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. 2006 Dec;80(23):11806-16.
doi: 10.1128/JVI.00466-06. Epub 2006 Sep 13.

ORF66 protein kinase function is required for T-cell tropism of varicella-zoster virus in vivo

Anne Schaap-Nutt  1 , Marvin Sommer, Xibing Che, Leigh Zerboni, Ann M Arvin
Affiliations

ORF66 protein kinase function is required for T-cell tropism of varicella-zoster virus in vivo

Anne Schaap-Nutt et al. J Virol. 2006 Dec.

Abstract

Several functions have been attributed to the serine/threonine protein kinase encoded by open reading frame 66 (ORF66) of varicella-zoster virus (VZV), including modulation of the apoptosis and interferon pathways, down-regulation of major histocompatibility complex class I cell surface expression, and regulation of IE62 localization. The amino acid sequence of the ORF66 protein contains a recognizable conserved kinase domain. Point mutations were introduced into conserved protein kinase motifs to evaluate their importance to ORF66 protein functions. Two substitution mutants were generated, including a G102A substitution, which blocked autophosphorylation and altered IE62 localization, and an S250P substitution, which had no effect on either autophosphorylation or IE62 localization. Both kinase domain mutants grew to titers equivalent to recombinant parent Oka (pOka) in vitro. pOka66G102A had slightly reduced growth in skin, which was comparable to the reduction observed when ORF66 translation was prevented by stop codon insertions in pOka66S. In contrast, infection of T-cell xenografts with pOka66G102A was associated with a significant decrease in infectious virus production equivalent to the impaired T-cell tropism found with pOka66S infection of T-cell xenografts in vivo. Disrupting kinase activity with the G102A mutation did not alter IE62 cytoplasmic localization in VZV-infected T cells, suggesting that decreased T-cell tropism is due to other ORF66 protein functions. The G102A mutation reduced the antiapoptotic effects of VZV infection of T cells. These experiments indicate that the T-cell tropism of VZV depends upon intact ORF66 protein kinase function.

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Figures

FIG. 1.
FIG. 1.
ORF66 kinase domain mutations and transfection results. (A) Multiple amino acid sequence alignment of catalytic domain residues of ORF66 and its alphaherpesvirus homologues. Residues mutated in ORF66 for these experiments are shown in black boxes with white lettering. The dots represent sequence not shown between the aligned sequences. Roman numerals above the sequence alignment refer to the five conserved protein kinase subdomains in which mutations were introduced in this study. Lines between ORF66 and homologous residues indicate amino acids that are identical among all of these genes, while stars indicate similar amino acids. (B) Locations of each mutation within the ORF66 kinase domain. Transfection results with cosmids carrying each single amino acid substitution are listed in the columns to the right of the diagram. In the first column, the number of transfections (TF) that yielded infectious virus for each mutant is shown, out of the total number of transfections that were performed with that mutated Spe23ΔAvr cosmid and yielded the control pOka virus. The second column shows the number of positive transfections out of the total number of transfections done with the mutated cosmid that contained a rescue insertion of intact ORF66.
FIG. 2.
FIG. 2.
Effects of ORF66 wild-type and mutated proteins on nuclear localization of IE62. Melanoma cells were transfected with pCMV62, expressing IE62, either alone (top row) or in combination with a threefold excess of ORF66 protein-expressing plasmid (pCR66-wt, pCR66-K122R, pCR66-D206N, pCR66-E210D, pCR66-D224N, or pCR66-P251S). Cells were fixed 48 h after transfection and analyzed by confocal microscopy for expression of ORF66 and IE62 proteins. IE62 was detected with a secondary FITC-conjugated antibody (green), and ORF66 protein was detected with a secondary Texas Red-conjugated antibody (red). Images of each row were merged with DAPI nuclear stain (third panel). Colocalizations of ORF66 kinase and IE62 appear yellow. Bars, 20 μm.
FIG. 3.
FIG. 3.
Effects of ORF66 mutations on VZV growth in vitro. Melanoma cells were inoculated on day 0 with 1 × 103 CFU of pOka or pOka ORF66 mutants. Two pOka66G102A and pOka66S250P mutant viruses were generated and tested independently. Aliquots were harvested daily for 6 days, and the number of infectious foci was determined by titration on melanoma cell monolayers. Each time point represents the mean of results for at least three wells. The x axis shows the days after inoculation when infected cell monolayers were harvested, and the y axis shows infectious foci per milliliter by infectious focus assay. One asterisk indicates that titers were significantly different from pOka (P < 0.05) at the same time point, while two asterisks indicate a difference with a P value of <0.001.
FIG. 4.
FIG. 4.
Localization of ORF66 and IE62 proteins in melanoma cells. (A) pOka and pOka ORF66 mutants were analyzed by confocal microscopy for expression of ORF66 and IE62 proteins. Melanoma cells were infected with 1 × 103 CFU of pOka, pOka66S, pOka66G102A, or pOka66S250P and fixed after 72 h. IE62 was detected with a secondary FITC-conjugated antibody (green), and ORF66 protein was detected with a secondary Texas Red-conjugated antibody (red). Images of each row were merged with DAPI nuclear stain (third panel). Colocalizations of ORF66 kinase and IE62 appear yellow. Bar, 10 μm. (B) Whole-cell lysate (left panels), cytoplasmic lysate (middle panels), and soluble nuclear lysate (right panels) were prepared from melanoma cells that were uninfected (lane 1) or infected with pOka (lane 2), pOka66S (lane 3), pOka66G102A (lane 4), or pOka66S250P (lane 5) for 48 h. In the top panels, IE62 is shown to migrate at approximately 175 kDa. The middle panels show ORF66 protein at approximately 48 kDa in whole-cell lysate and both cytoplasmic and nuclear fractions. α-Tubulin was detected in all cytoplasmic and whole-cell lysates but not in nuclear lysates, as expected.
FIG. 5.
FIG. 5.
pOka and ORF66 mutants generate complete virions in vitro. HEL fibroblasts were infected with 1 × 103 CFU of pOka, pOka66S, or pOka66G102A. Forty-eight hours postinfection, complete viral particles were observed by transmission electron microscopy in infected fibroblasts. A representative image of one cell infected with each virus is pictured, with at least one nucleocapsid in the nucleus (arrowheads) and two or more complete virions on the cell surface (arrows). The locations of the nucleus (Nuc) and cytoplasm (Cyt) are indicated. Magnification, ×10,000.
FIG. 6.
FIG. 6.
Autophosphorylation of the ORF66 kinase. Melanoma cells were left uninfected (lane 1) or infected with pOka (lanes 2 and 3), pOka66S (lane 4), pOka66G102A (lane 5), or pOka66S250P (lane 6). The cell lysate was immunoprecipitated with either polyclonal rabbit anti-ORF66 IgG (lanes 1, 3, 4, 5, and 6) or with rabbit preimmune IgG (lane 2). The immunoprecipitated proteins were incubated in kinase reaction buffer and subjected to SDS-PAGE. The arrow indicates the expected position of the autophosphorylated ORF66 protein.
FIG. 7.
FIG. 7.
Replication of VZV ORF66 mutants in T-cell xenografts in SCID-hu mice. T-cell xenografts were inoculated with HEL fibroblasts infected with pOka, pOka66S, or pOka66G102A on day 0 (inoculum titers are shown on the y axis as infectious foci/ml) and harvested at 10 to 14 or 18 to 21 days. T-cell suspensions were prepared, and titers were determined by infectious center assay. Each time point represents the mean number of plaques from two to four xenografts. Xenografts in which infectious virus was not recovered were excluded. One asterisk indicates that titers were significantly different from pOka (P < 0.05) at the same time point.
FIG. 8.
FIG. 8.
IE62 localization in T cells infected with pOka ORF66 mutants. T cells infected with pOka and pOka ORF66 mutants were analyzed by confocal microscopy for localization of IE62. Purified tonsil T cells were infected by coculture with fibroblast monolayers either uninfected (mock) or infected with pOka, pOka66S, pOka66G102A, or pOka66S250P. T cells were fixed and stained after 72 h. IE62 was detected with a secondary FITC-conjugated antibody (green, left panels), and gE was detected with a secondary Texas Red-conjugated antibody (red, middle panels). The cells were also stained with antibodies to CD3 and secondary Texas Red-conjugated antibody (red) to determine that most were CD3+ T cells; a representative picture is shown in the top row. Images of each row were merged with DAPI nuclear stain (third panel). Colocalizations of IE62 and gE appear yellow. Bars, 20 μm.
FIG. 9.
FIG. 9.
Flow cytometric analysis of apoptosis in T cells infected with pOka and ORF66 mutants. Purified human tonsil T cells were cocultured with fibroblasts that were either uninfected or infected with pOka, pOka66S, pOka66G102A, or pOka66S250P. The T cells were fixed and stained at 48 or 72 h postinfection (hpi) with antibodies to VZV proteins, CD3, and active caspase-3. Cells were gated on CD3-positive T cells and divided into VZV-positive and VZV-negative populations. Bars represent the percentage of caspase-3-positive cells in each category. One asterisk indicates a significant difference from the pOka value at the same time point (P < 0.05).
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