FIG. 10.

Protein composition of HSV particles made in KOS-infected Vero cells or Sf9 cells infected with recombinant baculoviruses expressing HSV proteins. Cells were harvested at 12 h (Vero cells) or 64 h (Sf9 cells) postinfection, and cell extracts were loaded onto 20 to 60% sucrose gradients. Following centrifugation, B capsids were harvested. If there was no visible band on the gradient, then the region of the gradient where B capsids should band was harvested. A portion of each sample was removed for Western analysis, and the remainder of the sample was treated with 2 M GuHCl as described in Materials and Methods. Following guanidine treatment, particles were purified by banding on a 20 to 60% sucrose gradients. Purified particles before (lanes 6 to 10) and after (lanes 1 to 5) GuHCl treatment were separated on SDS–12.5% polyacrylamide gels and immunoblotted. The blots were probed with antisera NC1, NC2, and NC5 (A), antiserum MCA406 (B), UL25 polyclonal antibody (C), UL6 polyclonal antibody (D), and UL9 monoclonal antibody (E). Samples include particles isolated from Vero cells infected with KOS (lanes 1 and 6), particles isolated from Sf9 cells infected with a mixture of recombinant baculoviruses expressing the six HSV-1 capsid proteins alone (lanes 5 and 10) or along with recombinant baculoviruses expressing the UL6, UL9, and UL25 proteins (lanes 4 and 9), and particles isolated from Sf9 cells infected with a single recombinant baculovirus expressing UL25 (A to C and E, lanes 2 and 7), UL9 (A to E, lanes 3 and 8), or UL6 (D, lanes 2 and 7). (C to E) Lane E contains total cell lysate of Sf9 cells infected with recombinant baculovirus expressing UL25 (C), UL6 (D), and UL9 (E). The position of the capsid proteins VP5, VP19C, and VP23 (A), scaffold proteins VP21 and VP22a (B), UL25 protein (C), UL6 protein (D), and UL9 protein (E) are indicated.