Fig. 1

Lactylation of the S protein promotes viral infection. a Lactylation occurs on the S protein of SARS-CoV-2. S/N/M/E-Flag plasmids were transfected into HEK293T cells for 48 h, after which the cells were harvested, and the lysates were coincubated with protein G-agarose and Flag antibodies to enrich the structural proteins. Protein lactylation was probed with a Pan-Kla antibody via western blot (WB). Exogenous lactate treatment increases the lactylation level of the S protein. S-Flag was transfected into HEK293T cells, and the cells were then treated with NaLa (25 mM, 50 mM) 12 h before harvest. At 48 h posttransfection, the cells were collected for IP analysis to examine the lactylation level of the S protein. 2-DG or oxamate treatment inhibits S protein lactylation. S-Flag was transfected into HEK293T cells for 36 h, after which the cells were treated with 2-DG (5 mM, 10 mM) or oxamate (10 mM, 20 mM) for another 12 h before harvest. S-WT/K424R/K776R/K1028R/3KR-Flag were transfected into HEK293T cells for 48 h, after which lactylation levels were detected. b S lactylation is essential for the infectivity of SARS-CoV-2 pseudoviruses. HEK293T-ACE2 cells were infected with S-WT or mutant pseudoviruses for 72 h. Infection efficacy was analyzed by measuring firefly luciferase activity relative to the S-WT level (set as 1) (n = 3). Statistical analysis was performed via one-way ANOVA. Exogenous NaLa treatment promoted SARS-CoV-2 infectivity, whereas 2-DG or oxamate treatment inhibits SARS-CoV-2 infectivity. HEK293T-ACE2 cells were infected with the SARS-CoV-2 BA.5 variant and then treated with NaLa (10 mM, 25 mM, or 50 mM), 2-DG (2 mM, 5 mM, or 10 mM) or oxamate (5 mM, 10 mM, or 20 mM). N protein expression levels in cell lysates and N mRNA levels in the cell supernatant were assayed 48 h later. c Representative images of H&E-stained lungs from differently treated mice. (scale bar, 100 μm). Weights of the mice monitored over the experimental duration. Viral RNA loads in mouse lungs were detected at 7 dpi by measuring the mRNA levels of M, N and E. d S-lactylation is crucial for S-mediated membrane fusion. HEK293T cells coexpressing S-WT/K424R/K776R/K1028R/3KR-Flag and GFP were cocultured with HEK293T-ACE2 cells. Cell fusion was measured by fluorescence microscopy after 24 h (scale bar, 50 μm). The fusion areas were quantitatively analyzed via ImageJ software, and statistical analysis was performed via two-way ANOVA (* for comparisons versus the control group-WT; # for comparisons versus the NaLa group-WT; and † for comparisons between selected groups). S mutants show reduced binding to ACE2 and TMPRSS2. ACE2-HA or TMPRSS2-myc were transfected into HEK293T cells along with S-WT-Flag or its mutants for 48 h, after which protein interactions were detected by co-IP. (All subfigures follow the same statistical criteria. ns, not significant, *p < 0.05 (#, †), **p < 0.01 (#, †), ***p < 0.001 (#, †), ****p < 0.0001 (#, †))