FIG 11 .
Expression of Grasp65-7A–GFP alters the envelope protein composition of extracellular virions. Transduced cells expressing either Grasp65wt-GFP or Grasp65-7A–GFP were plated and allowed to grow to confluence prior to infection with HCMV AD 169 at an MOI of 1. Extracellular virions were pelleted on day 4 p.i. by high-speed centrifugation through a 25% sorbitol cushion and analyzed by immunoblotting. (A) Expression of mutant Grasp65-7A alters virion composition. Panels show representative blots of envelope proteins (gB and gH), tegument proteins (pp150, pp65, and pp28), and minor capsid protein (pUL85) in extracellular particles collected from cells expressing Grasp65wt or Grasp65-7A. (B) Expression of Grasp65-7A leads to reduced incorporation of gH into extracellular particles compared to that in control samples. Plots show the densitometric ratio of the respective protein bands from Western blots of virions pelleted from infected cells expressing Grasp65-7A or Grasp65wt. Densitometry of the respective bands was accomplished by generating multiple exposures to create a linear range of densities and calculating density using Image Studio Lite version 5.2 software (Li-Cor). Ratios are expressed as mean values ± standard deviations. (C) The genome copy numbers were similar for pelleted virions prepared from supernatants of infected cell cultures expressing Grasp65wt (8.67log) and Grasp-7A (8.53log).