The antiviral effects of P-PCIs and ACG, a conventional antiviral drug, are additive. Vero cells were infected with wild-type HSV-1 (wild type [WT]) or an ACG-resistant mutant (dlPstTK⁻). Infected cells were treated with the concentrations of ACG indicated on the x axes, in the presence of the concentrations of Rosco (top graphs) (□, 0 μM; ▪, 10 μM; ▵, 30 μM) or Purv (bottom graphs) (□, 0 μM; ▪, 60 μM; ▵, 15 μM). Viral titers at 20 h postinfection are plotted against concentrations of ACG.

Proteins that bind to Purv in mock- or HSV-infected Vero or HEL cells. Protein extracts of mock- (M) or HSV-infected (H) Vero and HEL cells were incubated with Purv (P) or Me-Purv (Me) covalently linked to agarose beads. Purv-binding proteins were eluted, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and silver stained or immunoblotted with specific antibodies (see text for descriptions of antibodies used). The antibodies specific for cdk4, -6, and -9 did not recognize simian cdk4, -6, or -9 with high affinity and, thus, could not be used in Vero cells. (A) Vero cells were infected with wild-type HSV-1 and treated with the concentrations of drug indicated on the x axis. Viral titers at 20 h postinfection are plotted against concentrations of Me-Purv (▴), Purv (▵), or Rosco (□). (B) Silver-stained Purv-binding proteins in extracts of mock- (M) or HSV (H)-infected Vero and HEL cells. Molecular masses are indicated on the left in kilodaltons, and the ranges of molecular masses of the cdks targeted by Purv in vitro and their corresponding cyclins are indicated by the brackets on the right. No Vero cell protein bound to Me-Purv was detected (data not shown). Proteins in HEL cell extracts that bound to Purv, and not to Me-Purv, are indicated by white arrowheads on the right side of the gel. (C) Western blots of Purv-binding proteins in mock- (M)or HSV (H)-infected Vero cell extracts. Purv-binding proteins (P) or total proteins in cell extracts (Extract) were resolved by SDS-PAGE, transferred, and blotted with antibodies specific for HSV-1 proteins. Molecular masses are indicated on the left in kilodaltons. (D) Western blots of Purv- (P) or Me-Purv (Me)-binding proteins in mock- (M) or HSV (H)-infected Vero cell extracts. Purv- or Me-Purv-binding proteins or total proteins in cell extracts (Extract) were resolved by SDS-PAGE, transferred, and blotted with antibodies specific for cdk7, -5, -2, and -1 or erk2 as indicated on the right of each blot. Only the relevant sections of the blots are shown. Exposure times differed for each antibody. (E) Western blots of Purv- (P) or Me-Purv (Me)-binding proteins in mock- (M) or HSV (H)-infected HEL cell extracts. Purv- or Me-Purv-binding proteins or total proteins in cell extracts were resolved by SDS-PAGE, transferred, and blotted with antibodies specific for cdk7, -5, and -2, erk1 and -2, and cdk9, -6, -4, or -1 as indicated on the right side of each blot.

Rosco and Purv inhibit wild-type and drug-resistant strains of HSV-1 and -2, but not vaccinia virus or LCMV. (A) Vero cells infected with HSV-1, vaccinia virus, or LCMV were treated with increasing concentrations of Rosco or Purv. Inhibition of viral replication at 20 h postinfection, expressed as the log, is plotted against concentration of drug. ▵, vaccinia virus; ▴, LCMV; ▪, HSV-1. (B) Inhibition of cdk2/cyclinA phosphorylation of histone H1 by Rosco was evaluated in the presence of increasing concentrations of ATP, as indicated on the left of each gel. Kinase reactions were performed in vitro, phosphorylated histone H1 was then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gels were dried and exposed on a PhosphorImager. (C) Vero cells were infected with wild-type HSV-1 (KOS), wild-type HSV-2 (strain 186 or 333), or drug-resistant HSV-1 mutants (ACG^r5, dlPstTK⁻, and PAA^r5). Infected cells were treated with the indicated concentrations (on x axes) of PAA, ACG, Rosco, or Purv. Viral titers at 20 h postinfection were plotted against drug concentration. ▪, HSV-1 KOS; □, HSV-1 ACG^r5; ⋄, HSV-1 dlPstTK⁻; ▵, HSV-1 PAA^r5; ×, HSV-2 strain 333; ♦, HSV-2 strain 186.

Rosco inhibits replication of wild-type and drug-resistant strains of HIV-1 and enhances survival of cells in infected cultures. (A) CEMx174 cells were infected with HIV-1 and treated with Rosco. Levels of p24 at 2 and 4 days postinfection (dpi), expressed as picograms of p24 per 10⁶ viable cells, are plotted against drug concentration. The level of inoculum is indicated by the dashed line. Symbols for Rosco concentrations: □, 0 μM; ▪, 5 μM; ▵, 10 μM; ▴, 15 μM. (B) Mock- or HIV-1-infected cells were treated with Rosco and counted at 2 and 4 dpi. The fold change in the number of viable cells is plotted against days postinfection for each concentration of drug. Symbols for Rosco concentration are as described for panel A. (C) CEMx174 cells were infected with PI- or RTI-resistant HIV-1 strain L10R/M46I/L63P/V82T/I84V (13) or RT_MDR/MT-2 (35), respectively. Infected cells were treated with Rosco and levels of p24 per 10⁶ viable cells on day 4 pi are plotted against Rosco concentrations. ♦, wild type; □, PI^r; ▵, RTI^r. The levels of p24 produced by wild-type HIV-1 on day 4 pi in the presence of Rosco (see Fig. 4A) are included for comparison.

cdk7, but not cdk8 or erks, may mediate the anti-HSV effects of Rosco. (A) Vero cells were incubated in a low serum concentration and PD98059 for 4 days. Cells were infected with HSV-1, treated with PD98059 for 20 h, and harvested, and viral titers were determined (top panel). Another set of cells was stimulated with fresh serum and treated with PD98059, and the percentage of cell replication was determined (bottom panel). (B) RNAP II hypo- and hyperphosphorylated in vitro by cdk7 (▴)or cdk8 (□) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were dried and exposed on a PhosphorImager. Only the relevant sections of gels are shown (left panel). Phosphorylation levels were quantitated using ImageQuant software and are plotted against Rosco concentration (right panel). The IC₅₀ is indicated by the dashed line. The double lines on the y axes indicate a discontinuity on the scale. (C) RNAPII was phosphorylated by cdk7 in the presence of increasing concentrations of ATP and Rosco: ▪, 0 μM; ▵, 1 μM; ▴, 2 μM; □, 4 μM. A Lineweaver-Burk plot is presented; inhibition was shown to be competitive (left panel). The slopes of the regression functions presented in this graph were plotted against the concentration of Rosco to calculate the K_i (right panel).