A leucine zipper motif of a tegument protein triggers final envelopment of human cytomegalovirus

Abstract

The product of the human cytomegalovirus (HCMV) UL71 gene is conserved throughout the herpesvirus family. During HCMV infection, protein pUL71 is required for efficient virion egress and is involved in the final steps of secondary envelopment leading to infectious viral particles. We found strong indications for oligomerization of pUL71 under native conditions when recombinant pUL71 was negatively stained and analyzed by electron microscopy. Oligomerization of pUL71 during infection was further verified by native and reducing polyacrylamide gel electrophoresis (PAGE). By in silico analyses of the pUL71 sequence, we noticed a basic leucine zipper (bZIP)-like domain, which might serve as an oligomerization domain. We demonstrated the requirement of the bZIP-like domain for pUL71 oligomerization by coimmunoprecipitation and bimolecular fluorescence complementation using a panel of pUL71 mutants. These studies revealed that the mutation of two leucine residues is sufficient to abrogate oligomerization but that intracellular localization of pUL71 was unaffected. To investigate the relevance of the bZIP domain in the viral context, recombinant viruses carrying mutations identical to those in the panel of pUL71 mutants were generated. bZIP-defective viral mutants showed impaired viral growth, a small-plaque phenotype, and an ultrastructural phenotype similar to that of the previously described UL71 stop mutant virus. The majority of virus particles within the viral assembly compartment exhibited various stages of incomplete envelopment, which is consistent with the growth defect for the bZIP mutants. From these data we conclude that the bZIP-like domain is required for oligomerization of pUL71, which seems to be essential for correct envelopment of HCMV.

Fig 1

Analysis of pUL71 in infected cells. Extracts from AD169-infected (inf.) and mock-infected (mock) cells were harvested in 0.1% SDS and subjected to native 8% PAGE followed by immunostaining (native). Equivalent extracts were harvested in 0.1% SDS (nonreducing) or in 2% SDS–5% ME (reducing) prior to separation by SDS–8% PAGE. (A) Immunostaining was performed with pAbUL71 against pUL71. In addition, pp28 was detected using MAb5C3. (B) Extracts from 293T cells transfected with a construct expressing pUL71 (rpUL71) and mock-transfected cells (mock) were harvested in 0.1% SDS and subjected to native 8% PAGE followed by immunostaining (native). Equivalent extracts were harvested in 0.1% SDS (nonreducing) or in 2% SDS–5% ME (reducing) prior to separation by SDS–8% PAGE. Immunostaining was performed with pAbUL71 against pUL71. Molecular mass markers (M) are indicated on the right. Markers for native PAGE: β-galactosidase, 116 kDa; phosphorylase, 100 kDa; bovine serum albumin, 68 kDa; ovalbumin, 45 kDa; and carbonic anhydrase, 35 kDa.

Fig 2

Oligomerization potential of rpUL71. (A) Gel permeation chromatography analysis of rpUL71. rpUL71 purified in a single step was subjected to chromatography with a HiLoad 16/60 Superdex prep column using an ÄKTAFPLC system, and data were recorded at an absorbance of 280 nm. The column was calibrated with the following molecular mass markers: void volume (21 ml), ferritin (r_s = 6.10 nm; elution volume, 32 ml), and ovalbumin (OVA; r_s = 3.05 nm; elution volume, 55 ml). Five peaks were distinguished and termed peak 1 (maximum elution volume, 21.31 ml), peak 2 (maximum elution volume, 36.38 ml), peak 3 (maximum elution volume, 47.80 ml), peak 4 (maximum elution volume, 59.61 ml), and peak 5 (maximum elution volume, 71.42 ml). mAU, milli-absorbance units. (B) Analysis of the peak fractions by SDS-PAGE. The fractions peak 1 to peak 5 were separated by SDS-PAGE prior to Coomassie blue staining and immunoblot analysis with pAbUL71. Molecular mass markers are indicated on the left, and the position of rpUL71 is indicated on the right. (C) Electron microscopy analysis of purified rpUL71 from peak 3 negatively stained with 4% uranyl acetate. Molecular mass (in kilodaltons) can be calculated from particle size on the basis of the findings of Zipper et al. (49). Different high-molecular-mass forms of rpUL71, monomeric forms (a), dimers or trimers (b), and higher oligomeric forms (c and d), were obtained from the sample. The scale bars correspond to 20 nm. Incubation in phosphate buffer for 6 h led to predominantly monomeric forms (a) and dimers or trimers (b), but no higher-ordered structures (c and d) could be found. Incubation for 24 h led to predominantly monomeric forms (a), but dimers or trimers (b) and higher-ordered structures (c and d) could also be found.

Fig 3

Mutational analysis of the bZIP-like domain in pUL71. (A) Schematic representation of the leucine zipper-like motif (white bars). Exchanged amino acids are indicated in gray. Constructs 71_L34A, 71_L41A, and 71_L1 carry one or two single-nucleotide exchanges starting on heptad position 1 of the bZIP motif. Mutant 71_ΔLZ harbors a deletion of amino acids 34 to 41 of pUL71, representing the N-terminal part of the bZIP-like motif. Underlining indicates positions of the leucines (black) or the mutated leucines (grey). (B) Analysis of coiled-coil structures using PCOILS software. The position of the leucine zipper-like motif within the amino acid sequence is indicated by the red square. PCOILS uses sliding windows of 14 (green), 21 (blue), and 28 (red).

Fig 4

Analyzing the oligomerization potential of pUL71. (A) 293T cells were transfected with pcDNA71 (encoding 71_wt), pcDNA71_L34A (encoding 71_L34A), pcDNA71_L41A (encoding 71_L41A), pcDNA71_L1 (encoding 71_L1), or pcDNA71_ΔLZ (encoding 71_ΔLZ). Cell extracts were harvested 48 h posttranfection and subjected to immunostaining with the Xpress antibody. Molecular mass markers (M) are indicated on the left. (B) 293T cells were transfected with a combination of pcDNA71-xpress (encoding 71-wt-Xpress) and pcDNA71-myc (encoding 71_wt-myc), pcDNA71_L34A (encoding 71_L34A), pcDNA71_L41A (encoding 71_L41A), pcDNA71_L1-myc (encoding 71_L1-myc), or pcDNA71_ΔLZ-myc (encoding 71_ΔLZ-myc). As a control for self-interaction, cells were transfected with a combination of pcDNA71_L1-xpress (encoding 71_L1-Xpress) and pcDNA71_L1-myc (encoding 71_L1-myc) or pcDNA71_ΔLZ-xpress (encoding 71_ΔLZ-Xpress) and pcDNA71_ΔLZ-myc (encoding 71_ΔLZ-myc). Mock-infected cells or cells transfected with pcDNA71 or pcDNA71-myc together with the empty vector pHM1580 (Vector^myc) or pcDNA3.1+ (Vector^xpress) also served as controls. Cells were harvested 48 h after transfection and subjected to precipitation against MAbMyc_r. Extract and precipitate (IP myc) fractions were analyzed using the Xpress antibody (upper panels) and MAbMyc_r (lower panels). Between the left and right lanes in each panel, an additional lane was left empty to prevent contamination upon loading.

Fig 5

Analyzing oligomerization potential of pUL71 using BiFC. Cells were grown on coverslips prior to transfection with a combination of pcDNA71_YN (encoding 71_wt-YN) and construct pcDNA-YC (Vector-YC), pcDNA71_YC (encoding 71_wt-YC), pcDNA71_L1-YC (encoding 71_L1-YC), or pcDNA71_ΔLZ-YC (encoding 71_ΔLZ-YC). Additionally, cells were transfected with a construct expressing mCherry. Cells were fixed 14 h after transfection and analyzed using immunofluorescence. (A) Immunofluorescence data. YFP fluorescence is shown in green, and cell outlines are indicated in red using AxioVision software. (B) Quantification of BiFC results. I_BiFC is set to 100% for the 71_wt-YN/71_wt-YC interaction. Other interactions are set in ratio. Error bars on the histogram represent standard deviations of results from three independent experiments. The significance of results was determined using the paired Student t test. n, number of analyzed cells.

Fig 6

Construction of recombinant HCMV UL71 mutant genomes and representation of virus growth kinetics. (A) To investigate the function of the bZIP domain of pUL71 in the viral context, two different HCMV pUL71 mutant viruses as well as a revertant virus were constructed. The bZIP-like motif and the mutations are shown in red. (B) Protein expression of the different viral mutants 5 days postinfection. Immunostaining was performed using a polyclonal anti-pUL71 antibody (12), MAb65-33 (kindly provided by W. Britt, University of Alabama) against pp65, and MAb5C3 against pp28. (C) Growth kinetics of wild-type and mutant viruses. HFFs were infected using an MOI of 3 with the wild type, TBmut71-ΔLZ, TBmut71-L1, and TBresc71-LZ. Supernatants were harvested at the indicated time points, and progeny virus yields in the supernatants were determined by titration. Day 0 values represent the inoculum. Error bars in the histogram indicate the standard deviations of results from three independent experiments.

Fig 7

Analysis of cell-to-cell spread and intracellular localization of pUL71. (A) HFFs were infected with 100 PFU of the wild type, TBmut71-ΔLZ, TBmut71-L1, and TBresc71-LZ grown in overlay medium containing methyl cellulose (Methocel). The cells were fixed and subjected to immunofluorescence at day 9 p.i. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole), and infected cells were stained with MAbIE1 (red) against IE1. Representative micrographs of plaques produced by the wild type, TBmut71-ΔLZ, TBmut71-L1, and TBresc71-LZ are shown. (B) The plaque areas of individual plaques for each indicated virus were analyzed using a ×10 objective lens and the Axio Observer.Z1 fluorescence microscope. Plaque areas of at least 50 plaques of each virus were measured by the program ImageJ. The mean percentages of the areas and standard errors relative to the mean area of the wild-type virus plaques (set at 100%) are given. The experiment was repeated at least two times. The significance of the results compared to data for the wild type was determined by using an unpaired Student t test (TBmut71-ΔL1, P < 0.0001; TBmut71-L1, P < 0.0001; and TBresc71-LZ, P < 0.476). (C) HFFs were infected at an MOI of 1 with the wild type, TBmut71-ΔLZ, or TBmut71-L1. The spatial distribution of viral proteins was analyzed 120 h p.i. by confocal microscopy using a polyclonal anti-pUL71 antibody (12) (green) and MAb 3F12 (Virusys Corporation) directed against gB (red). (D) Association of bZIP mutants with purified extracellular virions. Infected cell extracts (lanes 1, 3, and 5) and extracellular virions (lanes 2, 4, and 6) were analyzed by immunoblotting with pAbUL71 and antibody against MCP. The molecular mass standards (M) are indicated on the left.

Fig 8

Electron microcopy analysis of ultrathin sections of HFFs infected with the wild type (A and C) or with TBmut71-L1 (B and D) at 5 days postinfection. Cells were prepared for electron microscopy by high-pressure freezing and freeze substitution. (A) Overview of a typical cytoplasmic assembly compartment of a wild-type HCMV-infected cell. (C) Higher magnification of a section of the assembly compartment of a wild-type virus-infected cell. Many wild-type virus particles are fully enveloped within single vesicles (arrowheads). (B) In TBmut71-L1 virus-infected cells, enlarged vesicles and accumulated virus particles can be observed in the assembly compartment. (D) These larger vesicles are used as multiple budding sites by TBmut71-L1 virus particles. Many virus particles have not completed their envelopment (arrows). Scale bars: 1 μm (A and B) and 400 nm (C and D).