Production of single‐cycle infectious SARS‐CoV‐2 through a trans‐complemented replicon

Abstract

Single‐cycle infectious virus can elicit close‐to‐natural immune response and memory. One approach to generate single‐cycle severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) is through deletion of structural genes such as spike (S) and nucleocapsid (N). Transcomplementation of the resulting ΔS or ΔN virus through enforced expression of S or N protein in the cells gives rise to a live but unproductive virus. In this study, ΔS and ΔN BAC clones were constructed and their live virions were rescued by transient expression of S and N proteins from the ancestral and the Omicron strains. ΔS and ΔN virions were visualized by transmission electron microscopy. Virion production of ΔS was more efficient than that of ΔN. The coated S protein from ΔS was delivered to infected cells in which the expression of N protein was also robust. In contrast, expression of neither S nor N was detected in ΔN‐infected cells. ΔS underwent viral RNA replication, induced type I interferon (IFN) response, but did not form plaques. Despite RNA replication in cells, ΔS infection did not produce viral progeny in culture supernatant. Interestingly, viral RNA replication was not further enhanced upon overexpression of S protein. Taken together, our work provides a versatile platform for development of single‐cycle vaccines for SARS‐CoV‐2.

Nsp1 facilitates SARS-CoV-2 replication through calcineurin-NFAT signaling

“…2A and B ). To further scrutinize the effect of Nsp1 on SARS-CoV-2 replication specifically, an S-deficient SARS-CoV-2 replicon (ΔS replicon) was employed ( 32 ). The ΔS replicon was transfected into VeroE6-TMPRSS2 cells, and vRNA samples were harvested on days 0, 1, and 2 for RT-qPCR of dsRED gene copies.…”

“…The grid was then washed with ddH 2 O droplet and excess liquid was removed by filter paper. The grid was then dried by air and examined with FEI TeCnai G2 20 scanning TEM at 100 kV ( 32 ).…”

Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety level 2 facilities

“…It enabled us to measure HiBiT luminescence without cell lysis. Other groups reported that replicons lacking only the S gene are applicable to the production of single-cycle infectious particles by the trans-complementation of the SARS-CoV-2 spike or other viral surface glycoproteins, such as the vesicular stomatitis virus glycoprotein (Cheung et al, 2022; Malicoat et al, 2022). The further application of our Δorf2 replicon to the production of single-cycle virus is of interest.…”