Fig. 1.

Expression of SARS-CoV genomic and messenger RNA in CHO cells transduced with a human lung cDNA library, and binding of SARS-CoV spike glycoprotein to cell membranes. (A) Multiplex RT-PCR at 1 and 16 h PI with SARS-CoV. Multiplex RT-PCR was performed as in ref. 17. Control CHO cells or CHO cells transduced with retrovirus pseudotypes containing a human lung cDNA library were inoculated with SARS-CoV or mock inoculated. The viral genomic and subgenomic RNAs and cellular mRNA encoding GAPDH were reverse transcribed and amplified. Negative images of the amplicons are shown. The bottom gel shows Vero E6 monkey kidney cells, a positive control for SARS-CoV infection, in which subgenomic viral RNA increased at 16 h PI. The minute amount of subgenomic viral RNA in the 1-h sample was probably due to a low level of subgenomic viral RNA contamination of the input virus. In the fourth lane of the upper gel, CHO library cells that were sorted twice for S₅₉₀ binding also showed an increase in subgenomic viral RNA at 16 h PI. These cells were further sorted for S₅₉₀ binding, and single-cell clones were produced. Four of these cell lines showed an increase in subgenomic viral RNA at 16 h PI: clones 2.15, 2.22, 2.27, and 2.37. The proviral integrants were amplified by RT-PCR of RNA from the cloned cell lines. Sequencing results in Table 1 showed that all four of the SARS-CoV-susceptible cell lines contained CD209L. Clone 2.27 cells that had the largest increase in subgenomic viral RNA were further studied. (B) Flow cytometric analysis of soluble SARS-CoV S₁₁₈₀ protein binding to CHO (Upper) and clone 2.27 (Lower) cells.