Examining the feasibility of replacing ORF3a with fluorescent genes to construct SARS-CoV-2 reporter viruses

The SARS-CoV-2 genome encodes at least nine accessory proteins, including innate immune antagonist and putative viroporin ORF3a. ORF3a plays a role in many stages of the viral replication cycle, including immune modulation. We constructed two recombinant (r)SARS-CoV-2 viruses in which the ORF3a gene was replaced with mScarlet (mS) or mNeonGreen (mNG), denoted as rSARS-CoV-2-Δ3a-mS and rSARS-CoV-2-Δ3a-mNG, respectively. rSARS-CoV-2-Δ3a-mNG generated a fluorescent signal after infection in both A549-ACE-2-TMPRSS2 (AAT) and Vero-E6-TMPRSS2 (VTN) cells, unlike rSARS-CoV-2-Δ3a-mS. rSARS-CoV-2-Δ3a-mS mS protein could be detected immunologically in VTN but not AAT cells, indicating the expression of a non-fluorescent mS protein. The analysis of the viral transcriptomes in infected AAT cells by nanopore direct RNA sequencing (dRNAseq) revealed that the level of mS transcript was below the limit of detection in AAT cells. rSARS-CoV-2-Δ3a-mNG virus was found to be genetically stable in AAT and VTN cells, but rSARS-CoV-2-Δ3a-mS acquired partial deletions of the mS gene during sequential passaging in VTN cells, creating the virus rSARS-CoV-2-Δ3a-ΔmS. The mS deletion in VTN cells removes the chromophore coding sequence, and this may explain the presence of a non-fluorescent mS protein detected in VTN cells. The rSARS-CoV-2-Δ3a-mNG, rSARS-CoV-2-Δ3a-mS and rSARS-CoV-2-Δ3a-ΔmS viruses all replicated to a lower titre and produced smaller plaques than the parental rSARS-CoV-2-S-D614G. Interestingly, the rSARS-CoV-2-Δ3a-ΔmS virus produced higher virus titres and larger plaque sizes than rSARS-CoV-2-Δ3a-mS. This suggested that both the insertion of mS coding sequence and the deletion of ORF3a coding sequence contributed to attenuation. In comparison with rSARS-CoV-2, the rSARS-CoV-2-Δ3a-mS and rSARS-CoV-2-Δ3a-mNG viruses showed increased sensitivity to pre-treatment of cells with IFN-α but did not exhibit a dose-dependent increase in replication in the presence of the Janus kinase-signal transducer and activator of transcription signalling pathway inhibitor, ruxolitinib. In conclusion, the replacement of the ORF3a coding sequence with those of fluorescent reporter proteins attenuated the replication of SARS-CoV-2 and its ability to effectively evade the innate immune response <i>in vitro</i>.