Impact of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole riboside and inhibitors of DNA, RNA, and protein synthesis on human cytomegalovirus genome maturation

Abstract

Herpesvirus genome maturation is a complex process in which concatemeric DNA molecules are translocated into capsids and cleaved at specific sequences to produce encapsidated-unit genomes. Bacteriophage studies further suggest that important ancillary processes, such as RNA transcription and DNA synthesis, concerned with repeat duplication, recombination, branch resolution, or damage repair may also be involved with the genome maturation process. To gain insight into the biochemical activities needed for herpesvirus genome maturation, 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) was used to allow the accumulation of human cytomegalovirus concatemeric DNA while the formation of new genomes was being blocked. Genome formation was restored upon BDCRB removal, and addition of various inhibitors during this time window permitted evaluation of their effects on genome maturation. Inhibitors of protein synthesis, RNA transcription, and the viral DNA polymerase only modestly reduced genome formation, demonstrating that these activities are not required for genome maturation. In contrast, drugs that inhibit both viral and host DNA polymerases potently blocked genome formation. Radioisotope incorporation in the presence of a viral DNA polymerase inhibitor further suggested that significant host-mediated DNA synthesis occurs throughout the viral genome. These results indicate a role for host DNA polymerases in genome maturation and are consistent with a need for terminal repeat duplication, debranching, or damage repair concomitant with DNA packaging or cleavage. Similarities to previously reported effects of BDCRB on guinea pig cytomegalovirus were also noted; however, BDCRB induced low-level formation of a supergenomic species called monomer+ DNA that is unique to human cytomegalovirus. Analysis of monomer+ DNA suggested a model for its formation in which BDCRB permits limited packaging of concatemeric DNA but induces skipping of cleavage sites.

FIG. 1.

Impact of metabolic inhibitors on HCMV genome maturation. (A) Cells were infected with HCMV at an MOI of 3 and treated with a variety of drug regimens and then harvested on the days p.i. indicated above each lane. Intracellular DNA species were separated by FIGE, transferred to nylon membranes, and hybridized with probe pON227 to specifically detect HCMV DNA forms. Control cultures to demonstrate the efficacy of BDCRB treatment were incubated in the absence of drug (Φ; harvested on days 3 and 6 p.i.), or in the presence of 10 μM BDCRB (harvested on day 3 p.i.). We conducted reversal experiments in which replicate cultures were first treated from days 0 to 3 p.i. with 10 μM BDCRB and then washed to remove the BDCRB and incubated with either medium alone (Φ) or medium containing 10 μM BDCRB, 50 μg/ml cycloheximide (CH), 5 μg/ml actinomycin D (ACTD), 200 μg/ml PFA, or 10 μg/ml aphidicolin (APH) until harvest on days 4, 5, or 6 p.i. (B) Additional inhibitors were tested using essentially the same protocol as described above. Controls were treated with no drug or with 10 μM BDCRB and harvested on day 3 p.i. Reversals were treated with 10 μM BDCRB for days 1 to 3 p.i. and then washed and incubated with medium alone (Φ) or medium containing 10 μM BDCRB, 10 μg/ml aphidicolin, 1.5 μg/ml HPMPA, 10 μg/ml PMEG, 50 μg/ml PMEA, 200 μg/ml PFA, 50 μg/ml GCV, 50 μg/ml CH, 5 μg/ml actinomycin D, 2 μg/ml α-amanitin (α-AMT), or 17 μg/ml DRB, until harvest on day 5. (C) Replicate infected cultures of those shown in panel B were treated with the same doses of the indicated drugs from the time of infection until harvesting on day 3 p.i. Arrows indicate the positions of concatemeric (concat.), monomer+ (M⁺), and 230-kb DNA forms.

FIG. 2.

Nucleotide incorporation into HCMV DNA in the presence of DNA polymerase inhibitors. Cells were mock infected or infected with HCMV at an MOI of 2 and incubated for 72 h. From 72 to 80 h p.i., the cultures were incubated in the presence of no drug (Φ), 20 μg/ml HPMPA, or 20 μg/ml PMEG. From 80 to 96 h p.i., the cultures were incubated with the same drugs in labeling medium containing [³²P]orthophosphate. DNA was prepared at 96 h p.i. and separated by FIGE. Concatemeric DNA was excised, digested with EcoRI, separated by agarose gel electrophoresis, and visualized by autoradiography of the dried gel. Uninfected confluent cell cultures (U) were similarly analyzed in the absence of drugs. The positions of molecular size markers are indicated to the left.

FIG. 3.

HCMV genome structure. Long and short arms are comprised of unique long (U_L) and unique short (U_S) regions flanked by inverted copies of the b sequence (gray boxes) and c sequence (open boxes). One to several reiterated copies of the a sequence are found at left, or long-arm ends (indicated by a_n) and in inverted orientation (indicated by a′_m) at a region designated the L/S junction where the long and short arms join. One or no copies of the a sequence are found at the right or short-arm end. Details of the termini are enlarged below. For simplicity, one a sequence (black box) is shown at the long-arm end, resulting in a 4.0-kb EcoRI W fragment. At the short-arm end, genomes designated type I lack an a sequence, resulting in a 6.5-kb HindIII Q₁ fragment, whereas genomes designated type II have one a sequence copy, resulting in a 7.1-kb HindIII Q₂ fragment (58). Thick bars indicate sequences used as hybridization probes that are cloned in the indicated plasmids.

FIG. 4.

Effects of BDCRB on the termini of HCMV DNA forms. Concatemer, monomer+, and 230-kb DNAs were excised from FIGE gels containing replicate samples of those used in the experiment shown in Fig. 1A. Each DNA sample was digested with EcoRI and HindIII, separated by agarose gel electrophoresis, transferred to nylon membranes, and sequentially hybridized with probes derived from plasmid pON2333 to detect short-arm-terminal fragments, plasmid pON227 to detect long-arm-terminal fragments, or plasmid pON226 to detect a sequence-containing fragments (for probe details, see Fig. 3). (A) Concatemeric DNAs; (B) 230-kb DNAs formed after removal of BDCRB; (C) 230-kb DNAs formed after 6 days in the continuous presence of BDCRB; (D) monomer+ DNA (M⁺) formed on day 4 p.i., 1 day after the removal of BDCRB. Numbers above lanes indicate days p.i. that samples were harvested; V, extracellular virion DNA produced in the absence of BDCRB; Φ, DNAs prepared after 3 days of culture in the absence of BDCRB; V6 (B), extracellular virion DNA prepared from culture supernatants of cells analyzed in the adjacent lane (i.e., cultured with BDCRB from days 1 to 3 and without BDCRB from days 3 to 6). The positions of molecular size markers are indicated to the right of each figure.

FIG. 5.

Models for genome maturation in the absence or presence of BDCRB. HCMV concatemers are illustrated, with short arms represented by open boxes and long arms represented by lines. A normal genome consists of one long arm and one short arm. (A) Normal cleavage in the absence of BDCRB. Starting with a short-arm concatemer end, packaging proceeds until one full short arm and one full long arm have entered the capsid. Cleavage then occurs at the left end of the long arm, releasing a normal genome within the capsid. (B) Premature cleavage in the presence of BDCRB. Packaging occurs as described for panel A, but cleavage occurs prior to the normal cleavage site, releasing a left-end-truncated genome within the capsid. (C) Monomer+ cleavage in the presence of BDCRB. Packaging occurs as described for panel A but continues past the normal cleavage site until an additional sort arm is packaged. Cleavage then occurs at the internal cleavage site located at the left end of the short arm, releasing a short-long-short monomer+ molecule within the capsid.

FIG. 6.

Effects of inhibitors on the termini of HCMV DNA forms. Replicates of samples shown in Fig. 1A that were treated with inhibitors after BDCRB removal were analyzed as described for Fig. 4. (A) Two hundred thirty-kilobase DNA formed in the presence of cycloheximide (CH) or PFA; (B) monomer+ (M⁺) DNA formed in the presence of cycloheximide (CH) or PFA; (C) concatemeric DNA formed in the presence of no drug (Φ), cycloheximide, actinomycin D (ACTD), PFA, or aphidicolin (APH). V, extracellular virion DNA produced in the absence of BDCRB (used as standards). The positions of molecular size markers are indicated to the right of each figure.