Human cytomegalovirus tegument protein pUL71 is required for efficient virion egress

Abstract

The human cytomegalovirus virion is composed of a DNA genome packaged in an icosahedral capsid, surrounded by a tegument of protein and RNA, all enclosed within a glycoprotein-studded envelope. Achieving this intricate virion architecture requires a coordinated process of assembly and egress. We show here that pUL71, a component of the virion tegument with a previously uncharacterized function, is required for the virus-induced reorganization of host cell membranes, which is necessary for efficient viral assembly and egress. A mutant that did not express pUL71 was able to efficiently accumulate viral genomes and proteins that were tested but was defective for the production and release of infectious virions. The protein localized to vesicular structures at the periphery of the viral assembly compartment, and during infection with a pUL71-deficient virus, these structures were grossly enlarged and aberrantly contained a cellular marker of late endosomes/lysosomes. Mutant virus preparations exhibited less infectivity per unit genome than wild-type virus preparations, due to aggregation of virus particles and their association with membrane fragments. Finally, mutant virus particles accumulated within the cytoplasm of infected cells and were localized to the periphery of large structures with properties of lysosomes, whose formation was kinetically favored in mutant-virus-infected cells. Together, these observations point to a role for pUL71 in the establishment and/or maintenance of a functional viral assembly compartment that is required for normal virion trafficking and egress from infected cells.

FIG 1

BADinUL71STOP generates fewer infectious progeny than BADwt. (A) Schematic of the BADinUL71STOP genome. An 11-base-pair translation stop cassette was inserted after nucleotide 36 of the UL71 ORF. (B) BADinUL71STOP produces >100-fold-fewer infectious progeny than BADwt. Infected fibroblasts and supernatants were harvested at the indicated times after infection at a multiplicity of 2 PFU/cell, and virus titers were determined by 50% tissue culture infective dose (TCID₅₀) assay. Inp, input virus. (C) Accumulation of RNA from neighboring genes is unaffected by the UL71STOP mutation. Fibroblasts were infected at a multiplicity of 2 PFU/cell with BADwt or BADinUL71STOP and harvested 72 h later. Total RNA was isolated. UL70 and UL72 message levels were quantified by reverse transcription-quantitative PCR (RT-qPCR) and normalized to actin RNA. Error bars represent standard errors of means calculated from technical repeats.

FIG 2

BADinUL71STOP produces a higher proportion of cell-associated progeny than BADwt but accumulates normal levels of viral DNA and proteins. Infections were performed at a multiplicity of 2 PFU/cell. (A) BADinUL71STOP produces >100-fold-fewer extracellular infectious viral progeny and ~10-fold-fewer cell-associated infectious viral progeny than BADwt. Cultures were harvested on the indicated days postinfection (dpi), and viral titers were determined by TCID₅₀ assay. Inp, input virus. Results are representative of an experiment conducted in duplicate with two separate isolates of BADinUL71STOP mutant virus. Error bars estimate standard deviations calculated from technical repeats. (B) Ratio of cell-associated to cell-free virus for mutant and wild-type viruses. The primary data displayed in panel A are plotted as ratios. Error bars represent standard errors of means calculated from technical repeats. (C) BADinUL71STOP-infected cells accumulate viral DNA normally. Fibroblasts were harvested at the indicated hour postinfection (hpi) with mutant or wild-type virus, and total cellular DNA was isolated. Viral genomes were quantified by qPCR with UL123-specific primers and normalized to cellular actin. Results are representative of an experiment conducted in duplicate with two separate isolates of BADinUL71STOP. Error bars represent standard errors of means calculated from technical repeats. (D) BADinUL71STOP-infected cells accumulate representative viral proteins from all kinetic classes normally. Cells were harvested at the indicated hpi, and whole-cell lysates were prepared. Proteins were analyzed by Western blotting using antibodies for IE1, pUL44, and pUL99. Tubulin was assayed as a loading control. Results are representative of an experiment conducted in duplicate with two separate isolates of BADinUL71STOP.

FIG 3

BADinUL71STOP virus spreads in a direct cell-to-cell manner. Infections with BADwt (A) or BADinUL71STOP (B) were performed at a multiplicity of 0.01 PFU/cell in the presence of nonsupplemented growth medium (untreated) or growth medium supplemented with 3% (vol/vol) CytoGam (+CytoGam). Nuclei were stained with DAPI (red), and infected cells were detected by probing for IE1 expression (green).

FIG 4

pUL71GFP partially colocalizes with pUL99 (pp28) and pUL55 (gB) in the viral assembly compartment (vAC). pUL71GFP (green) was tested for colocalization with pUL99 (red) (A) and pUL55 (red) (B). Fibroblasts were infected at a multiplicity of 1 PFU/cell and processed for immunofluorescence at the indicated time postinfection (hpi). DAPI-stained nuclei are blue in merged images. Results are representative of an experiment conducted with two separate isolates of BADinUL71GFP. Bars, 10 µm.

FIG 5

BADinUL71STOP-infected cells display altered vAC morphology. Fibroblasts were infected at a multiplicity of 1 PFU/cell with either BADwt or BADinUL71STOP virus and processed for immunofluorescence 96 h later. DAPI-stained nuclei are blue in merged images. (A) Compared to BADwt-infected cells (top panels), BADinUL71STOP-infected cells accumulate pUL99- and pUL55-containing vesicular structures that are enlarged (middle panels). A minor population of BADinUL71STOP-infected cells (<10%) display grossly enlarged vesicular structures that are ≥10 µm in diameter (bottom panels). Results are representative of experiments conducted in duplicate with two isolates of BADinUL71STOP. (B and C) Compared to BADwt-infected cells, the lysosomal marker LAMP1 (red) is aberrantly sorted into vesicular structures at the periphery of the vAC in BADinUL71STOP-infected cells. Arrows mark colocalization of pUL99 (B) (green) or pUL55 (C) (green) and LAMP1. LAMP1 was detected with either a rabbit polyclonal antibody (B) or a mouse monoclonal antibody (C). Results are representative of experiments conducted in triplicate with two separate isolates of BADinUL71STOP. (D) BADinUL71STOP-infected cells have enlarged, acidified structures in the perinuclear cytoplasmic region. At 96 hpi, live cells were stained with LysoTracker Green. Bars, 10 µm.

FIG 6

Purified BADinUL71STOP particles are aggregated and associated with membranes. (A) BADinUL71STOP particles sediment differently than wild-type virions. Virus particles from the supernatants of infected cultures or from cells plus supernatants were concentrated and then resolved by centrifugation through a glycerol tartrate gradient. (B) BADinUL71STOP genomes and infectivity sediment differently than those of wild type. Infectivity was measured for each isolated band species by TCID₅₀ assay, and viral genomes were quantified by qPCR using UL123-specific primers. (C) The BADinUL71STOP virion band contains elevated levels of representative virion proteins. Proteins in the indicated factions were assayed by Western blotting for capsid (pUL85), tegument (pUL83 and pUL99), and envelope (pUL55) proteins. Loading was normalized to viral genomes. (D) Electron micrographs of particles in gradient fractions. BADwt virions, BADwt dense bodies, and BADinUL71STOP virion aggregates were isolated from indicated gradient fractions and visualized by transmission electron microscopy. The areas marked by green boxes in the top panels are shown at higher magnification in the bottom panels.

FIG 7

At 96 hpi, BADinUL71STOP-infected cells contain large ICIs but few cytoplasmic virus particles. Cells were infected at a multiplicity of 2 with BADwt (A and B) or BADinUL71STOP (C and D). Arrows indicate cytoplasmic virus particles. Nu, nucleus; ICI, intracytoplasmic inclusion.

FIG 8

At 144 hpi, BADinUL71STOP-infected cells contain more cytoplasmic virus particles than BADwt-infected cells, many of which are associated with large ICIs. Cells were infected at a multiplicity of 2 with either BADwt (A and B) or BADinUL71STOP (C and D). Arrows indicate cytoplasmic virus particles. Nu, nucleus; ICI, intracytoplasmic inclusion.