Work

A nucleocapsid-tagged fluorescent pangolin coronavirus enables authentic-virus neutralization assays and localizes the antiviral activity of cepharanthine to viral entry

Year: 2026

Type: preprint

Abstract: Replication-competent coronaviruses carrying a fluorescently tagged structural protein are rare, and the only well-developed example tags the membrane (M) protein. Using the highly attenuated pangolin coronavirus GX_P2V(short_3UTR) as a backbone, we assembled a full-length cDNA clone by multifragment in vitro ligation and recovered GX_P2V-mBJ-N, a recombinant virus in which the bright monomeric green fluorescent protein mBaoJin is fused to the N terminus of the nucleocapsid (N) protein. The equivalent C-terminal fusion was not viable. GX_P2V-mBJ-N is infectious in Vero E6 cells but is attenuated and genetically unstable in the absence of exogenous N; supplying wild-type N in trans allows propagation to high titer with the fusion intact. Because mBaoJin is translated as part of N, fluorescence reports viral antigen synthesis directly, and infection can be quantified in a plate reader without immunostaining or plaque reduction. We applied the reporter virus in two settings. First, sera from ten healthy blood donors were titrated in parallel against GX_P2V-mBJ-N and a vesicular stomatitis virus (VSV)-based GX_P2V pseudovirus. The two assays agreed on rank order, but pseudovirus titers were about 10-fold higher overall and diverged by at least 70-fold in one donor, showing that the two formats are not interchangeable for individual samples. Second, time-of-addition experiments identified entry as the principal target of cepharanthine (EC50, 0.20 μM under continuous treatment), whereas preincubation of virions with 25 μM drug had no effect, excluding direct virion inactivation. GX_P2V-mBJ-N provides a practical authentic-virus platform for coronavirus serology and antiviral pharmacology.

Source: bioRxiv (Cold Spring Harbor Laboratory)

DOI: 10.64898/2026.08.16.742902