Fig 4.

The replicative cycle of US16-deficient viruses in endothelial cells is blocked at a stage prior to the expression of IE genes. (A) Expression of representative IE, E, and L proteins in endothelial cells infected with TR or TRΔUS16 viruses. HMVECs were grown to subconfluence and then mock infected (M) or infected with the parental RVTRwt or RVUS16stop (MOI, 1 PFU/cell). At the indicated times p.i., total cell extracts were prepared and analyzed by immunoblotting with anti-IEA, anti-UL44, or anti-UL99 MAb as described in Materials and Methods. Actin immunodetected with a MAb served as an internal control. (B) The US16 gene product is required for IE gene expression in endothelial cells. HMVECs were infected with RVTRwt or RVUS16stop (MOI, 0.1 PFU/cell). Total RNA was isolated at the indicated time p.i. and reverse transcribed. Real-time RT-PCR was carried out with the appropriate IE1, IE2, and β-actin primers to quantify the expression levels of IE1 and IE2 mRNA. For each time point, IE1 and IE2 mRNA levels were normalized according to the expression of the actin gene. The results were then analyzed using a standard-curve model, and the levels of IE1 and IE2 mRNAs were normalized to the levels of endogenous β-actin mRNA. The value at each time point was normalized to the value observed with cells infected with RVTRwt for 12 h, which was set at 1. Data are shown as means and SD. ***, P ≤ 0.001 versus calibrator sample.