Fig 8.

The HCMV ΔUS16 genome does not move to the nucleus in endothelial cells. (A) Separation of nuclear and cytoplasmic fractions. HMVECs were infected with equal numbers of RVTRwt or RVUS16stop virion particles. At 4 h p.i., infected cells were harvested, and nuclear and cytoplasmic extracts were prepared. Equal amounts of protein from the total cell lysate, the nuclear fraction, and the cytoplasmic fraction were then assayed for their pp65 content by immunoblotting. The purity was determined by immunoblotting for the nuclear ribonucleoprotein RNPA2 and tubulin. (B) Viral DNA in the nuclear fraction of HMVECs infected with RVTR or RVUS16stop. HMVECs were infected and fractionated as described for panel A. The amounts of viral DNA present in the nuclear fractions of infected cells were then quantified by real-time PCR using primers specific for the IE1 ORF and normalized to levels of the endogenous 18S gene. The data shown are the averages of three experiments plus SD. ***, P ≤ 0.001 compared to the amount of viral DNA measured in total extracts of cells infected with RVTRwt.