Cyclopropavir inhibits the normal function of the human cytomegalovirus UL97 kinase

Abstract

Cyclopropavir (CPV) is active against human cytomegalovirus (CMV), as well as both variants of human herpesvirus 6 and human herpesvirus 8. The mechanism of action of CPV against CMV is similar to that of ganciclovir (GCV) in that it is phosphorylated initially by the CMV UL97 kinase, resulting in inhibition of viral DNA synthesis. Resistance to CPV maps to the UL97 kinase but is associated primarily with H520Q mutations and thus retains good antiviral activity against most GCV-resistant isolates. An examination of CMV-infected cultures treated with CPV revealed unusual cell morphology typically associated with the absence of UL97 kinase activity. A surrogate assay for UL97 kinase activity confirmed that CPV inhibited the activity of this enzyme and that its action was similar to the inhibition seen with maribavir (MBV) in this assay. Combination studies using real-time PCR indicated that, like MBV, CPV also antagonized the efficacy of GCV and were consistent with the observed inhibition of the UL97 kinase. Deep sequencing of CPV-resistant laboratory isolates identified a frameshift mutation in UL27, presumably to compensate for a loss of UL97 enzymatic activity. We conclude that the mechanism of action of CPV against CMV is complex and involves both the inhibition of DNA synthesis and the inhibition of the normal activity of the UL97 kinase.

Fig. 1.

Inhibition of UL97 kinase activity by CPV. Transient expression of a pp65-GFP fusion in COS7 cells (green staining in the middle column) induces the formation of nuclear aggresomes, and coexpression of the CMV UL97 kinase (red staining in the first column) can inhibit their formation in a kinase-dependent manner (compare row A with the catalytically inactive UL97 mutant in row B). Pharmacologic inhibition of the UL97 kinase by 15 μM MBV also inhibits the ability of the kinase to prevent aggresome formation (row C) and leads to recruitment of the kinase to aggresomes (merged image in the last column with blue 4′,6-diamidino-2-phenylindole [DAPI] staining). Treatment with 15 μM CPV also inhibits the UL97 kinase and induces its recruitment into aggresomes (row D), whereas neither CDV nor GCV exhibits this activity at a concentration of 15 μM (rows E and F, respectively). UL97 with the V3656G mutation shows an inability to prevent aggresome formation (row G).

Fig. 2.

CPV increases the proportion of cells with aggresomes. COS7 cells coexpressing pp65-GFP and the UL97 kinase were treated with the compounds shown at a concentration of 15 μM. The number of cotransfected cells containing aggresomes was evaluated in three separate experiments, and the average proportion of cells is shown with the standard deviation of the data.

Fig. 3.

Combined efficacy against CMV as shown by real-time PCR. Infected HFF cells were treated with the combinations of compound concentrations shown, and genome copy numbers were determined by real-time PCR. (A) The matrix represents the combined GCV and MBV concentrations indicated on the axes, with the resulting reductions in the number of GE represented by the blue and red regions indicating >0.5 and >1 log₁₀ GE, respectively. (B) A plot of antagonism shows less-than-expected inhibition of viral replication (log₁₀ GE) at the combinations of compound concentrations indicated on the two horizontal axes; these data confirm that inhibition of the UL97 kinase by MBV antagonizes the antiviral activity of GCV. (C) Combinations of CPV and GCV were also evaluated by the same approach as in panel A, with genome copy number reductions of >1 and >2 log₁₀ GE represented by blue and red, respectively. (D) CPV also appeared to antagonize the antiviral activity of GCV when evaluated by the method used for panel B. Since the UL97 kinase is also required for the antiviral activity of CPV, the effect of MBV on its antiviral activity was also evaluated. (E) Reduction of genome copy numbers with CPV and MBV combinations was observed, with blue and red regions representing >1 and >2 log₁₀ GE, respectively. (F) As expected, MBV also antagonized the antiviral activity of CPV with an intensity greater than that observed for the other two combinations. The small areas depicted in gray in panels A, C, and E represent the few instances where the genome copy number is slightly greater than that of the virus control; this is due to the normal variability of the assay.

Fig. 4.

Inhibition of CMV replication by CPV and GCV. Dose-response curves for both CPV and GCV were obtained in combination studies. The curves shown represent the average data from three separate experiments and the standard deviations.