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. 2011 Sep;85(18):9437-46.
doi: 10.1128/JVI.05207-11. Epub 2011 Jul 6.

Interaction and interdependent packaging of tegument protein UL11 and glycoprotein e of herpes simplex virus

Jun Han  1 , Pooja Chadha, David G Meckes Jr, Nicholas L Baird, John W Wills
Affiliations

Interaction and interdependent packaging of tegument protein UL11 and glycoprotein e of herpes simplex virus

Jun Han et al. J Virol. 2011 Sep.

Abstract

The UL11 tegument protein of herpes simplex virus plays a critical role in the secondary envelopment; however, the mechanistic details remain elusive. Here, we report a new function of UL11 in the budding process in which it directs efficient acquisition of glycoprotein E (gE) via a direct interaction. In vitro binding assays showed that the interaction required only the first 28, membrane-proximal residues of the cytoplasmic tail of gE, and the C-terminal 26 residues of UL11. A second, weaker binding site was also found in the N-terminal half of UL11. The significance of the gE-UL11 interaction was subsequently investigated with viral deletion mutants. In the absence of the gE tail, virion packaging of UL11, but not other tegument proteins such as VP22 and VP16, was reduced by at least 80%. Reciprocally, wild-type gE packaging was also drastically reduced by about 87% in the absence of UL11, and this defect could be rescued in trans by expressing U(L)11 at the U(L)35 locus. Surprisingly, a mutant that lacks the C-terminal gE-binding site of UL11 packaged nearly normal amounts of gE despite its strong interaction with the gE tail in vitro, indicating that the interaction with the UL11 N terminus may be important. Mutagenesis studies of the UL11 N terminus revealed that the association of UL11 with membrane was not required for this function. In contrast, the UL11 acidic cluster motif was found to be critical for gE packaging and was not replaceable with foreign acidic clusters. Together, these results highlight an important role of UL11 in the acquisition of glycoprotein-enriched lipid bilayers, and the findings may also have important implications for the role of UL11 in gE-mediated cell-to-cell spread.

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Figures

Fig. 1.
Fig. 1.
Mapping of the gE-binding region within UL11. (A) Diagrams of UL11-GFP and a panel of deletion mutants that were used for GST-pulldown experiments. (B) GST and GST fused to the cytoplasmic tail of gE were expressed and purified from E. coli, followed by SDS-PAGE and Ponceau S staining. (C) Purified GST-gE.CT or GST-only were used in attempts to pull down full-length UL11-GFP and mutants from transfected Vero cell lysates. Proteins bound to the beads were separated by SDS-PAGE, transferred to nitrocellulose membrane, and subjected to Western blotting with rabbit anti-GFP antibodies (left panel). The right panel shows the expression level of UL11-GFP and its derivatives in Vero cells. A summary of the results is shown in panel A. WT, wild type.
Fig. 2.
Fig. 2.
Direct binding of UL11 to the cytoplasmic tail of gE. UL11 was expressed as a His6-tagged fusion protein. Purified GST-gE.CT or GST-only were incubated with 2-fold increasing amounts of purified His6-UL11 in NP-40 lysis buffer at room temperature for 5 h. Proteins bound to GST beads were washed with NP-40 buffer, separated by SDS-PAGE, transferred to nitrocellulose membrane, visualized by Ponceau S staining (A), and probed with rabbit anti-His6 antibodies (B).
Fig. 3.
Fig. 3.
Mapping of the UL11-binding region within the cytoplasmic tail of gE. (A) A series of gE-tail deletions were constructed as GST fusion proteins. (B) The recombinants were expressed and purified from E. coli. Vero cells transfected with UL11-GFP or GFP-only were lysed and incubated with the purified GST or GST-gE.CT proteins described above. Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and visualized by Ponceau S staining. (C) Detection of UL11-GFP with rabbit anti-GFP antibodies. (D) The purified GST fusion proteins were also tested for their ability to bind to purified His6-UL11. A summary of the binding results is shown in panel A.
Fig. 4.
Fig. 4.
Colocalization analysis of UL11 and gE. (A) Summary of the gE constructs used in the present study. Vero cells grown on coverslips were transfected with plasmids that express gE constructs alone (B) or cotransfected with a plasmid that expresses UL11-GFP (C). At 16 to 18 h posttransfection, the cells were fixed and permeabilized prior to staining with a gE mouse monoclonal antibody or a monoclonal antibody specific to the HA epitope. The primary antibodies were located with a secondary goat anti-mouse IgG antibody conjugated to Alexa 568. Nuclei were stained with DAPI (blue). The cells were viewed by confocal microscopy.
Fig. 5.
Fig. 5.
Mutant viruses used in the present study. A gE-tail truncation mutant was constructed by inserting a stop codon in US8 immediately after the coding sequence for the transmembrane domain. The UL11-null mutant (ΔUL11) was created by deleting the codons 31 to 96 but without altering the overlapping and essential UL12 gene. The mutants U1, UL11(Myr-), UL11(Myr-)-GFP, UL11(CCC-), UL11(CCC-)-GFP, and UL11(AC-)-GFP were constructed independently from the mutant ΔUL11 by replacing UL35 with the respective UL11 alleles. UL11 mutants LI-, AC-, nefAC, and furAC were made by deletions or substitutions as shown in the diagrams. UL11Δ71-96 was constructed by removing codons 71 to 96 and, similarly, replacement of codons 71 to 96 with the GFP-coding sequence was to construct UL11Δ71-96-GFP. The relative location of the BAC sequence is indicated.
Fig. 6.
Fig. 6.
Multistep growth curves of selected UL11 mutants. Vero cells were infected with the specified viruses at an MOI of 0.1 for 1 h at 37°C and then washed with citrate buffer to inactivate the viruses remaining on the cell surfaces. After being washed with DMEM, the cells were overlaid with 1 ml of DMEM containing 2% FBS. At the indicated times postinfection, media and cells from each well were harvested and analyzed by plaque assay to determine the intracellular and extracellular viral yields, which were added together to produce these graphs. (A) UL11 and gE-tail deletion viruses. (B) Acidic cluster mutants.
Fig. 7.
Fig. 7.
Interdependent packaging of UL11 and gE into virions. (A) Analysis of the virion compositions of wild type, gEΔCT, U1, and ΔUL11. Extracellular virions were purified from infected cell media, and virion proteins were separated by SDS-PAGE and probed with antibodies against VP5, gE, gD, VP22, UL16, and UL11 (left panels). The infected cell lysates were also analyzed for the expression levels of these proteins (right panels). (B) The amounts of gE, gD, VP16, VP22, UL16, and UL11 in the mutant virions were quantified and normalized to the level of the respective proteins in the wild-type virions. The data represent at least three independent experiments. WT, wild type.
Fig. 8.
Fig. 8.
Identification of the gE packaging determinant within UL11. (A) Wild type and UL11 mutants from infected-cell supernatants were pelleted through 30% sucrose cushions, and the virions were analyzed for the presence of VP5, gE, UL16, and UL11 by Western blotting. (B) Measurement of the intracellular expression levels of the corresponding proteins. (C) Quantitative analysis of gE in the UL11 mutant virions. The amount of gE in the various mutants was quantified and normalized to the level of wild-type gE. The data are representative of at least three independent experiments. WT, wild type.
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