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.2013 Jul;94(Pt 7):1647-1657.
doi: 10.1099/vir.0.052670-0. Epub 2013 Apr 11.

Vaccinia virus protein K7 is a virulence factor that alters the acute immune response to infection

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Vaccinia virus protein K7 is a virulence factor that alters the acute immune response to infection

Camilla T O Benfield et al. J Gen Virol.2013 Jul.

Abstract

Vaccinia virus (VACV) encodes many proteins that antagonize the innate immune system including a family of intracellular proteins with a B-cell lymphoma (Bcl)-2-like structure. One of these Bcl-2 proteins called K7 binds Toll-like receptor-adaptor proteins and the DEAD-box RNA helicase DDX3 and thereby inhibits the activation of NF-κB and interferon regulatory factor 3. However, the contribution of K7 to virus virulence is not known. Here a VACV lacking the K7R gene (vΔK7) was constructed and compared with control viruses that included a plaque purified wt (vK7), a revertant with the K7R gene reinserted (vK7-rev) and a frame-shifted virus in which the translational initiation codon was mutated to prevent K7 protein expression (vK7-fs). Data presented show that loss of K7 does not affect virus replication in cell culture or in vivo; however, viruses lacking the K7 protein were less virulent than controls in murine intradermal (i.d.) and intranasal (i.n.) infection models and there was an altered acute immune response to infection. In the i.d. model, vΔK7 induced smaller lesions than controls, and after i.n. infection vΔK7 induced a reduced weight loss and signs of illness, and more rapid clearance of virus from infected tissue. Concomitantly, the intrapulmonary innate immune response to infection with vΔK7 showed increased infiltration of NK cells and CD8⁺ T-cells, enhanced MHC class II expression by macrophages, and enhanced cytolysis of target cells by NK cells and VACV-specific CD8⁺ T-cells. Thus protein K7 is a virulence factor that affects the acute immune response to infection.

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Figures

Fig. 1.
Fig. 1.
VACV K7 expression occurs early after infection and is conserved among orthopoxviruses. (a) L929 cells were infected with vK7 at 10 p.f.u. per cell or mock-infected (M). Where indicated CHX or AraC was added throughout infection. At the indicated time points total RNA was extracted and 10 µg of RNA was separated by electrophoresis, transferred onto nylon membrane and probed with a32P-labelled K7-specific oligonucleotide. The position of the 0.5 kbp RNA marker is shown. (b) BSC-1 cells were infected with the indicated VACV or CPXV strains for 16 h and cell extracts were immunoblotted using K7-specific antiserum. Virus infection was monitored by immunoblotting to detect VACV D8 protein. WR, VACV strain WR; IHD, International Health Department. (c) BSC-1 cells were infected with the indicated VACVs at 1 p.f.u. per cell overnight or mock-infected. Cell lysates were immunoblotted using anti-alpha-tubulin, anti-K7 and mAb AB1.1 against VACV D8. The positions of molecular size markers are shown in kDa.
Fig. 2.
Fig. 2.
K7 is localized to the cytoplasm. (a) HeLa cells were mock-infected or infected with the indicated viruses at 10 p.f.u. per cell for 16 h. Nuclear and cytoplasmic fractions were then separated and analysed by SDS-PAGE and immunoblotting with the indicated antibodies. A fourfold greater proportion of the nuclear fraction was loaded than for the cytoplasmic fraction. The position of molecular size markers are shown in kDa. (b) HeLa cells were infected with vK7-HA or vΔK7 at 5 p.f.u. per cell for 5 h. After fixation and permeabilization, cells were stained with mouse anti-HA followed by anti-mouse Alexa Fluor 546 (red)-conjugated secondary antibody and viewed by confocal microscopy. Bar, 20 µm.
Fig. 3.
Fig. 3.
K7 is non-essential for VACV replication and spread in cell culture. (a) BSC-1 cells were infected with indicated viruses at 10 p.f.u. per cell and were harvested at the indicated times. Cells were disrupted by freeze–thawing and sonication and infectious virus was titrated by plaque assay. Data shown are mean±sd of 2 separate experiments. (b) Scatter plots with means (horizontal bars) of plaque sizes of vK7, vΔK7 and vK7-rev in BSC-1 cells infected with 50–100 p.f.u. per well and incubated for 2, 3 or 4 days. Plaque sizes (n = 15) were measured using ImagePro 4.0 analysis software.
Fig. 4.
Fig. 4.
K7 is a virulence factor in the i.d. infection model. C57BL/6 mice (n = 5 per group) were mock-infected or infected i.d. with 1×104 p.f.u. per ear of the indicated viruses into both ears. Lesion sizes were measured daily and data are expressed as mean±sem. Infection with vΔK7 induced a smaller lesion compared with vK7 and vK7-rev (*,P<0.05).
Fig. 5.
Fig. 5.
Deletion ofK7R causes attenuation in the i.n. infection model and more rapid viral clearance. (a, b) BALB/c mice (n = 5 per group) were mock-infected or infected i.n. with 7×103 p.f.u. per mouse and body weights (a) or signs of illness (described by Alcamí & Smith, 1992) (b) were measured daily. Body weight is expressed as the percentage of the mean weight of the same group of animals on day 0. (c) BALB/c mice (n = 5 per group) were mock-infected or infected i.n. with 1×104 p.f.u. per mouse. On the days indicated, infectious virus in lung cell extracts was measured by plaque assay. The Data shown are mean±sem. *, Significant difference between vΔK7 and other viruses (P<0.05).
Fig. 6.
Fig. 6.
K7 affects immune cell recruitment into the lung. Mice (n = 5 per group) were mock-infected or infected i.n. (a–d) with 1×104 p.f.u. On the days indicated, cells from lungs were washed, counted and stained for flow cytometry. Percentage of (a) leukocytes (as a proportion of all cells recovered), (b) total T-cells (as a proportion of all leukocytes), (c) surface expression of MHC class II (as a proportion of all macrophages) and (d) neutrophils (as a proportion of all cells recovered) in the lung. A forward/side scatter gate was applied to exclude cell debris and larger stromal cells from analysis. T-cells were defined as small, non-granular CD3+ cells. Macrophages were defined as large, granular F4/80+ and CD11b+ cells. Granulocytes were defined as granular Ly6G+ cells. Data are presented as mean±sd. Statistical analyses were by one-factor ANOVA with Bonferroni post-tests for pairwise comparisons; asterisks indicate significant difference between vΔK7 and other viruses: *,P<0.05; ***,P<0.001. BAL, bronchoalveolar lavage.
Fig. 7.
Fig. 7.
vΔK7 induces augmented intrapulmonary cytolysis by NK and CD8+ T-cells. BALB/c mice (n = 5 per group) were infected i.n. with the indicated viruses at 7×103 p.f.u. per mouse and lung tissue was harvested on day 6. Flow cytometry was used to define the percentage of NK cells (CD3−, DX5+; expressed as a proportion of all lymphocytes) (a) and T-cell subsets CD4+ and CD8+ (expressed as a proportion of all T-cells) (b). Chromium-release cytotoxicity assays were performed using total lung cell suspensions as effector cells. Yac-1 cells were used as NK cell targets (c) and VACV-infected P815 cells were used as CD8+ T-cell targets (d). Data are presented as mean±sd for cytometry (a, b) and mean±sem for cytotoxicity assays (c, d). Asterisks indicate significant difference between vΔK7 and other viruses: *,P<0.05; **,P<0.01; ***,P<0.001.
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