Biology subjects

Zhao, J.-J.

Publications and source records attributed to Zhao, J.-J..

2 recordsLinked to original sources

bioRxiv2026

Ribophorin-1 Governs Spike Abundance of Highly Pathogenic Coronaviruses by ER-associated degradation

The spike protein of highly pathogenic coronaviruses is indispensable for viral entry, pathogenesis, and immune evasion. However, specific host factors governing spike protein degradation and abundance on progeny virions remain largely uncharacterized. Here, we identify Ribophorin-1 (RPN1), a non-catalytic subunit of the oligosaccharyltransferase (OST) complex, as an ER-localized host restriction factor that selectively depletes spike to inhibit coronavirus infection. Conditional knockout of Rpn1 in mouse lung markedly exacerbates SARS-CoV-2 pathogenesis, increasing viral load, immune cell infiltration, and syncytium formation. In cells, RPN1 knockdown enhances susceptibility to diverse variants, whereas its overexpression attenuates infection. Nano-flow cytometry, cryo-EM and LSCM show that RPN1 reduces spike abundance on progeny virions, resulting in reduction in subsequent syncytium formation across SARS-CoV, SARS-CoV-2, and MERS-CoV. Mechanistically, RPN1 promotes VCP/p97-dependent retrotranslocation and AMFR-mediated ubiquitination of spike protein. Heterologous expression of RPN1s functional domain (residues 180-307) confers significant protection against SARS-CoV-2 both in vitro and in vivo. Collectively, our findings define an RPN1-initiated ERAD pathway that selectively targets coronavirus spike for proteasomal destruction, and provide proof-of-concept evidence that targeting this RPN1-dependent pathway represents a promising broad-spectrum antiviral strategy.

microbiology↗

bioRxiv2021

GREPore-seq: A Robust Workflow to Detect Changes after Gene Editing through Long-range PCR and Nanopore Sequencing

To achieve the enormous potential of gene-editing technology in clinical therapies, both the on-target and unintended editing consequences need to be thoroughly evaluated. However, there is a lack of a comprehensive, pipelined, large-scale and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR-Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. To overcome the high error rates and indels of nanopore sequencing, we developed a pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can detect NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also identifies HDR-mediated large gene knock-in, which excellently correlates with FACS analysis data. Low-level plasmid backbone insertion after HDR editing was also detected. We have established a practical workflow to identify genetic changes, including quantifying dsODN insertions, knock-ins, plasmid backbone insertions, and large fragment deletions after CRISPR editing. This toolkit for nanopore sequencing of pooled long amplicons should have broad applications in assessing on-target HDR editing and inadvertent large indels of over 1 kb. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq).

bioinformatics↗