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. 2006 Nov;80(22):11105-14.
doi: 10.1128/JVI.00846-06. Epub 2006 Sep 6.

Kaposi's sarcoma-associated herpesvirus virions inhibit interferon responses induced by envelope glycoprotein gpK8.1

Stuart T Perry  1 , Teresa Compton
Affiliations

Kaposi's sarcoma-associated herpesvirus virions inhibit interferon responses induced by envelope glycoprotein gpK8.1

Stuart T Perry et al. J Virol. 2006 Nov.

Abstract

The Kaposi's sarcoma-associated herpesvirus (KSHV) envelope glycoprotein gpK8.1 contributes to cellular attachment through binding cell surface heparan sulfate proteoglycans. By using a soluble recombinant form of gpK8.1, we discovered that a consequence of gpK8.1 interaction with human fibroblasts is the induction of an antiviral response, as characterized by the activation of interferon regulatory factor 3 (IRF-3), production of interferon beta (IFN-beta), and expression of interferon-stimulated antiviral genes. In contrast, neither IFN-beta expression nor a functional antiviral response is observed in cells treated with KSHV virions. The interferon response induced by soluble gpK8.1 can be inhibited by simultaneous treatment with UV-inactivated virions, while the induction of an indicator inflammatory cytokine, interleukin-6, was readily evident in the response to both gpK8.1 and KSHV. In addition, KSHV virions abrogate gpK8.1-mediated activation of IRF-3, an early transcriptional regulator for cellular antiviral responses. Although innate immune responses are initiated during contact between gpK8.1 and cellular receptor(s), these results suggest that the virion contains one or more structural elements that selectively repress an effective antiviral response while allowing cellular responses favorable to the KSHV life cycle.

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Figures

FIG. 1.
FIG. 1.
Expression of soluble gpK8.1 protein. (A) Full-length gpK8.1 is 228 aa long, including a signal sequence and a transmembrane domain. gpK8.1-S contains the N-terminal 196 aa of gpK8.1 and six-His at the C terminus. (B) 293T cells were transfected with pTriEx plasmid encoding either full-length gpK8.1 (lane 1) or soluble gpK8.1 lacking the transmembrane domain (lane 2). gpK8.1 was detected by Western blotting with an anti-gpK8.1 rabbit polyclonal antibody (a gift from J. Jung). (C) Fractions were collected during the purification of gpK8.1-S and analyzed by SDS-PAGE and Coomassie blue staining. Lane 1, supernatant; lane 2, column flowthrough; lane 3, imidazole wash; lane 4, elution. *, gpK8.1-S.
FIG. 2.
FIG. 2.
KSHV induces ISG transcription via gpK8.1. (A) NHDF cells were mock treated or treated with gpK8.1-S (10 μg/ml) or UV-KSHV (6.7 × 108 genomes/ml). Total RNA was harvested at 2-h, 6-h, 12-h, 18-h, and 24-h time points, and RT-PCR analysis was performed with primers specific for ISG56, OAS, IFN-β, and GAPDH. PCR products were analyzed on a 1% agarose gel and visualized by ethidium bromide staining. (B) NHDF cells were mock treated or treated with gpK8.1-S (10 μg/ml) in the presence (+) of increasing amounts of soluble heparin sulfate (10 μg/ml, 25 μg/ml, 50 μg/ml, and 100 μg/ml) or polymyxin B (50 μg/ml and 10 μg/ml) or treated with poly(I:C) (50 μg/ml) in the presence or absence (−) of 100 μg/ml soluble heparin. Samples were harvested and analyzed as described above. (C) NHDF cells were mock infected and treated with increasing concentrations of UV-inactivated KSHV or soluble gpK8.1 as indicated. At 18 h posttreatment, the medium was analyzed for secreted IFN-β by ELISA. The results shown are representative of at least three independent RT-PCR experiments. Error bars indicate standard deviations.
FIG. 3.
FIG. 3.
(A) IL-6 is upregulated in response to KSHV and glycoprotein gpK8.1. NHDF cells were mock treated or treated with gpK8.1-S (10 μg/ml) or UV-KSHV (6.7 × 108 genomes/ml). After 6 h of incubation, total RNA was harvested and RT-PCR analysis was performed with primers specific for IL-6. (B) NHDF cells were mock infected and treated with increasing concentrations of UV-inactivated KSHV or soluble gpK8.1 as indicated. At 18 h posttreatment, the medium was analyzed for secreted IL-6 by ELISA. Error bars indicate standard deviations.
FIG. 4.
FIG. 4.
KSHV inhibits gpK8.1-mediated ISG transcription. NHDF cells were mock treated, treated with gpK8.1-S (10 μg/ml), UV-KSHV (6.7 × 108 genomes/ml), or gpK8.1-S and KSHV together. After 6 h incubation, total RNA was harvested and RT-PCR analysis was performed with primers specific for ISG54, IFN-β, and GAPDH. −, absence of; +, presence of.
FIG. 5.
FIG. 5.
IRF-3 is activated in response to glycoprotein gpK8.1. (A) NHDF cells were mock treated, infected with UV-inactivated HCMV (multiplicity of infection of 1), or treated with gpK8.1-S (10 μg/ml) or UV-KSHV (6.7 × 108 genomes/ml) in the presence of cycloheximide (100 μg/ml). At 6 h posttreatment, whole-cell extracts were prepared and analyzed by immunoblotting with an anti-IRF-3 antibody. (B) IRF-3 translocates to the nucleus in response to glycoprotein gpK8.1. NHDF cells were (a) mock treated, (b) treated with gpK8.1-S (5 μg/ml), (c) infected with UV-KSHV (4 × 108 genomes/ml), or (d) treated with both gpK8.1 and KSHV in the presence of cycloheximide (100 μg/ml). At 6 h posttreatment, cells were fixed and cellular localization of IRF-3 was determined by indirect immunofluorescence. (C) NHDF cells were treated with gpK8.1-S or UV-KSHV as described above. Mixed samples were treated either by adding gpK8.1 and UV-KSHV simultaneously or by adding UV-KSHV for 30 min pre- or postaddition of gpK8.1 as indicated. IRF-3 was detected by indirect immunofluorescence, and nuclei were scored for IRF-3 staining in multiple fields of view (n > 100 nuclei) for each sample. Error bars indicate standard deviations.
FIG. 6.
FIG. 6.
Cells establish an antiviral state in response to gpK8.1. (A) NHDF cells were treated with gpK8.1 at the concentrations indicated and UV-KSHV (4.7 × 108, 4.7 × 107, 4.7 × 106 genomes/ml) or IFN-α/β (100 U/ml; Biosource). After 6 h of incubation, the cells were washed and challenged with VSV (100 PFU/well) for 1 h. At 36 h post-VSV challenge, plaques were visualized with crystal violet. The mock-infected well was not challenged with VSV. (B) NHDF cells were mock treated, treated with gpK8.1-S (5 μg/ml), or treated with both UV-KSHV (4.7 × 108 genomes/ml) and gpK8.1-S and challenged with VSV as described above.
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