Phosphorylation by the varicella-zoster virus ORF47 protein serine kinase determines whether endocytosed viral gE traffics to the trans-Golgi network or recycles to the cell membrane

Abstract

Like all alphaherpesviruses, varicella-zoster virus (VZV) infection proceeds by both cell-cell spread and virion production. Virions are enveloped within vacuoles located near the trans-Golgi network (TGN), while in cell-cell spread, surface glycoproteins fuse cells into syncytia. In this report, we delineate a potential role for serine/threonine phosphorylation of the cytoplasmic tail of the predominant VZV glycoprotein, gE, in these processes. The fact that VZV gE (formerly called gpI) is phosphorylated has been documented (E. A. Montalvo and C. Grose, Proc. Natl. Acad. Sci. USA 83:8967-8971, 1986), although respective roles of viral and cellular protein kinases have never been delineated. VZV ORF47 is a viral serine protein kinase that recognized a consensus sequence similar to that of casein kinase II (CKII). During open reading frame 47 (ORF47)-specific in vitro kinase assays, ORF47 phosphorylated four residues in the cytoplasmic tail of VZV gE (S593, S595, T596, and T598), thus modifying the known phosphofurin acidic cluster sorting protein 1 domain. CKII phosphorylated gE predominantly on the two threonine residues. In wild-type-virus-infected cells, where ORF47-mediated phosphorylation predominated, gE endocytosed and relocalized to the TGN. In cells infected with a VZV ORF47-null mutant, internalized VZV gE recycled to the plasma membrane and did not localize to the TGN. The mutant virus also formed larger syncytia than the wild-type virus, linking CKII-mediated gE phosphorylation with increased cell-cell spread. Thus, ORF47 and CKII behaved as "team players" in the phosphorylation of VZV gE. Taken together, the results showed that phosphorylation of VZV gE by ORF47 or CKII determined whether VZV infection proceeded toward a pathway likely involved with either virion production or cell-cell spread.

FIG. 1.

Detection of gE, gI, and ORF47 in infected cell lysates. Lane 1: wild-type VZV rOka lysate immunoblotted for VZV gI. Lane 2: wild-type VZV rOka lysate immunoblotted for VZV gE. Lane 3: VZV rOka-47S, the ORF47-null mutant, lysate immunoblotted for VZV gI. Lane 4: VZV rOka-47S lysate immunoblotted for VZV gE. Note the VZV gE dimer expressed in rOka-47S (left arrow). Lane 5: ORF47.12 transfected HeLa cell lysate, immunoprecipitated with antibody to the epitope tag, and immunoblotted for ORF47. Note the ORF47.12 monomer (54 kDa) and dimer (110 kDa) (arrows). Lane 6: Coprecipitation of ORF47 with VZV gE from infected cells. VZV-32-infected cell lysate, immunoprecipitated for VZV gE, immunoblotted for ORF47. Both the ORF47 monomer and dimer coprecipitated with VZV gE. Lane 7: rOka-47S lysate immunoblotted for ORF47. Lane 8: rOka-47S lysate immunoprecipitated for VZV gE and immunoblotted for ORF47. Abbreviations: IP, immunoprecipitation; WB, Western blotting, ppt, precipitated total lysate.

FIG. 2.

Phosphorylation of VZV gE by ORF47 kinase and CKII. Immunoprecipitated VZV gE was added to in vitro kinase assays specific for either ORF47.12 or CKII. Shown are vector-transfected negative control (V+E), ORF47.12 kinase assays with 1 mM (+h) or 2 mM (++h) heparin, and CKII-specific kinase assays with 1 mM (+h) or 2 mM (++h) heparin. This experiment was repeated three times with similarly proportional results, and a representative sample is shown. bk, background. Error bars, standard deviations.

FIG. 3.

Differential phosphorylation of residues within the gE acidic cluster by ORF47 or CKII. The serines and/or threonines in the gE acidic cluster were replaced with alanines, and these point mutants were added to ORF47- or CKII-specific in vitro kinase assays. All ORF47.12 reactions included 2 mM heparin. CKII reactions did not include heparin. Abbreviations: SSTT, wild-type gE (EDSESTDTEEE), SSAA, alanines were substituted for the C-terminal threonines (EDSESADAEEE); AATT, alanines were substituted for the N-terminal serines (EDAEATDTEEE); AAAA, alanines were substituted for all four phosphorylatable residues (EDAEAADAEEE). Note that 47wt indicates the amount of radioactivity incorporated into wild-type gE by ORF47 kinase. This experiment was repeated three times with similarly proportional results, and a representative sample is shown. bk, background. Error bars, standard deviations.

FIG. 4.

Endocytosis of VZV gE in cells infected with rOka or rOka-47S. (A, C, and E) rOka-infected cells; (B, D, and F) rOka-47S-infected cells. (A and B) Endocytosis at 0-min time point showing labeled gE (red) on the plasma membrane. Actin (green) was visualized with phalloidin, and the nuclei (blue) were visualized with TOTO-3. (C and D) Endocytosis after 60 min. Note the presence of red in the centers of the syncytia in the rOka sample but not in the centers of the rOka-47S sample. (E and F) Analysis of pixel intensity using the Zeiss software. White arrows in panels C and D overlie the analyzed lines. Note the high levels of red pixel intensity in the rOka sample but not in the rOka-47S sample.

FIG. 5.

Localization of endocytosed gE and TGN markers in cells infected with rOka or rOka-47S. Endocytosis of gE was analyzed after 60 min. (A) rOka-infected cells; (B) rOka-47S-infected cells. Individual channels are shown on the left; merged channels are shown on the right. In VZV-infected cells, the TGN (red) relocalizes to the center of the syncytium. Note that gE (green) also relocalized to the TGN (yellow) in rOka-infected cells but not in rOka-47S-infected cells, where the TGN remained a red color.

FIG. 6.

Endocytosis assay of the VZV gI glycoprotein. (A) VZV rOka, 0 min of endocytosis; (B) VZV rOka, 60 min of endocytosis; (C) VZV rOka-47S, 0 min of endocytosis; (D) VZV rOka-47S, 60 min of endocytosis. Note the signals in the centers of the syncytia in panels B and D.

FIG. 7.

DRB inhibition of CKII in cells infected with rOka or rOka-47S. Antibody uptake endocytosis assays were performed to investigate the effect of DRB. Red indicates gE endocytosis, green indicates phalloidin staining of the actin cytoskeleton, and blue indicates nuclear stain. Numbers atop the columns indicate duration of endocytosis. (A to C) rOka-infected cells with DRB at 0 (A), 5 (B) or 45 (C) μM. (D to F) rOka-47S-infected cells with DRB at 0 (D) 5 (E), or 45 (F) μM.

FIG. 8.

Differences in cell-to-cell spread between VZV rOka and VZV rOka-47S. Confocal images of VZV-infected cells were analyzed over time. The first, third, and fifth rows show samples infected with VZV rOka (wt). The remaining rows show samples infected with VZV rOka-47S. All viral proteins were visualized as green, and nuclei were visualized as blue. In the top two rows, MAb 6B5 recognizes gI. In the middle two rows, MAb 5C6 recognizes IE62. In the bottom two rows, MAb 251 recognizes the ORF33 serine protease associated with viral capsids.

FIG. 9.

Identification of VZV IE62 in polykaryons. The 36-hpi micrographs immunolabeled for IE62 shown in Fig. 8 were enlarged in order to better illustrate the differences between cells infected with rOka and rOka-47S. To accentuate the contrast, the nuclei were pseudocolored magenta and the IE62 proteins were colored green. The numbers beneath each micrograph indicate the percentage of IE62 pixels exhibiting the highest intensity, as determined by a previously described assay (38). Note that VZV rOka-47S infection led to the formation of larger, more irregular syncytia, while those of rOka were smaller and circular.

FIG. 10.

Effect of phosphorylation by ORF47 kinase and CKII on gE localization. At the plasma membrane (PM), VZV gE associates with and is phosphorylated by either VZV ORF47 or CKII. VZV gE is internalized in early endosomes (EE). If VZV gE is phosphorylated on S593 or S595 (predominantly by ORF47), PACS-1 is postulated to bind the gE acidic cluster in the secondary endosomes (SE) and gE is transported to the TGN. If VZV gE is phosphorylated on T596 or T598 (half the time in ORF47-associated gE, predominantly in CKII-associated gE), PACS-1 is postulated to bind less well and gE recycles back to the plasma membrane.