Cellular Vimentin Regulates Construction of Dengue Virus Replication Complexes through Interaction with NS4A Protein

Teo, Su Hui Catherine; Chu, Justin Jang Hann

doi:10.1128/jvi.01249-13

Cited by 126 publications

(107 citation statements)

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“…Such events were proposed to anchor the viral replication complex to the ER membranes (Miller et al, 2007). Its N-terminal portion has also been reported to have multiple functions, such as facilitating NS4A homo-oligomerization (Stern et al, 2013) and interacting with cellular vimentin to regulate viral replication during DENV infection (Teo & Chu, 2014). The hydrophobic regions are proposed to be responsible for inducing membrane rearrangements, similar to those observed in infected cells, in order to anchor the viral replication complex to the ER membranes (Miller et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Genetic interaction between NS4A and NS4B for replication of Japanese encephalitis virus

Deng

et al. 2015

Journal of General Virology

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Flavivirus NS4A and NS4B are important membrane proteins for viral replication that are assumed to serve as the scaffold for the formation of replication complexes. We previously demonstrated that a single Lys-to-Arg mutation at position 79 in NS4A (NS4A-K79R) significantly impaired Japanese encephalitis virus (JEV) replication. In this study, the mutant virus was subject to genetic selection to search for the potential interaction between NS4A and other viral components. Sequencing of the recovered viruses revealed that, in addition to an A97E change in NS4A itself, a Y3N compensatory mutation located in NS4B had emerged from independent selections. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that both adaptive mutations greatly restored the replication defect caused by NS4A-K79R. Our results, for the first time to our knowledge, clearly showed the genetic interaction between NS4A and NS4B, although the mechanism underlying their interaction is unknown.

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Section: Introductionmentioning

confidence: 99%

Genetic interaction between NS4A and NS4B for replication of Japanese encephalitis virus

Deng

et al. 2015

Journal of General Virology

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“…NS4A has also been shown to antagonize the host immune response (7) and to induce autophagy (22). Known interactors of DENV type 2 (DENV-2) NS4A include the host protein vimentin (23) and the viral protein NS4B (24). Miller and colleagues have proposed a membrane topology model of DENV NS4A (Fig.…”

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confidence: 99%

Determinants of Dengue Virus NS4A Protein Oligomerization

Lee

Xie

Zou

et al. 2015

J Virol

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Flavivirus NS4A protein induces host membrane rearrangement and functions as a replication complex component. The molecular details of how flavivirus NS4A exerts these functions remain elusive. Here, we used dengue virus (DENV) as a model to characterize and demonstrate the biological relevance of flavivirus NS4A oligomerization. DENV type 2 (DENV-2) NS4A protein forms oligomers in infected cells or when expressed alone. Deletion mutagenesis mapped amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]) of NS4A as the major determinant for oligomerization, while the N-terminal 50 residues contribute only slightly to the oligomerization. Nuclear magnetic resonance (NMR) analysis of NS4A amino acids 17 to 80 suggests that residues L31, L52, E53, G66, and G67 could participate in oligomerization. Ala substitution for 15 flavivirus conserved NS4A residues revealed that these amino acids are important for viral replication. Among the 15 mutated NS4A residues, 2 amino acids (E50A and G67A) are located within TMD1. Both E50A and G67A attenuated viral replication, decreased NS4A oligomerization, and reduced NS4A protein stability. In contrast, NS4A oligomerization was not affected by the replication-defective mutations (R12A, P49A, and K80A) located outside TMD1. trans complementation experiments showed that expression of wild-type NS4A alone was not sufficient to rescue the replication-lethal NS4A mutants. However, the presence of DENV-2 replicons could partially restore the replication defect of some lethal NS4A mutants (L26A and K80A), but not others (L60A and E122A), suggesting an unidentified mechanism governing the outcome of complementation in a mutant-dependent manner. Collectively, the results have demonstrated the importance of TMD1-mediated NS4A oligomerization in flavivirus replication. Many flaviriruses are significant human pathogens, including the four serotypes of dengue virus (DENV). DENV infection causes an acute and systemic disease that may often be asymptomatic but can lead to clinical symptoms ranging from fever to lifethreatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (1, 2). DENV is a global public health threat, with an estimated 390 million human infections per year, of which 96 million show clinical manifestations (3). Prior infection with a particular DENV serotype does not always protect against the other three serotypes (1) and is often associated with increased disease severity in the second infection (4). There are currently no licensed vaccines or antiviral drugs for either prophylaxis or treatment of DENV infection. In light of this lack of weaponry in our fight against DENV, there is an ongoing urgent need for the development of effective antivirals against the virus. Understanding the molecular details of viral replication will greatly facilitate vaccine and therapeutic development.DENV is a positive-strand RNA virus with a genome approximately 11 kb in length. The viral genome has one open reading frame flanked by 5= and 3= untranslated ...

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“…(ii) Interaction between DENV NS4A and cellular vimentin regulates the formation of virus replication complexes (23). (iii) Flavivirus NS4A protein induces autophagy to prevent cell death and facilitate viral replication (24).…”

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confidence: 99%

Characterization of Dengue Virus NS4A and NS4B Protein Interaction

Zou

Xie

Wang

et al. 2015

J Virol

127

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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Cellular Vimentin Regulates Construction of Dengue Virus Replication Complexes through Interaction with NS4A Protein

Cited by 126 publications

References 50 publications

Genetic interaction between NS4A and NS4B for replication of Japanese encephalitis virus

Genetic interaction between NS4A and NS4B for replication of Japanese encephalitis virus

Determinants of Dengue Virus NS4A Protein Oligomerization

Characterization of Dengue Virus NS4A and NS4B Protein Interaction

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