Objective  To investigate functions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural protein ORF3a and ORF3b in host cells.

Methods  The assembly and release of SARS-CoV-2 pseudovirus were detected by immunofluorescence assay; real-time polymerase chain reaction and western blot were used to detect the effects of ORF3a and ORF3b on expression of IFN-β and inflammatory factors. Western blot was used to detect the effects of ORF3a and ORF3b on protein expression in interferon signaling pathway.

Results  Fluorescence microscopic results showed that the number of packaged and released pseudovirus particles in human lung cell line A549 A549-angiotensin converting enzyme 2 (ACE2)cells overexpressing ACE2 infected by SARA-COV-2 pseudovirus and transfected ORF3a expression vector and ORF3b expression vector was higher than those of untreated cells and empty vector cells, indicating that ORF3a and ORF3b could promote the assembly and release of SARA-COV-2 particles; A549 cells were stimulated with ploy (I ∶ C) and then transfected with ORF3a or ORF3b expression plasmid. The expression of IFN-β in above cells was significantly lower than that single ploy (I ∶ C) stimulated A549cells(P < 0.001), indicating that the transfected expression of ORF3a and ORF3b could significantly inhibit IFN-β expression in cells induced by ploy (I ∶ C). Western blot assay showed that the expressions of key proteins such as MyD88 and TRAF6 in interferon signaling pathway were inhibited after cells dealing with IFN-β and transfected with ORF3a or ORF3b as compared with treated by IFN-β cells alone; inflammatory cytokines levels such as (tumor necrosis factor-α, interleukin-1β, interleukin-6) in A549 cells transfected with ORF3a or ORF3b expression vector were significantly increased in ORF3a or ORF3b expression host cells compared with the empty vector A549 cells(P < 0.01 or P < 0.001).

Conclusion  Non-structural protein ORF3 can promote viral assembly and release of SARS-CoV-2, induce host inflammatory response, and inhibit host interferon expression.