four DENV serotypes, and 3) epitope mapping at the domain or residue level to the DENV E protein. We combined epitope mapping and activity information to determine a residue-level index of epitope propensity and cross-reactivity and generated detailed composite epitope maps of primary and secondary antibody responses. We found differing patterns of epitope-specificity between primary and secondary infections, where secondary responses target a distinct subset of epitopes found in the primary response. We found that secondary infections were marked with an enhanced response to cross-reactive epitopes, such as the fusion-loop and E-dimer region, as well as increased cross-reactivity in what are typically more serotype-specific epitope regions, such as the domain I-II interface and domain III.Conclusions/significanceOur results support the theory that pre-existing cross-reactive memory B cells form the basis for the secondary antibody response, resulting in a broadening of the response in terms of cross-reactivity, and a focusing of the response to a subset of epitopes, including some, such as the fusion-loop region, that are implicated in poor neutralization and antibody-dependent enhancement of infection.

Dataset Information

Annexin II as a Dengue Virus Serotype 2 Interacting Protein Mediating Virus Interaction on Vero Cells.

Publications

Annexin II as a Dengue Virus Serotype 2 Interacting Protein Mediating Virus Interaction on Vero Cells.

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