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Comprehensive Interactome Analysis Reveals that STT3B Is Required for N-Glycosylation of Lassa Virus Glycoprotein.

ABSTRACT: Lassa virus (LASV) is the causative agent of a fatal hemorrhagic fever in humans. The glycoprotein (GP) of LASV mediates viral entry into host cells, and correct processing and modification of GP by host factors is a prerequisite for virus replication. Here, using an affinity purification-coupled mass spectrometry (AP-MS) strategy, 591 host proteins were identified as interactors of LASV GP. Gene ontology analysis was performed to functionally annotate these proteins, and the oligosaccharyltransferase (OST) complex was highly enriched. Functional studies conducted by using CRISPR-Cas9-mediated knockouts showed that STT3A and STT3B, the two catalytically active isoforms of the OST complex, are essential for the propagation of the recombinant arenavirus rLCMV/LASV glycoprotein precursor, mainly via affecting virus infectivity. Knockout of STT3B, but not STT3A, caused hypoglycosylation of LASV GP, indicating a preferential requirement of LASV for the STT3B-OST isoform. Furthermore, double knockout of magnesium transporter 1 (MAGT1) and tumor suppressor candidate 3 (TUSC3), two specific subunits of STT3B-OST, also caused hypoglycosylation of LASV GP and affected virus propagation. Site-directed mutagenesis analysis revealed that the oxidoreductase CXXC active-site motif of MAGT1 or TUSC3 is essential for the glycosylation of LASV GP. NGI-1, a small-molecule OST inhibitor, can effectively reduce virus infectivity without affecting cell viability. The STT3B-dependent N-glycosylation of GP is conserved among other arenaviruses, including both the Old World and New World groups. Our study provided a systematic view of LASV GP-host interactions and revealed the preferential requirement of STT3B for LASV GP N-glycosylation.IMPORTANCE Glycoproteins play vital roles in the arenavirus life cycle by facilitating virus entry and participating in the virus budding process. N-glycosylation of GPs is responsible for their proper functioning; however, little is known about the host factors on which the virus depends for this process. In this study, a comprehensive LASV GP interactome was characterized, and further study revealed that STT3B-dependent N-glycosylation was preferentially required by arenavirus GPs and critical for virus infectivity. The two specific thioredoxin subunits of STT3B-OST MAGT1 and TUSC3 were found to be essential for the N-glycosylation of viral GP. NGI-1, a small-molecule inhibitor of OST, also showed a robust inhibitory effect on arenavirus. Our study provides new insights into LASV GP-host interactions and extends the potential targets for the development of novel therapeutics against Lassa fever in the future.

SUBMITTER: Zhu S

PROVIDER: S-EPMC6854512 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Comprehensive Interactome Analysis Reveals that STT3B Is Required for N-Glycosylation of Lassa Virus Glycoprotein.

Zhu Shenglin S Wan Weiwei W Zhang Yanjun Y Shang Weijuan W Pan Xiaoyan X Zhang Lei-Ke LK Xiao Gengfu G

Journal of virology 20191113 23

Lassa virus (LASV) is the causative agent of a fatal hemorrhagic fever in humans. The glycoprotein (GP) of LASV mediates viral entry into host cells, and correct processing and modification of GP by host factors is a prerequisite for virus replication. Here, using an affinity purification-coupled mass spectrometry (AP-MS) strategy, 591 host proteins were identified as interactors of LASV GP. Gene ontology analysis was performed to functionally annotate these proteins, and the oligosaccharyltrans ...[more]

PMID: 31511384

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Project description:Lassa virus (LASV) is an enveloped RNA virus endemic to West Africa and responsible for severe cases of hemorrhagic fever. Virus entry is mediated by the glycoprotein complex consisting of a stable-signal peptide, a receptor-binding subunit, GP1, and a viral-host membrane fusion subunit, GP2. Several cellular receptors can interact with the GP1 subunit and mediate viral entry, including alpha-dystroglycan (?DG) and lysosome-associated membrane protein 1 (LAMP1). In order to define the regions within GP1 that interact with the cellular receptors, we implemented insertional mutagenesis, carbohydrate shielding, and alanine scanning mutagenesis. Eighty GP constructs were engineered and evaluated for GP1-GP2 processing, surface expression, and the ability to mediate cell-to-cell fusion after low-pH exposure. To examine virus-to-cell entry, 49 constructs were incorporated onto vesicular stomatitis virus (VSV) pseudoparticles and transduction efficiencies were monitored in HAP1 and HAP1-?DAG1 cells that differentially produce the ?DG cell surface receptor. Seven constructs retained efficient transduction in HAP1-?DAG1 cells yet poorly transduced HAP1 cells, suggesting that they are involved in ?DG utilization. Residues H141, N146, F147, and Y150 cluster at the predicted central core of the trimeric interface and are important for GP-?DG interaction. Additionally, H92A-H93A, 150HA, 172HA, and 230HA displayed reduced transduction in both HAP1 and HAP1-?DAG1 cells, despite efficient cell-to-cell fusion activity. These mutations may interfere with interactions with the endosomal receptor LAMP1 or interfere at another stage in entry that is common to both cell lines. Insight gained from these data can aid in the development of more-effective entry inhibitors by blocking receptor interactions.IMPORTANCE Countries in which Lassa virus is endemic, such as Nigeria, Sierra Leone, Guinea, and Liberia, usually experience a seasonal outbreak of the virus from December to March. Currently, there is neither a preventative vaccine nor a therapeutic available to effectively treat severe Lassa fever. One way to thwart virus infection is to inhibit interaction with cellular receptors. It is known that the GP1 subunit of the Lassa glycoprotein complex plays a critical role in receptor recognition. Our results highlight a region within the Lassa virus GP1 protein that interacts with the cellular receptor alpha-dystroglycan. This information may be used for future development of new Lassa virus antivirals.

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Comprehensive Interactome Analysis Reveals that STT3B Is Required for N-Glycosylation of Lassa Virus Glycoprotein.

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Comprehensive Interactome Analysis Reveals that STT3B Is Required for N-Glycosylation of Lassa Virus Glycoprotein.

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