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Membrane topology and function of dengue virus NS2A protein.

ABSTRACT: Flavivirus nonstructural protein 2A (NS2A) is a component of the viral replication complex that functions in virion assembly and antagonizes the host immune response. Although flavivirus NS2A is known to associate with the endoplasmic reticulum (ER) membrane, the detailed topology of this protein has not been determined. Here we report the first topology model of flavivirus NS2A on the ER membrane. Using dengue virus (DENV) NS2A as a model, we show that (i) the N-terminal 68 amino acids are located in the ER lumen, with one segment (amino acids 30 to 52) that interacts with ER membrane without traversing the lipid bilayer; (ii) amino acids 69 to 209 form five transmembrane segments, each of which integrally spans the ER membrane; and (iii) the C-terminal tail (amino acids 210 to 218) is located in the cytosol. Nuclear magnetic resonance (NMR) structural analysis showed that the first membrane-spanning segment (amino acids 69 to 93) consists of two helices separated by a "helix breaker." The helix breaker is formed by amino acid P85 and one positively charged residue, R84. Functional analysis using replicon and genome-length RNAs of DENV-2 indicates that P85 is not important for viral replication. However, when R84 was replaced with E, the mutation attenuated both viral RNA synthesis and virus production. Remarkably, an R84A mutation did not affect viral RNA synthesis but blocked intracellular formation of infectious virions. Collectively, the mutagenesis results demonstrate that NS2A functions in both DENV RNA synthesis and virion assembly/maturation. The topology model of DENV NS2A provides a good starting point for studying how flavivirus NS2A modulates viral replication and evasion of host immune response.

SUBMITTER: Xie X

PROVIDER: S-EPMC3624351 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Membrane topology and function of dengue virus NS2A protein.

Xie Xuping X Gayen Shovanlal S Kang CongBao C Yuan Zhiming Z Shi Pei-Yong PY

Journal of virology 20130213 8

Flavivirus nonstructural protein 2A (NS2A) is a component of the viral replication complex that functions in virion assembly and antagonizes the host immune response. Although flavivirus NS2A is known to associate with the endoplasmic reticulum (ER) membrane, the detailed topology of this protein has not been determined. Here we report the first topology model of flavivirus NS2A on the ER membrane. Using dengue virus (DENV) NS2A as a model, we show that (i) the N-terminal 68 amino acids are loca ...[more]

PMID: 23408612

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Project description:The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMS1 to -8) and participates in RNA replication, virion assembly, and host antiviral response. However, the roles of specific amino acid residues within the pTMS regions of NS2A during the viral life cycle are not clear. Here, we explore the function of DENV NS2A by introducing a series of alanine substitutions into the N-terminal half (pTMS1 to -4) of the protein in the context of a DENV infectious clone or subgenomic replicon. Six NS2A mutants (NM5, -7, -9, and -17 to -19) around pTMS1 and -2 displayed a novel phenotype showing a >1,000-fold reduction in virus yield, an absence of plaque formation despite wild-type-like replicon activity, and infectious-virus-like particle yields. HEK-293 cells infected with the six NS2A mutant viruses failed to cause a virus-induced cytopathic effect (CPE) by MitoCapture staining, cell proliferation, and lactate dehydrogenase release assays. Sequencing analyses of pseudorevertant viruses derived from lethal-mutant viruses revealed two consensus reversion mutations, leucine to phenylalanine at codon 181 (L181F) within pTMS7 of NS2A and isoleucine to threonine at codon 114 (I114T) within NS2B. The introduction of an NS2A-L181F mutation into the lethal (NM15, -16, -25, and -33) and CPE-defective (NM7, -9, and -19) mutants substantially rescued virus infectivity and virus-induced CPE, respectively, whereas the NS2B-L114T mutation rescued the NM16, -25, and -33 mutants. In conclusion, the results revealed the essential roles of the N-terminal half of NS2A in RNA replication and virus-induced CPE. Intramolecular interactions between pTMSs of NS2A and intermolecular interactions between the NS2A and NS2B proteins were also implicated.IMPORTANCE The characterization of the N-terminal (current study) and C-terminal halves of DENV NS2A is the most comprehensive mutagenesis study to date to investigate the function of NS2A during the flaviviral life cycle. A novel region responsible for virus-induced cytopathic effect (CPE) within pTMS1 and -2 of DENV NS2A was identified. Revertant genetics studies implied unexpected relationships between various pTMSs of DENV NS2A and NS2B. These results provide comprehensive information regarding the functions of DENV NS2A and the specific amino acids and transmembrane segments responsible for these functions. The positions and properties of the rescuing mutations were also revealed, providing important clues regarding the manner in which intramolecular or intermolecular interactions between the pTMSs of NS2A and NS2B regulate virus replication, assembly/secretion, and virus-induced CPE. These results expand the understanding of flavivirus replication. The knowledge may also facilitate studies of pathogenesis and novel vaccine and antiflaviviral drug development.

Project description:UnlabelledFlavivirus nonstructural protein 2A (NS2A) plays important roles in both viral RNA synthesis and virion assembly. The molecular details of how the NS2A protein modulates the two distinct events have not been defined. To address this question, we have performed a systematic mutagenesis of NS2A using dengue virus (DENV) serotype 2 (DENV-2) as a model. We identified two sets of NS2A mutations with distinct defects during a viral infection cycle. One set of NS2A mutations (D125A and G200A) selectively abolished viral RNA synthesis. Mechanistically, the D125A mutation abolished viral RNA synthesis through blocking the N-terminal cleavage of the NS2A protein, leading to an unprocessed NS1-NS2A protein; this result suggests that amino acid D125 (far downstream of the N terminus of NS2A) may contribute to the recognition of host protease at the NS1-NS2A junction. The other set of NS2A mutations (G11A, E20A, E100A, Q187A, and K188A) specifically impaired virion assembly without significantly affecting viral RNA synthesis. Remarkably, mutants defective in virion assembly could be rescued by supplying in trans wild-type NS2A molecules expressed from a replicative replicon, by wild-type NS2A protein expressed alone, by a mutant NS2A (G200A) that is lethal for viral RNA synthesis, or by a different mutant NS2A that is defective in virion assembly. In contrast, none of the mutants defective in viral RNA synthesis could be rescued by trans-complementation. Collectively, the results indicate that two distinct sets of NS2A molecules are responsible for DENV RNA synthesis and virion assembly.ImportanceDengue virus (DENV) represents the most prevalent mosquito-borne human pathogen. Understanding the replication of DENV is essential for development of vaccines and therapeutics. Here we characterized the function of DENV-2 NS2A using a systematic mutagenesis approach. The mutagenesis results revealed two distinct sets of NS2A mutations: one set of mutations that result in defects in viral RNA synthesis and another set of mutations that result in defects in virion assembly. trans-Complementation analysis showed that mutants defective in viral RNA synthesis could not be rescued by wild-type NS2A; in contrast, mutants defective in virion assembly could be successfully rescued by wild-type NS2A or even by a mutant NS2A that is incompetent to support viral RNA synthesis. These results support a model in which two distinct sets of NS2A molecules are responsible for DENV RNA synthesis (located in the viral replication complex) and virion assembly (located in the virion assembly/budding site). The study confirms and extends our understanding of the two critical roles of flavivirus NS2A in viral RNA synthesis and virion assembly.

Project description:Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.

Dataset Information

Membrane topology and function of dengue virus NS2A protein.

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Membrane topology and function of dengue virus NS2A protein.

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