Effects of A159-K160 deletion and mutation on replication and infectivity of IBV.

<p>a. In vitro transcripts derived from wild type, MΔ5 and Mm1 full-length IBV clones were electroporated into Vero cells. At 24 and 72 hours post-electroporation, total RNA were extracted from the cells and specific primers were used to detect the minus RNA (upper panel) and subgenomic mRNA 4 (lower panel) by RT-PCR. b. H1299 cells expressing wild type IBV M and MΔ5 constructs with a vaccinia virus system were infected with IBV at a multiplicity of infection of approximately 0.5 PFU/cell. Cells were collected at 24 hours post-infection and polypeptides were analyzed by Western blot with anti-IBV M (second panel), anti-IBV N (third panel) and anti-actin (bottom panel) antibodies. H1299 cells expressing wild type IBV M and MΔ5 constructs with a vaccinia virus system without IBV infection harvested at 40 hours post-transfection were also analyzed by Western blot with anti-IBV M antibodies (top panel). c. Interaction of Mm1 mutant M protein with IBV E protein. H1299 cells expressing the E (lane 1), M (lane 2) and E+M (lane 3) and E+Mm1 were harvested at 24 hours post-transfection and lysed. The total cell lysates were either detected directly by Western blot with anti-E antibodies (top panel) or immunoprecipitated with anti-M antibodies. The precipitates were analyzed by Western blot with anti-M (middle panel) and anti-E (bottom panel) antibodies, respectively.