Dimerization of Flavivirus NS4B Protein

Zou, Jing; Xie, Xuping; Lee, Le Tian; Chandrasekaran, Ramya; Reynaud, A.; Yap, Li Jian; Wang, Qing Yin; Dong, Hansong; Kang, CongBao; Yuan, Zhiming; Lescar, Julien; Shi, Pei–Yong

doi:10.1128/jvi.02782-13

Cited by 87 publications

(93 citation statements)

References 37 publications

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“…For NS4B protein, two bands, representing a monomer (28 kDa) and dimer (56 kDa), were observed. The result is consistent with our previous result that NS4B could dimerize (36). Western blotting showed that antibodies against DENV-2 NS4A or NS4B could specifically detect various oligomeric forms of recombinant proteins (Fig.…”

Section: Resultssupporting

confidence: 82%

“…In addition to the DENV-2 NS4A-NS4B interaction, recent studies showed that DENV-2 NS4A or NS4B alone could oligomerize (20,36). The latter findings were confirmed by the current result that recombinant NS4A of DENV-2 formed high-order oligomers and that recombinant NS4B formed dimers on SDS-PAGE gels (Fig.…”

Section: Discussionsupporting

confidence: 80%

“…Therefore, only the monomer of DENV-2 NS4A could bind to NS4B. For NS4B, the cytosolic loop (residues 129 to 165) and the C-terminal region (residues 166 to 248) are responsible for DENV-2 NS4B dimerization (36). NS4B cytosolic loop residues 129 to 146 are required for both DENV-2 NS4B dimerization and NS4A-NS4B interaction.…”

Section: Discussionmentioning

confidence: 99%

“…NS4B cytosolic loop residues 129 to 146 are required for both DENV-2 NS4B dimerization and NS4A-NS4B interaction. Since C-terminal residues 166 to 248 alone of NS4B are sufficient for dimerization (36), NS4B could dimerize through residues 166 to 248 of its C terminus and simultaneously interact with NS4A through its residues 84 to 146. Thus, both the monomeric and dimeric forms of NS4B of DENV-2 could bind to NS4A.…”

Section: Discussionmentioning

confidence: 99%

“…DENV-2 NGC NS4B protein was expressed and purified by following a previously described protocol (36). A similar protocol was used to express and purify DENV-2 NS4A protein with some modifications.…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

Characterization of Dengue Virus NS4A and NS4B Protein Interaction

Zou

Xie

Wang

et al. 2015

J Virol

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125

107

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Flavivirus 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 K d (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 ␣1, comprised of residues 17 to 32, and helix ␣2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix ␣3 (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.T he four serotypes of dengue virus (DENV-1 to DENV-4) are the causati...

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Section: Resultssupporting

confidence: 82%

Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Characterization of Dengue Virus NS4A and NS4B Protein Interaction

Zou

Xie

Wang

et al. 2015

J Virol

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125

107

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Secondary Structure and Membrane Topology of the Full‐Length Dengue Virus NS4B in Micelles

Wong

Lee

et al. 2016

Angewandte Chemie

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Dengue virus nonstructural protein 4B (NS4B) is a membrane protein consisting of 248 residues with a crucial role in virus replication and interference with the host innate immunity. The dengue virus serotype 3 NS4B was reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Backbone resonance assignment of NS4B was obtained using conventional solution NMR experiments. Further studies suggested that NS4B contained eleven helices and six of them form five potential transmembrane regions. This study provides atomic level information for an important drug target to control flavivirus infections.

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