A human cytomegalovirus gO-null mutant fails to incorporate gH/gL into the virion envelope and is unable to enter fibroblasts and epithelial and endothelial cells

Abstract

Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRDeltagO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRDeltagO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRDeltagO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRDeltagO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRDeltagO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of gH/gL complexes into the HCMV envelope. Moreover, these data suggest that it is gH/gL, and not gH/gL/gO, that is present in virions and is required for infection of fibroblasts and epithelial and endothelial cells. Our observations that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial/endothelial cells differ from models for other beta- and gammaherpesviruses that use one of two different gH/gL complexes to enter different cells.

FIG. 1.

Construction of a HCMV TR gO-null mutant. (A) BAC-TR containing the entire genome of HCMV clinical strain TR (except for where the BAC sequence replaces US2-US5) was previously described (34). The UL73 (gN), UL74 (gO), and UL75 (gH) genes are depicted. The N-terminal 1,141 nucleotides of UL74 (beginning at the gO start codon and extending to codon 380) were replaced by homologous recombination with a kanamycin resistance (Kan^r) cassette flanked by FRT sites producing BAC-TRΔgO Kan^r. The replacement did not affect the UL73 or UL75 promoters, coding sequences, or poly(A) sites. Following induction of Flp recombinase in bacteria, the Kan^r cassette was excised, leaving a single FRT site in place of the N-terminal UL74 sequences (BAC-TRΔgO). (B) Southern blot analyses of BAC clones. BAC-TR (wild type [wt]), BAC-TRΔgO Kan^r clones 2 and 3, and BAC-TRΔgO clones 2.1 and 3.1 were digested with EcoRI. This produces a 32.4-kb fragment for wild-type UL74 but a 4.5-kb fragment when the Kan^r cassette is inserted. Flp recombination produced clones 2.1 and 3.1 that lacked both Kan^r and UL74 sequences. The blots were probed with either gO sequences or Kan^r sequences. (C) Expression of gH, gL, gB, gO, and gN proteins in wild-type TR and TRΔgO-infected NHDF after 8 days infection by Western blotting.

FIG. 2.

Entry of TRΔgO into fibroblasts with and without PEG treatment. Multiwell dishes of NHDF were incubated with extracellular HCMV particles concentrated by pelleting from fibroblast culture supernatants. Wild-type (wt) TR and TRΔUL131 were used at 1 PFU/cell. A similar quantity of TRΔgO extracellular virions (based on quantifying genomes using qPCR) or 10 times (10×) that amount of TRΔgO were also incubated with cells. These viruses were centrifuged with cells at 800 × g for 1 h. Some of the wells were subsequently treated with 44% PEG for 30 s and then immediately washed (+ PEG). After 48 h, cells were fixed, permeabilized, and stained for HCMV immediate-early (IE) protein 86 (IE86). Numbers indicate the average number of IE86⁺ cells in three replicate wells.

FIG. 3.

Entry of TRΔgO into epithelial cells with and without PEG treatment. Multiwell dishes of ARPE-19 epithelial cells were incubated with the following extracellular HCMV particles: wild-type (wt) TR corresponding to 10 PFU (defined using fibroblasts)/ARPE-19 cell or a similar quantity of TRΔgO virus particles (based on quantifying genomes using qPCR). The virus and cells were centrifuged at 800 × g for 1 h at 15°C. Some of the wells were subsequently incubated with 44% PEG for 30 s and then immediately washed. After 48 h, cells were fixed, permeabilized, and stained for IE86. Numbers indicate the average number of IE86⁺ cells in three wells.

FIG. 4.

Entry of TRΔgO into endothelial cells with and without PEG treatment. Multiwell dishes of HPV-AEC endothelial cells were incubated with the following extracellular HCMV particles: wild-type (wt) TR or TRΔ131 using 1 PFU (defined using fibroblasts)/endothelial cell or a similar quantity (1×) of TRΔgO virus particles (based on quantifying genomes using qPCR) or 10 times that quantity of TRΔgO particles (10×). The cells and viruses were centrifuged at 800 × g for 1 h at 15°C. Some of the wells were subsequently incubated with 44% PEG for 30 s and then immediately washed. After 48 h, cells were stained for IE86. Numbers indicate the average number of IE86⁺ cells in three wells.

FIG. 5.

The cell-to-cell spread of TRΔgO in fibroblasts and epithelial cells. NHDF, MRC-5 fibroblasts, and ARPE-19 epithelial cells were infected with wild-type (wt) TR or TRΔgO using ∼100 to 200 PFU/well. Cells and virus were centrifuged at 800 × g for 1 h at 15°C, all wells were treated with 44% PEG for 30 s, and then the cells were washed and incubated in the presence of HCMV neutralizing antibodies for 10 or 20 days. Cells were then fixed and stained for IE86. The numbers of infected cells in 10 representative plaques were counted, and the average numbers are shown. Bar represents 100 μm.

FIG. 6.

Release of TRΔgO from fibroblasts into extracellular supernatants. (A) MRC-5 fibroblasts that had been transfected with BAC-TRΔgO or BAC-TR were trypsinized and plated with other MRC-5 cells. This allowed for the spread of TRΔgO, such that ∼10% of these cells showed a CPE and expressed IE86. After 10 days, culture supernatants from these TRΔgO-infected fibroblasts and supernatants from wild-type TR-infected fibroblasts (in which all the cells were infected) were subjected to quantitative PCR to enumerate HCMV genomes. (B) NHDF were infected with wild-type TR or TRΔgO using low-speed centrifugation, followed by PEG enhancement of entry, such that approximately 20% of the cells were infected by both viruses following 2 days of infection. Infected cell supernatants were collected 2, 4, 6, and 8 days postinfection and treated with DNase, and viral DNA was isolated and quantified by qPCR. d, day.

FIG. 7.

Electron microscopy of wild-type TR- and TRΔgO-infected fibroblasts. NHDF were infected with wild-type TR or TRΔgO by using low-speed centrifugation and PEG treatment. Under these conditions, ∼10 to 20% of TRΔgO-infected cells displayed IE86 expression by day 2, whereas all the cells were infected with wild-type TR. After 7 days, cells were fixed, stained, and analyzed by transmission electron microscopy. cp, cytoplasm; pm, plasma membrane; arrowheads, enveloped virus particles.

FIG. 8.

Analyses of HCMV proteins in TRΔgO extracellular virus particles. NHDF were infected with wild-type TR or TRΔgO using low-speed centrifugation and PEG to enhance entry. After 8 days, virus particles were prepared from cell culture supernatants by centrifugation through 20% sorbitol cushions. The quantities of the virus particles per sample were made similar by measuring viral genomes using qPCR. A cell lysate from wild-type TR-infected cells was loaded as a positive control for gO immunoblotting (TR wt cell). Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to Immobilon membranes, and Western blotted for gO, gH, gL, gN, UL130, gB, the major capsid protein (MCP), or tegument protein pp65. Lighter and darker exposures are shown for gH and gL.