Dataset Information

TurboID-based proximity labeling identifies reticulon-like protein B2 as a pro-viral factor coopted for the biogenesis of viral replication organelles in plants

ABSTRACT: Although intensive studies have been performed to identify and characterize host factors involved in viral replication complex formation, the high dynamism of this process and the insoluble properties of the complex make it difficult to be analyzed comprehensively using traditional affinity purification methods. TurboID-based proximity labeling emerges as a robust tool for probing molecular interactions in planta, yet there is no report on the application of TurboID on the study of plant virus replication. Here, we used Beet black scorch virus (BBSV), an endoplasmic reticulum-replicating virus, as a model, and systematically investigated the composition of BBSV virus replication complex (VRC) by fusing the TurboID enzyme to viral replicase protein p23. Among the 185 identified p23-proximal proteins, reticulon family proteins showed high reproducibility in the mass spectrometry datasets. We focused on the reticulon-like protein B2 (RTNLB2) and demonstrated its pro-viral functions in BBSV replication. Mechanistic analysis revealed that RTNLB2 binds to p23, induces the ER membrane curvature and constricts ER tubules to facilitate the assembly of BBSV VRC. Our comprehensive proximal interactome analysis of BBSV VRC provides a resource for understanding of plant viral replication and offers new insights into the formation of membrane scaffolds for viral RNA synthesis.

ORGANISM(S): Nicotiana Benthamiana

SUBMITTER: Yongliang Zhang

PROVIDER: PXD040073 | iProX | Sun Feb 12 00:00:00 GMT 2023

REPOSITORIES: iProX

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Publications

RETICULON-LIKE PROTEIN B2 is a proviral factor co-opted for the biogenesis of viral replication organelles in plants.

Zhang Qianshen Q Wen Zhiyan Z Zhang Xin X She Jiajie J Wang Xiaoling X Gao Zongyu Z Wang Ruiqi R Zhao Xiaofei X Su Zhen Z Li Zhen Z Li Dawei D Wang Xiaofeng X Zhang Yongliang Y

The Plant cell 20230801 8

Endomembrane remodeling to form a viral replication complex (VRC) is crucial for a virus to establish infection in a host. Although the composition and function of VRCs have been intensively studied, host factors involved in the assembly of VRCs for plant RNA viruses have not been fully explored. TurboID-based proximity labeling (PL) has emerged as a robust tool for probing molecular interactions in planta. However, few studies have employed the TurboID-based PL technique for investigating plant ...[more]

PMID: 37216674

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Project description:Upon infection, viruses remodel host cells to create an environment hospitable to viral replication. In response, host cells have evolved to sense infection and rapidly induce pathways to limit viral replication. One strategy used by both players is to regulate the host cell proteome for their benefit. To characterize these changes, we used a mass spectrometry strategy to define alterations in the viral and host proteome during infection of human cells with West Nile virus (WNV). Our studies reveal previously uncharacterized post-translational modifications (PTMs) on viral proteins, including phosphorylation sites on the WNV nonstructural proteins, NS3 and NS5, that impact viral RNA replication. Additionally, we monitored changes in host protein abundance and found that while 82 proteins had increased expression upon WNV infection, only five of these are canonical interferon stimulated genes (ISGs). Our data suggests that the majority of the upregulated non-ISGs are regulated at the translational, rather than transcriptional level, defining a new group of virus-induced proteins (VIPs). HERPUD1 is a stress-response protein that was induced at both the transcriptional and translational level. We show HERPUD1 restricts WNV replication, likely through a mechanism independent of its previously defined role in ER-associated degradation (ERAD). Our phosphoproteomics studies revealed eight phosphorylation sites on viral proteins, including a site on the WNV helicase/protease NS3 that is important for viral replication. Further, we identified XXX phosphorylation sites on host proteins with increased abundance and XXX sites with decreased abundance during WNV infection. We used bioinformatics to identify phosphosites predicted to increase host protein activity and found two antiviral host kinases that are activated by phosphorylation during WNV infection. First, we identified a phosphorylation site at S108 of AMPKβ1, a non-catalytic subunit that is thought to regulate activity of the AMPK complex. We also found that a group I p21-activated kinase, PAK2, is phosphorylated at S141. This phosphorylation event in the autoinhibitory domain activates PAK2, which restricts viral protein translation. Altogether, our work contributes to our understanding of the interplay between host and virus while providing a resource to define the changes to the proteome that regulate viral infection.

Dataset Information

TurboID-based proximity labeling identifies reticulon-like protein B2 as a pro-viral factor coopted for the biogenesis of viral replication organelles in plants

Publications

RETICULON-LIKE PROTEIN B2 is a proviral factor co-opted for the biogenesis of viral replication organelles in plants.

Similar Datasets

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