FIG. 2.

Vps4E228Q (Vps4EQ) or Vps24-Red does not affect the early steps of HSV-1 replication. (A to D) 293T cells were transfected with plasmids expressing the dominant negative forms of Vps4 (B) or Vps24 (D) cellular protein or their respective empty vector, pBJ5 (A) or pDsRed2-N1 (pReD) (C). Twelve hours after transfection, the cells were infected with the R8102 recombinant HSV-1, carrying the lacZ gene under the control of the ICP-27 immediate promoter (3 PFU/cell) (5). Six hours later, the infected cells were stained with X-Gal. The panels illustrate light microscope images (magnification, ×1.5). (E) Quantification of HSV-1 genomic copies by real-time PCR assay. 293T cells were transfected with the constructs expressing either Vps4E228Q or Vps24-Red or the corresponding empty vectors. Twelve hours posttransfection, the cells were infected with HSV-1 (10 PFU/cell). Twenty-four hours later, the amount of viral DNA was evaluated by real-time PCR assay. Results are expressed as numbers of viral DNA copies per cell. (F and G) Total RNA was extracted from 293T cells transfected with the indicated plasmids. The RT-PCR assay was performed with primers specific for VP16 and gD (F) or the β-actin genes (G). Lanes: 1, 293T cells transfected with pBJ5 and infected with HSV-1; 2, 293T cells transfected with pBJ5-Vps4E228Q and infected with HSV-1; 3, 293T cells transfected with pDsRed-Vps24 and infected with HSV-1; C+, Vero cells infected with HSV-1; C−, uninfected 293T cells; B, no-template control; MW, molecular weight markers. (H) 293T cells transfected with the indicated plasmids were infected with HSV-1 strain F, labeled with [³⁵S]methionine, and harvested 24 h after infection. Radiolabeled proteins were detected by autoradiography following SDS-PAGE. u.i., uninfected 293T cells.