Nafamostat-Mediated Inhibition of SARS-CoV-2 Ribosomal Frameshifting Is Insufficient to Impair Viral Replication in Vero Cells. Comment on Munshi et al. Identifying Inhibitors of -1 Programmed Ribosomal Frameshifting in a Broad Spectrum of Coronaviruses. Viruses 2022, 14, 177

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has been reported to have caused 18 [...].

Figure 1

Nafamostat inhibits SARS-CoV-2 −1PRF. (A) Top panel: Schematic illustration of the SARS-CoV-2 genome, including the site of −1PRF and the frameshifting stimulation element (FSE). Bottom panel: For quantification of −1PRF, the FSE sequence, including the heptanucleotide slippery sequence, and the three-stemmed pseudoknot sequence (GenBank: NC_004718) were inserted between the coding sequences for Renilla reniformis luciferase (RLuc) (0 reading frame) and firefly luciferase (FLuc) (−1 reading frame). As a consequence, FLuc is only translated if −1PRF occurs, with the ratio between FLuc and RLuc activities indicating the efficiency of −1PRF. In order to determine the maximum signal for frameshifting, the heptanucleotide slippery sequence and the FSE were removed and FLuc was set into the same reading frame as RLuc (0 frame) (LucMax reporter). (B) Vero 76 cells were treated with the indicated concentrations of Nafamostat for 1 h. DMSO treatment served as a control. Subsequently, the treated cells were transfected with a plasmid encoding the SARS-CoV-2 −1PRF reporter cassette or were cotransfected with the reporter plasmid and pQCXIP-plasmids encoding SFL or SFLS under the control of a CMV promotor using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). To exclude unspecific effects of Nafamostat on translation (0 frame product) cells were transfected with LucMax reporter and were treated with Nafamostat as described for the −1PRF reporter. At 12 h post-transfection, the medium was replaced by fresh medium again containing Nafamostat at the indicated concentrations and cells were incubated for an additional 36 h. At 48 h post-transfection, RLuc and FLuc signals were quantified using a luminometer. The ratio of FLuc versus RLuc signals measured for the −1PRF reporter and the LucMax reporter was determined. Next, the results obtained for the −1PRF reporter were normalized against the respective results measure for the LucMax reporter. Finally, relative frameshifting measured upon treatment with DMSO was set as 100%. The average of six biological replicates carried out with technical triplicates is shown. Error bars indicate the standard error of the mean (SEM). Statistical significance was assessed by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (*, p ≤ 0.05; ***, p ≤ 0.001).

Figure 2

Nafamostat inhibits SARS-CoV-2 infection of Calu-3 but not Vero 76 cells. Vero 76 and Calu-3 cells were incubated with the indicated 10-fold serial dilutions of Nafamostat for 1 h prior to infection with SARS-CoV-2, Pango lineage B.1.513, at a multiplicity of infection of 0.01 for 1 h. After virus inoculation, cells were washed and further incubated with Nafamostat for 24 h. Virus-containing supernatants were harvested and viral titers were determined by titration on Vero E6 cells. Titers are shown as plaque forming units (PFU)/mL. The graphs show mean ± SEM of three independent biological replicates. Statistical significance of differences between viral titers of control-treated cells (DMEM) and cells incubated with Nafamostat was analyzed by one-way analysis of variance (ANOVA) with Dunnett’s posttest (p > 0.05, not significant [not indicated]; ***, p ≤ 0.001).