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. 2009 Apr;83(8):3891-903.
doi: 10.1128/JVI.01251-08. Epub 2009 Feb 4.

Human cytomegalovirus glycoprotein B is required for virus entry and cell-to-cell spread but not for virion attachment, assembly, or egress

Marisa K Isaacson  1 , Teresa Compton
Affiliations

Human cytomegalovirus glycoprotein B is required for virus entry and cell-to-cell spread but not for virion attachment, assembly, or egress

Marisa K Isaacson et al. J Virol. 2009 Apr.

Abstract

Glycoprotein B (gB) homologs are conserved throughout the family Herpesviridae and appear to serve essential, universal functions, as well as specific functions unique to a particular herpesvirus. Genetic analysis is a powerful tool to analyze protein function, and while it has been possible to generate virus mutants, complementation of essential virus knockouts has been problematic. Human cytomegalovirus (HCMV) gB (UL55) plays an essential role in the replication cycle of the virus. To define the function(s) of gB in HCMV infection, the BAC system was used to generate a recombinant virus in which the UL55 gene was replaced with galK (pAD/CreDeltaUL55). UL55 deletions in the viral genome have been made before, demonstrating that UL55 is an essential gene. However, without being able to successfully complement the genetic defect, a phenotypic analysis of the mutant virus was impossible. We generated fibroblasts expressing HCMV gB that complement pAD/CreDeltaUL55 and produce infectious virions lacking the UL55 gene but containing wild-type gB on the virion surface (DeltaUL55-gB HCMV). This is the first successful complementation of an HCMV mutant with a glycoprotein deleted. To characterize DeltaUL55 infection in the absence of gB, noncomplementing cells were infected with DeltaUL55-gB virus. All stages of gene expression were detected, and significant amounts of DNase-resistant viral DNA genomes, representing whole intact virions, were released into the infected cell supernatant. Gradient purification of these virions revealed they lacked gB but contained other viral structural proteins. The gB-null virions were able to attach to the cell surface similarly to wild-type gB-containing virions but were defective in virus entry and cell-to-cell spread. Glycoprotein B-null virions do, however, contain infectious DNA, as IE gene expression can be detected in fibroblasts following treatment of attached gB-null virions with a membrane fusion agent, polyethylene glycol. Taken together, our results indicate that gB is required for virus entry and cell-to-cell spread of the virus. However, HCMV gB is not absolutely required for virus attachment or assembly and egress from infected cells.

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Figures

FIG. 1.
FIG. 1.
Recombination replacing the HCMV UL55 gB gene with galK in pAD/Cre. (A) The galK recombination cassette was PCR amplified using primer pair A (Table 1), adding 50 bp of flanking gB sequence to either end of the galK gene. The ∼2.7-kb UL55 gB gene of pAD/Cre was replaced via recombination with the ∼1.2-kb galK gene, generating pAD/CreΔUL55. Recombinants were selected with chloramphenicol on minimal-medium plates containing galactose. (B) To revert pAD/CreΔUL55, a 2.7-kb wild-type UL55 recombination cassette was recombined into pAD/CreΔUL55, generating pAD/CreΔUL55R. Recombinants were selected with chloramphenicol on minimal-medium plates containing deoxygalactose. Correct recombination for all BACs was confirmed via PCR (C), EcoRI digestion (D), and Southern blotting (E and F). (C) PCR primer pair C (Table 1) corresponds to UL56 and to the 3′ end of UL55 that was not replaced during recombination generating 2.8- or 1.5-kb PCR fragments in pAD/Cre and pAD/CreΔUL55R or pAD/CreΔUL55, respectively. (D) The loss of an EcoRI site in UL55 following recombination leads to the disappearance of a 7.3-kb fragment (arrow), which is restored in pAD/CreΔUL55R. Southern blot probes to UL55 (E) or galK (F) detected these genes (arrows) in pAD/Cre and pAD/CreΔUL55R or pAD/CreΔUL55, respectively.
FIG. 2.
FIG. 2.
Generation and growth kinetics analysis of HCMV from electroporation of BAC plasmids into fibroblasts. NHDF were electroporated in the absence of a BAC plasmid (Mock) or with pAD/Cre, pAD/CreΔUL55, or pAD/CreΔUL55R. At 14 days postelectroporation, IE gene expression was detected via indirect immunofluorescence (A), and 1 week later, the cells were stained with crystal violet to detect plaque formation (B). NHDF were infected at an MOI of 5 (C) or 0.01 (D) with virus generated from electroporation of pAD/Cre (⧫) or pAD/CreΔUL55R (○). Virus-containing supernatant was removed and replaced with fresh medium supplemented with 2% FBS. At time zero and various times postinfection, supernatant was collected and titrated on NHDF. The values represent averages and standard deviations from three separate infections during one experiment.
FIG. 3.
FIG. 3.
gB expression in NHDF and complementation of pAD/CreΔUL55. (A) Following transduction of NHDF with recombinant (-GFP or -gB) retrovirus, cells were collected and lysates were analyzed by SDS-PAGE, along with HCMV AD169-infected cell lysate (HCMV Lysate). The gel was transferred to nitrocellulose and immunoblotted to detect gB using antibody 58-15. (B) Transduced cells were plated on coverslips, and gB was detected via indirect immunofluorescence using antibody 27-78. (C) NHDF-GFP or NHDF-gB were electroporated in the absence of BAC plasmid or with pAD/Cre or pAD/CreΔUL55. Approximately 3 weeks postelectroporation, the cells were stained with crystal violet to visualize plaque formation. (D) NHDF on coverslips were infected with supernatant from the BAC electroporations, and 24 h postinfection, IE gene expression was detected via indirect immunofluorescence. (E) Single-step growth curve comparing ΔUL55-gB virus (▪) replication on NHDF-gB to HCMV revertant virus (•) on NHDF-gB. The cells were infected at an MOI of 5.0. At various times postinfection, cells and supernatants were collected and titrated on NHDF-gB or -GFP. The values represent averages from duplicate infections in one experiment.
FIG. 4.
FIG. 4.
HCMV gB is not required for virion egress. (A) Infectious AD169 HCMV or ΔUL55-gB virus generated from propagation on NHDF-gB and purified by sorbitol cushioning was used to infect noncomplementing NHDF. Infected cell lysate was collected 6 days postinfection and analyzed by immunoblotting to detect IE, early (UL44), and late (pp28) gene expression during a single round of virus replication. (B) NHDF-GFP or NHDF-gB were mock infected or infected with HCMV AD169 or ΔUL55-gB at an MOI of 1 PFU/cell. Six days postinfection, the supernatant was collected and cell debris was removed by centrifugation, the supernatant was DNase I treated, and HCMV DNA was quantitated by real-time PCR to determine the number of DNase-resistant genomes that were released into the supernatant following infection. The error bars indicate standard deviations for three experiments. (C) Infected cell supernatants from ΔUL55-gB infection of NHDF-GFP or NHDF-gB were concentrated over a 20% sorbitol cushion, followed by gradient purification in a 20 to 70% sorbitol step gradient. Virus bands were extracted, and HCMV structural proteins, including gB, gH, pp150, pp71, and pp28, were detected by Western immunoblotting.
FIG. 5.
FIG. 5.
gB-null HCMV can attach to cells and contains infectious HCMV DNA. (A) gB from equivalent numbers of purified HCMV AD169 and ΔUL55 gB virions was analyzed by immunoblotting. (B) NHDF were treated with AD169, ΔUL55-gB, or ΔUL55 gB-null virus or virus pretreated with soluble heparin at 4°C for 1 h. The cells were washed three times with serum-free DMEM, the cell lysate was collected, DNA was extracted, and the viral genomes were quantitated by real-time PCR. The graph is representative of one of three similar experiments. The error bars represent standard deviations from triplicate samples in one experiment. (C) NHDF were infected with HCMV AD169, ΔUL55-gB, or ΔUL55 gB-null virus. The supernatant was removed, and the cells were washed three times with serum-free DMEM or serum-free DMEM containing decreasing concentrations of PEG (50, 25, and 12.5%). DMEM supplemented with 2% FBS was added back to the cells, and 24 h postinfection, IE gene expression was detected by indirect immunofluorescence.
FIG. 6.
FIG. 6.
gB is required for cell-to-cell spread. NHDF-GFP or NHDF-gB were infected with dilutions of HCMV ΔUL55-gB virus. The virus inoculum was removed, and the cells were overlaid with plaquing medium. Approximately 3 weeks postinfection, the cells were stained with crystal violet to visualize plaque formation.
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