Fig. 1.

NSP1 and NSP15 stably expressing A549 ​cells lines. Phylogenetic analysis of the SARS-CoV-2 (A) NSP1 and NSP15 as performed using MEGAX software. (B) NSP1 and NSP15, part of the ORF1a and 1b were subcloned into a pCDNA3.1/V5–HIS-TOPO plasmid (HindIII and EcoRV restriction sites) and transfected into A549 ​cells. Expression of (C) NSP1 and (D) NSP15 was confirmed by total RNA extraction (polymerase chain reaction; PCR) and total protein extraction (Western blot). (E) Representative pictures of confocal microscopy imaging with maximum projections of z-stacks for each channel demonstrating the subcellular localization of expressed NSP1 (anti-V5 antibody; 488 ​nm) and NSP15 (anti-V5 antibody; 488 ​nm) and lysosome-associated membrane protein 1 (LAMP-1) (red) in stably expressing A549 ​cells. Maximum projections of all combined channels (merge) were analyzed using LAS AF software to determine the relative fluorescence intensity (arbitrary units [A.U]) profiles in stably expressing cell line for anti-V5 (green) and DAPI (blue) and LAMP-1 (red) along the white line path (micrometers) indicated in the corresponding ROIs (merge) demonstrating overlapping signals. Validation of colocalization efficiencies by Manders’ colocalization coefficient as analyzed using ImageJ 72 ​h. Colocalizations between (F) NSP1-anti-V5 (M1) and LAMP-1 (M2) and (G) NSP15-anti-V5 (M1) and LAMP-1 (M2) represent the overlap of M1 as the denominator and vice versa (n ​= ​50). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)