Fig. 1.

Determination of coronaviral poly(A) tail length in BCoV-infected BHK-21 cells. a Strategy for determining coronaviral poly(A) tail length. Total cellular RNA from virus-infected cells was collected, decapped, and head-to-tail ligated. The ligated viral RNA was used as template for RT with 5′ UTR-specific primer 2, and PCR was performed with 3′ UTR-specific primer 1 and primer 2. The synthesized RT-PCR product contained the sequences from BCoV 3′ UTR, poly(A) tail and BCoV 5′ UTR. b The RT-PCR products synthesized with RNA from BCoV inoculum (lane 2) and with total cellular RNA collected from mock-infected BHK-21 cells (lane 3) and BCoV-infected BHK-21 cells at different time points of infection (lanes 5–13) by the method described in a. c The length of the poly(A) tail as determined by sequencing the RT-PCR products from samples obtained from b, lane 2, and lanes 5–13. The length of poly(A) tail at 1 hpi was compared with that at different time points of infection for statistical analysis. d Quantitation analysis of the RT-PCR products shown in b, lanes 5–13. e Left panel expression of BCoV N protein as analyzed by Western blot analysis. Right panel quantitation analysis of BCoV N protein at different time points of infection. M ds DNA size markers in nt pairs, In inoculum. Values in c–e represent the mean ± SD of three individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001