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Secretory Carrier Membrane Protein 3 Interacts with 3A Viral Protein of Enterovirus and Participates in Viral Replication.

ABSTRACT: Picornaviruses are a diverse and major cause of human disease, and their genomes replicate with intracellular membranes. The functionality of these replication organelles depends on the activities of both viral nonstructural proteins and co-opted host proteins. The mechanism by which viral-host interactions generate viral replication organelles and regulate viral RNA synthesis is unclear. To elucidate this mechanism, enterovirus A71 (EV-A71) was used here as a virus model to investigate how these replication organelles are formed and to identify the cellular components that are critical in this process. An immunoprecipitation assay was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify 172 cellular proteins and four viral proteins associating with viral 3A protein. Secretory carrier membrane protein 3 (SCAMP3) was one of the host proteins we selected for further investigation. Here, we demonstrate by immunoprecipitation assay that SCAMP3 associates with 3A protein and colocalizes with 3A protein during virus infection. SCAMP3 knockdown or knockout in infected cells decreases synthesis of EV-A71 viral RNA, viral proteins, and viral growth. Furthermore, the viral 3A protein associates with SCAMP3 and phosphatidylinositol-4-kinase type III β (PI4KIIIβ) as shown by immunoprecipitation assay and colocalizes to the replication complex. Upon infection of cells with a SCAMP3 knockout construct, PI4KIIIβ and phosphatidylinositol-4-phosphate (PI4P) colocalization with EV-A71 3A protein decreases; viral RNA synthesis also decreases. SCAMP3 is also involved in the extracellular signal-regulated kinase (ERK) signaling pathway to regulate viral replication. The 3A and SCAMP3 interaction is also important for the replication of coxsackievirus B3 (CVB3). SCAMP3 also associates with 3A protein of CVB3 and enhances viral replication but does not regulate dengue virus 2 (DENV2) replication. Taken together, the results suggest that enterovirus 3A protein, SCAMP3, PI4KIIIβ, and PI4P form a replication complex and positively regulate enterovirus replication. IMPORTANCE Virus-host interaction plays an important role in viral replication. 3A protein of enterovirus A71 (EV-A71) recruits other viral and host factors to form a replication complex, which is important for viral replication. In this investigation, we utilized immunoprecipitation combined with proteomics approaches to identify 3A-interacting factors. Our results demonstrate that secretory carrier membrane protein 3 (SCAMP3) is a novel host factor that associates with enterovirus 3A protein, phosphatidylinositol-4-kinase type III β (PI4KIIIβ), and phosphatidylinositol-4-phosphate (PI4P) to form a replication complex and positively regulates viral replication. SCAMP3 is also involved in the extracellular signal-regulated kinase (ERK) signaling pathway to regulate viral replication.

SUBMITTER: Lu JY

PROVIDER: S-EPMC8552740 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Like other enteroviruses, enterovirus 71 (EV71) relies on phosphatidylinositol 4-kinase III? (PI4KB) for genome RNA replication. However, how PI4KB is recruited to the genome replication sites of EV71 remains elusive. Recently, we reported that a host factor, ACBD3, is needed for EV71 replication by interacting with viral 3A protein. Here, we show that ACBD3 is required for the recruitment of PI4KB to RNA replication sites. Overexpression of viral 3A or EV71 infection stimulates the interaction of PI4KB and ACBD3. Consistently, EV71 infection induces the production of phosphatidylinositol-4-phosphate (PI4P). Furthermore, PI4KB, ACBD3, and 3A are all localized to the viral-RNA replication sites. Accordingly, PI4KB or ACBD3 depletion by small interfering RNA (siRNA) leads to a reduction in PI4P production after EV71 infection. I44A or H54Y substitution in 3A interrupts the stimulation of PI4KB and ACBD3. Further analysis suggests that stimulation of ACBD3-PI4KB interaction is also important for the replication of enterovirus 68 but disadvantageous to human rhinovirus 16. These results reveal a mechanism of enterovirus replication that involves a selective strategy for recruitment of PI4KB to the RNA replication sites.IMPORTANCE Enterovirus 71, like other human enteroviruses, replicates its genome within host cells, where viral proteins efficiently utilize cellular machineries. While multiple factors are involved, it is largely unclear how viral replication is controlled. We show that the 3A protein of enterovirus 71 recruits an enzyme, phosphatidylinositol 4-kinase III?, by interacting with ACBD3, which alters cellular membranes through the production of a lipid, PI4P. Consequently, the viral and host proteins form a large complex that is necessary for RNA synthesis at replication sites. Notably, PI4KB-ACBD3 interaction also differentially mediates the replication of enterovirus 68 and rhinovirus 16. These results provide new insight into the molecular network of enterovirus replication.

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.

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Secretory Carrier Membrane Protein 3 Interacts with 3A Viral Protein of Enterovirus and Participates in Viral Replication.

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