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. 2001 Feb 13;98(4):1895-900.
doi: 10.1073/pnas.98.4.1895.

Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation

D G Wolf  1 , C T Courcelle, M N Prichard, E S Mocarski
Affiliations

Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation

D G Wolf et al. Proc Natl Acad Sci U S A. .

Abstract

The human cytomegalovirus UL97 kinase, an important target of antiviral therapy, has an impact on at least two distinct phases of viral replication. Compared with wild-type virus, the UL97 deletion mutant exhibits an early replication defect that reduces DNA accumulation by 4- to 6-fold, as well as a late capsid maturation defect responsible for most of the observed 100- to 1000-fold reduction in replication. Block-release experiments with the antiviral 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole revealed an important role for UL97 kinase in capsid assembly. Although cleavage of concatemeric DNA intermediates to unit-length genomes remained unaffected, progeny mutant virus maturation was delayed, with accumulation of progeny at significantly reduced levels compared with wild type after release of this block. Transmission electron microscopy confirmed the aberrant accumulation of empty A-like capsids containing neither viral DNA nor an internal scaffold structure, consistent with a failure to stably package DNA in mutant virus-infected cells. The function of UL97 in DNA synthesis as well as capsid assembly suggests that protein phosphorylation mediated by this herpesvirus-conserved kinase increases the efficiency of these two distinct phases of virus replication.

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Figures

Figure 1
Figure 1
Accumulation of viral DNA in cells infected with UL97 mutant RCΔ97.08 (Δ97) and parental strain AD169. (A) DNA accumulation curves of DNA copies per well from QC-PCR (0, 24, 48 hpi) and blot hybridization (72, 96, 168 hpi) after infection of HFs with an moi of 1. ○, AD169; ■, RCΔ97.08. (B) DNA blot hybridization to detect levels of viral DNA after a 96-h PFA-induced DNA synthesis block (D) and release (R) for a subsequent 24, 48, 72, or 96 h. Total cellular DNA was digested with BamHI and blot hybridized to detect a 2.3-kb viral BamHI fragment.
Figure 2
Figure 2
Immunoblot analysis of proteins expressed during RCΔ97.19 (Δ97) or AD169 infection of HFs (moi of 1). The four panels (from Top to Bottom) show detection of IE1 and IE2 antigens with the use of RαIE1-IE2, ppUL44 antigen with the use of monoclonal antibody 1202, pp65 (ppUL83), antigen with the use of monoclonal antibody 1205B, and cellular actin loading control with the use of goat polyclonal antiserum. Size markers (in kDa) are indicated on the right. M, mock-infected.
Figure 3
Figure 3
DNA blot hybridization showing accumulation and cleavage of viral DNA in cells infected with UL97 mutant RCΔ97.08 (Δ97) and parental strain AD169 (moi of 0.1) after BDCRB drug block (D) and release (R) at the indicated times. Total cellular DNA was digested with EcoRI and subjected to blot analysis to detect L-S junction (upper arrow) and L terminal (lower arrow) fragments, with the use of radiolabeled probe pON227.
Figure 4
Figure 4
Growth curves for RCΔ97.08 and AD169. (A) Growth curves during BDCRB block-release. HFs were infected (moi of 0.1), and supernatant progeny virus were harvested at the indicated times after infection or after drug release (R) and titered on HF monolayers. (B) Growth curves for RCΔ97.08 and AD169. HFs were infected (moi of 1), and supernatant progeny virus was harvested at the indicated times after infection and titered on HF monolayers. ■, RCΔ97.08; ○, AD169.
Figure 5
Figure 5
Analysis of total and encapsidated viral DNA in AD169-, RCΔ97.08-, and RCΔ97.19-infected cells. (A) DNA blot hybridization showing accumulation of total and DNase-resistant viral DNA in cells infected with mutant RCΔ97.08 (Δ97) and parental strain (moi of 1). Total infected cell DNA was isolated before or after DNase digestion (+DNase) at the indicated times (hpi). Viral DNA digestion and hybridization were carried out to identify junction and terminal EcoRI fragments (arrows) as indicated for Fig. 3. M, mock-infected. (B) Quantitative analysis of total and DNase-resistant (+DNase) viral DNA in AD169-, RCΔ97.08-, and RCΔ97.19- (Δ97) infected cells at the times indicated. Quantitation was carried out with the use of blot hybridization for all samples, except for the DNase-resistant mutant DNA, and QC-PCR was used for all samples, yielding values similar to blots at higher DNA levels. The mean DNA copy number values, derived from three independent experiments, are displayed. Empty bars, total AD169 DNA; stippled bars, DNase-resistant AD169 DNA; black bars, total RCΔ97 strains DNA; hatched bars, DNase-resistant RCΔ97 strains DNA.
Figure 6
Figure 6
Transmission electron micrographs of virus-infected cells at 72 hpi. (A and B) HFs infected with mutant RCΔ97.08. (C and D) HFs infected with strain AD169. (E) U373-vec cells infected with mutant RCΔ97.08. (F) UL97–9 cells infected with mutant RCΔ97.08. Arrows in B and E point to empty A capsids. Arrows in D and F point to DNA-containing capsids. [Magnification: A and C, ×3,800; B, D–F, ×35,000. Size bars: A and C, 5 μm; B, D–F, 1 μm.]
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