The herpes simplex virus 1 UL51 gene product has cell type-specific functions in cell-to-cell spread

Abstract

The herpes simplex virus 1 (HSV-1) UL51 gene encodes a 244-amino-acid (aa) palmitoylated protein that is conserved in all herpesviruses. The alphaherpesvirus UL51 (pUL51) protein has been reported to function in nuclear egress and cytoplasmic envelopment. No complete deletion has been generated because of the overlap of the UL51 coding sequence 5' end with the UL52 promoter sequences, but partial deletions generated in HSV and pseudorabies virus (PrV) suggest an additional function in epithelial cell-to-cell spread. Here we show partial uncoupling of the replication, release, and cell-to-cell spread functions of HSV-1 pUL51 in two ways. Viruses in which aa 73 to 244 were deleted from pUL51 or in which a conserved YXXΦ motif near the N terminus was altered showed cell-specific defects in spread that cannot be accounted for by defects in replication and virus release. Also, a cell line that expresses C-terminally enhanced green fluorescent protein (EGFP)-tagged pUL51 supported normal virus replication and release into the medium but the formation of only small plaques. This cell line also failed to support normal localization of gE to cell junctions. gE and pUL51 partially colocalized in infected cells, and these two proteins could be coimmunoprecipitated from infected cells, suggesting that they can form a complex during infection. The cell-to-cell spread defect associated with the pUL51 mutation was more severe than that associated with gE-null virus, suggesting that pUL51 has gE-independent functions in epithelial cell spread.

Importance: Herpesviruses establish and reactivate from lifelong latency in their hosts. When they reactivate, they are able to spread within their hosts despite the presence of a potent immune response that includes neutralizing antibody. This ability is derived in part from a specialized mechanism for virus spread between cells. Cell-to-cell spread is a conserved property of herpesviruses that likely relies on conserved viral genes. An understanding of their function may aid in the design of vaccines and therapeutics. Here we show that one of the conserved viral genes, UL51, has an important role in cell-to-cell spread in addition to its previously demonstrated role in virus assembly. We find that its function depends on the type of cell that is infected, and we show that it interacts with and modulates the function of another viral spread factor, gE.

FIG 1

Construction of recombinant viruses. (A) Schematic diagram of the HSV-1(F) genome (line 1) and of the recombinant viruses constructed for this study. The positions of the terminal and internal repeats that flank the long genome component (TRL and IRL, respectively) and the short genome component (IRS and TRS, respectively) are indicated with gray bars. (Line 2) The structures of the wild-type sequences in the regions of UL51 and US8 are shown. (Line 3) The UL51Δ73–244 virus carries a stop codon and a kanamycin resistance cassette in place of the sequences coding for amino acids 73 to 244 of pUL51. (Line 4) The UL51-FLAG virus carries a FLAG tag at the C terminus of UL51 followed by a kanamycin resistance cassette. (Line 5) The UL51(Y19A)-FLAG virus was constructed by mutating the Y19 codon in the context of the UL51-FLAG virus shown in line 4. (Line 6) The FLAG-gE virus was constructed by the insertion of a FLAG-coding sequence between the codons for amino acids 20 and 21 of gE. This was predicted to yield an N-terminally FLAG-tagged gE protein after signal peptide cleavage. (Line 7) The UL51-HA/FLAG-gE virus was constructed by introducing an HA epitope-coding sequence at the C terminus of the UL51 protein-coding sequence in the context of the FLAG-gE virus shown in line 6. (Line 8) the ΔgE virus was constructed by scarless removal of the sequences encoding amino acids 1 to 335 of gE. (B) Expression of UL51 by mutant recombinant viruses. Lysates from Vero cells infected for 16 h with the indicated viruses were probed for either ICP27 to control for the extent of infection and loading (top) or UL51 using anti-UL51 polyclonal antiserum (bottom). (C) Expression of epitope-tagged proteins by recombinant viruses. Lysates from Vero cells infected for 16 h with the indicated viruses were probed for gE (top), the FLAG epitope (middle), or UL51 (bottom). (D) Expression of UL51 by a complementing cell line. Lysates of either Vero or UL51-complementing cells that had been infected with the indicated viruses were probed with anti-UL51 polyclonal antiserum. WB, Western blot.

FIG 2

Growth and spread of UL51 deletions on Vero and HEp-2 cells. (A) Single-step growth of BAC-derived HSV-1(F), UL51-FLAG, and two independently isolated UL51 deletion viruses was measured on Vero cells. Stocks were prepared from the total infected culture (cells and medium). (B) Virus released into the medium during the single-step growth experiment shown in panel A. (C) Sizes of plaques formed by wild-type and mutant viruses on Vero cells. Plaque areas were measured 2 days following low-multiplicity infection as described in Materials and Methods. Each oval represents the area of a single plaque. Twenty plaques were measured for each virus. Note that the y axis has a logarithmic scale. (D) Same as panel C except that plaques were measured on Vero and UL51-complementing cells, as indicated below the graph. (G to H) Same as panels A to C except that measurements were performed by using HEp-2 cells. Note that the y axis in panel F has a linear scale. For replication and release measurements (A, B, E, and F), each point represents the mean of three independent experiments, and the error bars represent the ranges of values obtained. Statistical significance for replication and release experiments, where noted in the text, was determined by using a Student t test, as implemented in Microsoft Excel. Panels C and F are each representative of three independent experiments. The differences in plaque sizes between the HSV-1(F) BAC and the UL51 deletion mutants shown in panel G are significant, with P values of <0.01 determined by using a Kolmogorov-Smirnov test.

FIG 3

Alignment of N-terminal sequences of UL51 homologs from human herpesviruses. Homologs of UL51 from all herpesviruses for which sequences are available were aligned by using the MUSCLE sequence alignment program (52). The alignment from the N terminus of the human herpesvirus homologs is shown. The positions of the conserved cysteine residue that is the palmitoylation site (26) and of the conserved YXXΦ motif are boxed. VZV, varicella-zoster virus; Kaposi's sarcoma-associated herpesvirus; HHV6, human herpesvirus 6.

FIG 4

Growth and spread of UL51(Y19A) mutants on Vero and HEp-2 cells. (A) Single-step growth of UL51-FLAG and two independently isolated UL51(Y19A) mutant viruses measured on Vero cells. Stocks were prepared from the total infected culture (cells and medium). (B) Virus released into the medium during the single-step growth experiment shown in panel A. (C) Sizes of plaques formed by control and mutant viruses. Twenty plaques were measured for each virus. Note that the y axis has a logarithmic scale. (D to F) Same as panels A to C except that measurements were performed with HEp-2 cells. Note that the y axis in panel F has a linear scale. For replication and release measurements (A, B, D, and E), each point represents the mean of three independent experiments, and the error bars represent the ranges of values obtained. Panels C and F are each representative of three independent experiments. The differences in plaque sizes between UL51-FLAG and the UL51(Y19A) mutants shown in panel F are significant, with P values of <0.01.

FIG 5

Growth, release, and spread of HSV-1(F) on pUL51-EGFP-expressing cells. (A) Single-step growth and supernatant virus curves for HSV-1(F) on Vero cells (circles) and a stably transfected clonal Vero cell line that expresses pUL51-EGFP in response to infection. (B) Sizes of plaques formed by HSV-1(F) on Vero or pUL51-EGFP-expressing cells. Horizontal bars indicate the median plaque sizes. Data from one of three representative experiments are shown. The difference in plaque sizes is significant, with a P value of <0.001 determined by using a Kolmogorov-Smirnov test.

FIG 6

Change in gE localization in pUL51-EGFP-expressing cells. Localizations of pUL51-EGFP, pUL51-FLAG, and gE were determined 16 h after infection of Vero (A) or pUL51-EGFP-expressing (B) cells with the UL51-FLAG virus. pUL51-FLAG was detected with anti-FLAG antibody (blue), and gE was detected with mouse monoclonal anti-gE (red). Arrowheads point to sites of gE staining at cell junctions.

FIG 7

Morphology of syncytial HSV-1(F) variants on Vero and pUL51-EGFP-expressing cells. Representative plaques immunostained by using an anti-gD monoclonal antibody are shown.

FIG 8

Copurification of gE and pUL51. Images of Western blots are shown. (A) Flag-tagged gE was purified from lysates of Vero cells infected with the indicated viruses using anti-FLAG magnetic beads, and samples of the unfractionated lysates and of the purified proteins were separated by SDS-PAGE, blotted onto nitrocellulose, and probed as indicated at the left. (B) Same as panel A except that FLAG-tagged pUL51 was purified.

FIG 9

Comparison of spread phenotypes of gE and UL51 deletions. Plaques formed by each of the indicated viruses on Vero cells were measured and plotted as described in the legend of Fig. 2. Dark bars represent the median plaque size. The difference between the HSV-1(F) BAC and the gE-null viruses was significant, with a P value of <0.001.

FIG 10

Schematic drawing of a possible mechanism for pUL51 function. Exposure of pUL51 on the exterior face of cytoplasmic membranes positions it to participate in multiple functions late in infection. It is positioned to interact with other tegument components to facilitate secondary envelopment. It may also mark the exterior of transport vesicles that bud from the envelopment compartment and interact with cell-specific cargo adapters to facilitate trafficking of virion proteins, including gE, or the virions themselves for CCS or for release.