key: cord-0968618-b793k9fo authors: Zhang, Xianwen; Liu, Yang; Liu, Jianying; Bailey, Adam L.; Plante, Kenneth S.; Plante, Jessica A.; Zou, Jing; Xia, Hongjie; Bopp, Nathen; Aguilar, Patricia; Ren, Ping; Menachery, Vineet D.; Diamond, Michael S.; Weaver, Scott C.; Xie, Xuping; Shi, Pei-Yong title: A trans-complementation system for SARS-CoV-2 date: 2021-01-19 journal: bioRxiv DOI: 10.1101/2021.01.16.426970 sha: 16465f48dabdecd499bff8c666bac2108102463e doc_id: 968618 cord_uid: b793k9fo The biosafety level-3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research and countermeasure development. Here we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing a deletion of ORF3 and envelope gene, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round, but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. The results suggest that the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development. We generated and characterized a trans-complementation system for SARS-CoV-2. The 1 9 0 system produced a high yield of single-round infectious Δ ORF3-E virion that could be used for 1 9 1 neutralization and antiviral testing. An mNG reporter was introduced into the Δ ORF3-E virion to 1 9 2 indicate viral replication. Depending on research needs, other reporter genes, such as luciferase 1 9 3 or GFP, could be engineered into the system. A reliable high-throughput neutralization assay is 1 9 4 important for COVID-19 vaccine evaluation and for studying the kinetics of neutralizing antibody 1 9 5 levels in post-vaccinated and naturally infected people (4, 22, 23) . Three types of cell-based 1 9 6 high-throughput neutralization assays currently are available: (i) pseudovirus assay, which 1 9 7 expresses SARS-CoV-2 S protein alone on heterologous viruses, can be performed at BSL-2 1 9 8 laboratories (24, 25); (ii) a reporter SARS-CoV-2 assay, which must be performed at BSL-3 1 9 9 laboratories, represents authentic viral infection (3, 5, 6, 26) ; (iii) bona fide fully infectious SARS-2 0 0 CoV-2 by focus reduction neutralization test (24). The Δ ORF3-E mNG virion combines the 2 0 1 advantages of each assay type by recapitulating the authentic viral infection for a single round, 2 0 2 thus supporting its use at BSL2 laboratories. The Δ ORF3-E mNG virion can be readily adapted 2 0 3 to investigate vaccine-elicited neutralization against newly emerged SARS-CoV-2 isolates, such 2 0 4 as the rapidly spreading United Kingdom and South African strains (27, 28) , by swapping or 2 0 5 mutating the S gene. The trans-complementation system can also be used for high-throughput antiviral can be used to identify inhibitors of all steps of SARS-CoV-2 infection cycle, including virion 2 1 1 assembly and release; this system also allows selection for resistance to inhibitors for mode-of-2 1 2 action studies. In addition, the single-round Δ ORF3-E virion could be developed as a safe 2 1 3 vaccine platform, as previously reported for other coronaviruses (29, 30). Our results support that the trans-complementation system can be performed safely in 2 1 5 BSL-2 laboratories. (i) The system produced single-round infectious Δ ORF3-E mNG virion that 2 1 6 does not infect normal cells for multiple rounds and thus cannot spread in vitro or in vivo. (ii) The 2 1 7 system did not produce WT virus, even after multiple independent selections. (iii) Although an 2 1 8 adaptive mutation in M protein was selected to confer multi-round infection on normal cells, the 2 1 9 replication level of such virion (i.e., S-IV-P5-Vero-P2) was barely detectable, with infectious 2 2 0 titers >100,000-fold lower than the WT SARS-CoV-2. The molecular mechanism of how S-IV-2 2 1 P5-Vero-P2 could infect cells for multiple rounds without the ORF3 and E proteins remains to be 2 2 2 defined. Previous studies showed that deletion of both ORF3 and E genes was lethal for SARS- mNG RNA as accessory proteins are not essential for viral replication (1). One limitation of our study is the use of Vero E6 cells for constructing the Vero-ORF3-E hACE2 or Vero-TMPRSS2-hACE2 cells. In summary, we have developed a trans-complementation system for SARS-CoV-2 that 2 3 8 likely can be performed at BSL-2 laboratories for COVID-19 research and countermeasure 2 3 9 development. Thus, the experimental system could be used by researchers in industry, 2 4 0 academia, and government laboratories who lack access to a BSL-3 facility. total cellular mRNA was isolated and subject to RT-PCR with primers EcoR1-mCherry-F and 3 1 8 BamH1-Cov-optORF3-R (Table S2) , followed by cDNA sequencing of the ORF3-E genes. SARS-CoV-2, the N gene was PCR-amplified by primers CoV-T7-N-F and polyT-N-R (Table 3 3 2 S2) from a plasmid containing the F7 fragment (2); the PCR product was then used for in vitro 3 3 3 transcription using the T7 mMessage mMachine kit (Ambion). To assess viral RNA levels, a quantitative RT-PCR assay was conducted using an iTaq normalizing the infection rates of serum-treated groups to those of non-serum-treated controls. The curves of the relative infection rates versus the serum dilutions (log10 values) were plotted 3 8 0 using Prism 9 (GraphPad, San Diego, CA). A nonlinear regression method was used to 1 7 Micrographs were taken using a JEM 1400 (JEOL USA Inc.). Multiple randomly selected fields 4 0 4 were imaged. Bioinformatics analysis. Fluorescence images were processed using ImageJ (41). Virus sequences were download from the NCBI database and aligned using Geneious software. The results presented in the study are available upon request from the corresponding 4 2 0 authors. The mNG reporter SARS-CoV-2 has been deposited to the World Reference Center for 4 2 1 Emerging Viruses and Arboviruses (https://www.utmb.edu/wrceva) at UTMB for distribution. mNG virion for two rounds as described in Fig. 1G . Viral RNAs were extracted from the second- Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis Primer name Sequences (5′ to 3′) pcov-F56-F1 TATACGAAGTTATATTCGATGCGGCCGCGTCTCAGAGTGCTTTGGTTTAT GATAATAAG pncov-R5