FIG 1.
Transfection with the hCD26 transgene construct, pCAGGS.hCD26, effectively converts nonsusceptible mouse fibroblastic 17CL-1 cells to become fully supportive of productive MERS-CoV infection. (A) Schematic diagram of hCD26 expressing vector cassette, designated pCAGGS.hCD26. The hCD26 gene was cloned in this vector by restriction digestion with EcoRI at the multiple cloning site (MCS) which is driving the expression in the presence of chicken β-actin promoter. Introns and polyadenylation signals were included in the expressing cassette to enhance the transcription and increase the mRNA stability of transgenes. These elements were cloned into the plasmid such that the transgene could be expressed in one piece from the plasmid backbone by digestion with restriction enzymes (38, 39). Confluent parental and 17CL-1 cells stably transfected with the hCD26 transgene construct were analyzed for the expression of hCD26 with a goat anti-hCD26 antibody using indirect immunofluorescent staining (B) and Western blot analysis (C). The expression of hCD26 antigen was detected exclusively in 17CL-1/hCD26 cells as the green fluorescent protein with an expected size of 110 kDa. (D to F) To assess the permissiveness of 17CL-1/hCD26 cells to MERS-CoV infection, confluent 17CL-1 and 17CL-1/hCD26 cells, grown in 12-well plates, were infected with either MERS-CoV/EMC-2012 or rMERS-CoV/RFP at an MOI of 1 and monitored for CPE for 2 days after infection (D and E), and yields of infectious progeny virus were titrated at 1 and 2 dpi (F; the dashed line indicates the detection limit). The data shown are representative of at least two independently conducted experiments. The infectious virus titer (log10 TCID50/ml) is expressed as the mean ± the standard error of triplicate samples. ***, P ≤ 0.001 (determined using the Student t test, comparing 17CL-1 and 17CL-1/hCD26 cells).