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. 2008 Aug;82(15):7653-65.
doi: 10.1128/JVI.00311-08. Epub 2008 May 21.

Varicella-zoster virus open reading frame 66 protein kinase is required for efficient viral growth in primary human corneal stromal fibroblast cells

Angela Erazo  1 , Michael B Yee, Nikolaus Osterrieder, Paul R Kinchington
Affiliations

Varicella-zoster virus open reading frame 66 protein kinase is required for efficient viral growth in primary human corneal stromal fibroblast cells

Angela Erazo et al. J Virol. 2008 Aug.

Abstract

Varicella-zoster virus (VZV) open reading frame 66 (ORF66) encodes a serine/threonine protein kinase that is not required for VZV growth in most cell types but is needed for efficient growth in T cells. The ORF66 kinase affects nuclear import and virion packaging of IE62, the major regulatory protein, and is known to regulate apoptosis in T cells. Here, we further examined the importance of ORF66 using VZV recombinants expressing green fluorescent protein (GFP)-tagged functional and kinase-negative ORF66 proteins. VZV virions with truncated or kinase-inactivated ORF66 protein were marginally reduced for growth and progeny yields in MRC-5 fibroblasts but were severely growth and replication impaired in low-passage primary human corneal stromal fibroblasts (PCF). To determine if the growth impairment was due to ORF66 kinase regulation of IE62 nuclear import, recombinant VZVs that expressed IE62 with alanine residues at S686, the suspected target by which ORF66 kinase blocks IE62 nuclear import, were made. IE62 S686A expressed by the VZV recombinant remained nuclear throughout infection and was not packaged into virions. However, the mutant virus still replicated efficiently in PCF cells. We also show that inactivation of the ORF66 kinase resulted in only marginally increased levels of apoptosis in PCF cells, which could not fully account for the cell-specific growth requirement of ORF66 kinase. Thus, the unique short region VZV kinase has important cell-type-specific functions that are separate from those affecting IE62 and apoptosis.

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Figures

FIG. 1.
FIG. 1.
IE62 cellular distribution and GFP expression in VZV-infected cells. MRC-5 cells (A) or PCF cells (B) were infected at an MOI of 0.0005 with MeWo cell-grown VZV.GFP-66 (a to c), VZV.GFP-66kd expressing complete GFP-tagged kinase-inactivated ORF66 protein (d to f), VZV.GFP-66s expressing GFP tagged to residues 1 to 84 of ORF66 (g to i), or VZV.GFP-66Rsc (j to l). Cells were fixed with 4% paraformaldehyde at 3 days p.i. and immunostained with rabbit anti-IE62, which was then detected with anti-rabbit Alexa Fluor 546 (a, d, g, and j). ORF66 expression was determined using GFP autofluorescence (b, e, h, and k). The merge panels (c, f, i, and l) are overlays of GFP (green), IE62 (red), and nuclei stained with Hoechst dye (blue). Fluorescence images were taken using a 40× objective.
FIG. 1.
FIG. 1.
IE62 cellular distribution and GFP expression in VZV-infected cells. MRC-5 cells (A) or PCF cells (B) were infected at an MOI of 0.0005 with MeWo cell-grown VZV.GFP-66 (a to c), VZV.GFP-66kd expressing complete GFP-tagged kinase-inactivated ORF66 protein (d to f), VZV.GFP-66s expressing GFP tagged to residues 1 to 84 of ORF66 (g to i), or VZV.GFP-66Rsc (j to l). Cells were fixed with 4% paraformaldehyde at 3 days p.i. and immunostained with rabbit anti-IE62, which was then detected with anti-rabbit Alexa Fluor 546 (a, d, g, and j). ORF66 expression was determined using GFP autofluorescence (b, e, h, and k). The merge panels (c, f, i, and l) are overlays of GFP (green), IE62 (red), and nuclei stained with Hoechst dye (blue). Fluorescence images were taken using a 40× objective.
FIG. 2.
FIG. 2.
Progeny virus growth curves reveal a requirement for ORF66 for VZV growth in PCF cells. (A) Growth curves were performed as detailed in Materials and Methods, and the titers of the progeny VZV in MRC-5 cells (top) and PCF cells (bottom) are shown. Confluent monolayers were infected at an MOI of 0.001 with VZV.GFP-66, VZV.GFP-66kd, VZV.GFP-66s, or VZV.GFP-66Rsc. The inoculum (Inoc) of each virus was immediately determined upon the setup of the studies at time 0 h. At 24, 48, 72, and 96 h postinfection, infected cells were trypsinized and titrated onto subconfluent monolayers of MeWo cells. Plaque formation and enumeration were assessed by fluorescence microscopy at 4 to 5 days postinfection. The x-axis values represent hours postinfection when cells were harvested, and y-axis values represent average total numbers of infectious centers at each time point. Each data point was determined in quadruplicate, and results represent the means ± standard errors of these values. (B) GFP-positive infected cell numbers fail to increase in PCF cells if the ORF66 protein is disrupted. MRC-5 cells (top) or PCF cells (bottom) in confluent monolayers in 35-mm dishes were infected with VZV.GFP-66, VZV.GFP-66kd, or VZV.GFP-66s at an MOI of 0.001 or mock infected. On the indicated day after infection indicated (x axis), cells were harvested and counted by flow cytometry, gating for GFP-positive cells. The ratio of GFP-positive cells to the total number of cells in the harvested monolayers is expressed as a percentage (y axis). Values represent the means ± standard errors of three parallel but independent values.
FIG. 3.
FIG. 3.
DNA characterization of BAC DNA and of VZV-infected cell DNA of the recombinants VZV.71.S686A and VZV.IE62(S686A)2. (A) Schematic representation of the mutagenesis of IE62 residue S686. The top portion represents IE62 and its nuclear localization signal (NLS), with the presence of the existing AgeI sites shown. Part of the DNA sequence and encoded amino acids of the target region in the parental DNA and in mutant DNA are shown underneath. The DNA sequence of the alterations induced by mutagenesis that introduce the novel AgeI site is underlined. (B) A 1.2% agarose gel showing DNA fragments generated by AgeI digestion of the VZV BAC DNAs derived from pOka and BACs containing the mutation in ORF71(S) or in both ORF62 and ORF71 (Dbl). M is the DNA size marker (1Kb plus; Invitrogen). The abundant 5-kbp DNA seen in the mutant BACs represents the pBAD-I-SceI plasmid used to select for loss of the Kanr cassette. The positions of the 1,018-bp fragment in the pOka BAC and the resultant smaller digestion fragments in the mutants are shown by arrows. (C) Southern blot of rVZV-infected MeWo cell genomic DNA, digested with AgeI and probed with a [α-32P]dCTP-labeled 1,018-bp fragment obtained from an AgeI digest of pKCMV62. The autoradiograph shows DNA of VZV pOka, VZV.71.S686A, and VZV.IE62(S686A)2. The approximate sizes of DNA markers are indicated to the left.
FIG. 4.
FIG. 4.
IE62 localizes to the nucleus in VZV.IE62(S686A)2-infected cells. (A) Scanned images of VZV.GFP-66 and VZV.IE62(S686A)2-infected MeWo cell plaques, immunostained with rabbit anti-IE62 and mouse anti-MCP antibody, which were then detected with anti-rabbit Alexa Fluor 546 or anti-mouse Alexa Fluor 488, respectively. The merge panels are overlays of IE62 (red) and MCP (green). (B) Immunofluorescence images of VZV.IE62(S686A)2-infected MRC-5 (top row) or PCF cells (bottom row) immunostained for IE62 (red). Nuclei detected using Hoechst stain (blue) are displayed in the merged images. All cells were fixed at 3 days p.i. with 4% paraformaldehyde, and images were taken using a 40× objective.
FIG. 5.
FIG. 5.
VZV.IE62(S686A)2 does not incorporate IE62 into the virion tegument. (A) Sypro ruby staining of purified VZV virion particles harvested from infected MeWo cells following separation on a 4 to 15% Tris-HCl linear gradient SDS gel. A MeWo-infected cell extract is also shown (cell ext), with arrows indicating two suspected forms of IE62. Virion particles were obtained from cells infected with VZV.GFP-66 (P) or VZV.IE62(S686A)2 (686). Virions are shown following purification after the 5 to 15% Ficol gradient step (Ficol 1) and after the second fractionation on 10 to 50% sucrose gradients (Sucrose2). Arrows depict the 175-kDa protein in the VZV.GFP-66 virion fractions. (B) Immunoblot analysis of cell extracts and sucrose gradient-purified virions that were equalized based on the abundance of ORF10 protein and then probed with rabbit antibodies to VZV proteins derived from ORFs 4, 9, 10, 29, 47, 61, 62, and 63 and β-actin. The HSV homologues of some VZV proteins are indicated in brackets. M indicates the marker lane.
FIG. 6.
FIG. 6.
Progeny growth curves of VZV.IE62(S686A)2, VZV.GFP-66, and ORF66 mutants in PCF cells. Growth curves were determined as detailed in the legend for Fig. 2, using confluent PCF cell monolayers infected with VZV.GFP-66, VZV.GFP-66kd, VZV.GFP-66s, or VZV.IE62(S686A)2 after infection at an MOI of 0.001. To quantify plaques of VZV.IE62(S686A)2, cells were fixed in 4% paraformaldehyde at 4 to 5 days p.i., permeabilized, and immunostained with primary rabbit anti-62 antibodies so that plaques could be enumerated using fluorescent microscopy. Inoculant data values represent direct seeding onto MeWo cells on the same day PCF cells were infected. The x-axis values represent hours p.i. when cells were harvested, and y-axis values represent infectious centers. Each data point was done in quadruplicate and represents the mean ± standard error of these values. Growth curves are representative of two independent experiments.
FIG. 7.
FIG. 7.
Apoptosis in VZV PCF and MRC-5 cells infected with VZV and kinase-negative mutants. MRC-5 (top) and PCF cells (bottom) were infected at an MOI of 0.02 with VZV.GFP-66, VZV.GFP-66kd, or VZV.GFP-66s or mock infected. At 24, 48, and 72 h p.i., cells were carefully harvested and stained with APC-Annexin V and the viability stain 7-AAD. Cells for analysis were gated on GFP-positive cells for infection. The x axis shows the fraction of GFP-positive cells that stained APC-Annexin V positive as a percentage of total GFP-positive cells. Results represent means ± standard errors of values from three separate but identical experiments. Statistical analysis was done with Student's t test. Asterisks represent the level of statistical significance: *, P < 0.05; **, P < 0.001 compared to VZV.GFP-66 values.
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