Human cytomegalovirus UL97 kinase and nonkinase functions mediate viral cytoplasmic secondary envelopment

Abstract

Previous studies have revealed critical roles for the human cytomegalovirus (HCMV) UL97 kinase in viral nuclear maturation events. We have shown recently that UL97 affects the morphology of the viral cytoplasmic assembly compartment (AC) (M. Azzeh, A. Honigman, A. Taraboulos, A. Rouvinski, and D. G. Wolf, Virology 354:69-79, 2006). Here, we employed a comprehensive ultrastructural analysis to dissect the impact of UL97 on cytoplasmic steps of HCMV assembly. Using UL97 deletion (ΔUL97) and kinase-null (K355M) mutants, as well as the UL97 kinase inhibitor NGIC-I, we demonstrated that the loss of UL97 kinase activity resulted in a unique combination of cytoplasmic features: (i) the formation of pp65-rich aberrant cytoplasmic tegument aggregates, (ii) distorted intracytoplasmic membranes, which replaced the normal architecture of the AC, and (iv) a paucity of cytoplasmic tegumented capsids and dense bodies (DBs). We further showed that these abnormal assembly intermediates did not result from impaired nuclear capsid maturation and egress per se by using 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl) benzimidizole (BDCRB) to induce the artificial inhibition of nuclear maturation and the nucleocytoplasmic translocation of capsids. The specific abrogation of UL97 kinase activity under low-multiplicity-of-infection conditions resulted in the improved release of extracellular virus compared to that of ΔUL97, despite similar rates of viral DNA accumulation and similar effects on nuclear capsid maturation and egress. The only ultrastructural correlate of the growth difference was a higher number of cytoplasmic DBs, tegumented capsids, and clustered viral particles observed upon the specific abrogation of UL97 kinase activity compared to that of ΔUL97. These combined findings reveal a novel role for UL97 in HCMV cytoplasmic secondary envelopment steps, with a further distinction of kinase-mediated function in the formation of the virus-induced AC and a nonkinase function enhancing the efficacy of viral tegumentation and release.

FIG. 1.

Transmission electron microscopy of ΔUL97 (a and c)- and AD169 (b and d)-infected HFF at 96 hpi. The arrow in panel b points to viral particles clustered within cytoplasmic vacuoles. The arrowheads point to dense bodies. The arrows in panel d point to tegumented C capsids. E, electron-dense structures; M, ovoid membrane stacks; GA, Golgi apparatus; NU, nucleus. Cells in the presented micrographs were fixed by high-pressure freezing.

FIG. 2.

Immunoelectron microscopy detection of HCMV proteins in ΔUL97-infected HFF at 96 hpi. Ultrathin sections prepared following high-pressure freezing were subjected to immunogold labeling with anti-pp65 (a), pp28 (b and c), and gB (d) antibodies. The specific staining of the cytoplasmic electron-dense structures (a and b) and ovoid membrane stacks (c and d) is shown.

FIG. 3.

Transmission electron microscopy of AD169-infected HFF treated with BDCRB and NGIC-I at 96 hpi. (a and b) Nucleus; (c, e, and f) cytoplasm; (d) intercellular space; (b to d) BDCRB-treated infected HFF; (e and f) BDCRB- and NGIC-I-treated infected HFF. Arrows in panel a point to C capsids. Arrows in panels c and d point to dense bodies. E, electron-dense structures; M, ovoid membrane stacks. Cells in the presented micrographs were fixed by standard fixation.

FIG. 4.

Viral growth and DNA accumulation curves for ΔUL97, T2819, and AD169. HFF were infected at an MOI of 0.1 PFU/cell, and titers of the cell-associated virus in infected cells (a), the resulting progeny virus in the supernatant (b and e), or the accumulation of viral DNA in cell lysates (c and d) were determined at the indicated times. As indicated in panel b, growth curves also were obtained during treatment with NGIC-1 (broken lines). Panels d and e show the viral DNA accumulation (d) and growth curves (e) during BDCRB block release.

FIG. 5.

Transmission electron microscopy of UL97 kinase-abrogated HCMV-infected HFF at 96 hpi. (A) T2819-infected HFF showing nucleus (a) and cytoplasm (b and d). Arrows in panel c point to dense bodies. (B) AD169-infected cells treated with NGIC-I. Arrows in panel a point to dense bodies. E, electron-dense structures. M, ovoid membrane stacks. Cells in the presented micrographs were fixed by standard fixation.