Inhibition of TANK binding kinase 1 by herpes simplex virus 1 facilitates productive infection - PubMed (original) (raw)
Inhibition of TANK binding kinase 1 by herpes simplex virus 1 facilitates productive infection
Yijie Ma et al. J Virol. 2012 Feb.
Abstract
The γ(1)34.5 protein of herpes simplex viruses (HSV) is essential for viral pathogenesis, where it precludes translational arrest mediated by double-stranded-RNA-dependent protein kinase (PKR). Paradoxically, inhibition of PKR alone is not sufficient for HSV to exhibit viral virulence. Here we report that γ(1)34.5 inhibits TANK binding kinase 1 (TBK1) through its amino-terminal sequences, which facilitates viral replication and neuroinvasion. Compared to wild-type virus, the γ(1)34.5 mutant lacking the amino terminus induces stronger antiviral immunity. This parallels a defect of γ(1)34.5 for interacting with TBK1 and reducing phosphorylation of interferon (IFN) regulatory factor 3. This activity is independent of PKR. Although resistant to IFN treatment, the γ(1)34.5 amino-terminal deletion mutant replicates at an intermediate level between replication of wild-type virus and that of the γ(1)34.5 null mutant in TBK1(+/+) cells. However, such impaired viral growth is not observed in TBK1(-/-) cells, indicating that the interaction of γ(1)34.5 with TBK1 dictates HSV infection. Upon corneal infection, this mutant replicates transiently but barely invades the trigeminal ganglia or brain, which is a difference from wild-type virus and the γ(1)34.5 null mutant. Therefore, in addition to PKR, γ(1)34.5 negatively regulates TBK1, which contributes viral replication and spread in vivo.
Figures
Fig 1
An outline of type I IFN responses. Upon virus infection, TLR3 on the endosomal membrane, RIG-I, MDA5, and DNA sensors in the cytoplasm are available to detect viral nucleic acids. These sensors transmit signals to TBK1 via multiple adaptor proteins; TBK1 subsequently phosphorylates transcription factor IRF-3/7, leading to production of IFN-α/β, RANTES, and ISGs. Secreted IFN-α/β acts in an autocrine or paracrine manner, where it induces the expression of a wide array of ISGs. Collectively, these gene products promote the establishment of an antiviral state. PKR, which is constitutively expressed in normal cells, is also upregulated by IFN-α/β. Once bound to viral dsRNA, PKR is activated to phosphorylate eIF2α and shuts off protein synthesis.
Fig 2
(A) Schematic representation of the genome structure and sequence arrangements of HSV-1(F) and related mutants. Boxes on the top line denote the inverted repeats flanking the unique long and unique short sequences, represented by thin lines. The expanded sections below show the γ134.5 loci, where thin lines represent DNA sequences retained in each virus. HSV-1(F) is wild-type virus, whereas R3616 lacks the entire γ134.5 coding region. H1001 has a deletion of the amino terminus of γ134.5, and H1002 is a repair virus that harbors the wild-type γ134.5 gene. (B) Expression of different γ134.5 variants. Vero cells were infected with the indicated viruses at 0.05 PFU per cell. At 24 h after infection, lysates of cells were subjected to immunoblotting with polyclonal antibody against γ134.5. Size markers are listed on the left.
Fig 3
The γ134.5 protein inhibits the induction of antiviral genes via the amino terminus. Mouse embryonic fibroblasts (MEFs) were either mock infected or infected with HSV-1(F), R3616, H1001, or H1002 (5 PFU/cell). At indicated time points, total RNA extracted from cells was subjected to quantitative real-time PCR amplification for expression of IFN-β (A), ISG56 (B), ISG54 (C), or RANTES (D). The data were normalized to that for 18S rRNA, and fold induction was calculated as described in Materials and Methods. Results are expressed as fold activation with standard deviations among triplicate samples.
Fig 4
The amino terminus of γ134.5 is required to inhibit IRF3 phosphorylation. (A) MEFs were infected with indicated viruses at 5 PFU/cell, and cell lysates were subjected to immunoblotting analysis with antibodies against IRF3, phosphorylated IRF3 (Ser396), and ICP27, respectively, at 3 h and 6 h postinfection. (B) Quantification of IRF3 phosphorylation. The protein bands shown in panel A were quantified using NIH ImageJ software. The data are presented as the relative amount of phosphorylated IRF3 normalized to the total level of IRF3 in each sample, with mock infection arbitrarily set at 1.0.
Fig 5
(A) Effect of γ134.5 variants on IRF3 phosphorylation by TBK1. 293T cells were transfected with HA-TBK1 along with empty vector, FLAG-γ134.5, or FLAG-ΔN146. At 40 h after transfection, aliquots of cell lysates were processed for protein expression with antibodies against FLAG and HA. In parallel, lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were incubated with GST-IRF3 (amino acids [aa] 380 to 427) and ATP for kinase assays. Samples were subjected to electrophoresis and probed with antibodies against phosphorylated IRF3, FLAG, and HA, respectively. (B) Quantitation of in vitro kinase assays. The relative kinase activity was expressed as a ratio of phosphorylated IRF3 to TBK1 in the immunoprecipitate, with the control TBK1 arbitrarily set to 100 (NIH ImageJ software). The data represent four independent experiments with standard deviations (*, P < 0.05). (C) Interactions of γ134.5 variants with TBK1. 293T cells were cotransfected with FLAG-γ134.5 or FLAG-ΔN146 along with an empty vector, FLAG-TBK1. At 36 h after transfection, lysates of cells were immunoprecipitated with anti-HA antibody. Samples were processed for immunoblotting analysis with antibodies against FLAG and HA, respectively. (D and E) Effects of γ134.5 variants on promoter activation by TBK1. 293T cells were transfected with an empty vector or a plasmid expressing HA-γ134.5, FLAG-TBK1, FLAG-ΔN146, or FLAG-γ134.5, along with an IFN-β or ISG56 reporter. At 36 h posttransfection, cells were harvested for luciferase assays. Results are expressed as fold activation with standard deviations among triplicate samples.
Fig 6
(A) HSV γ134.5 inhibits IFN-β expression independently of PKR. PKR−/− MEFs were either mock infected or infected with indicated viruses (5 PFU/cell). At 3 and 6 h after infection, total RNA extracted from cells was subjected to quantitative real-time PCR amplification. The level of IFN-β mRNA was normalized to the level of 18S rRNA, and fold induction was calculated as described in Materials and Methods. Results are representative of three independent experiments with standard deviations among triplicate samples. (B) Deletion of the γ134.5 amino terminus has no effect on HSV resistance to IFN-α. Vero cells were untreated or pretreated with IFN-α (Sigma) for 20 h. Cells were then infected with indicated viruses at 0.05 PFU per cell, and viral yields were determined at 48 h postinfection.
Fig 7
Viral replication in TBK1+/+ or TBK1−/− cells. Cells were infected with indicated viruses (0.05 PFU/cell). At 24 h after infection, virus yields were titrated on Vero cells. Data are averages for three independent experiments with standard deviations.
Fig 8
(A) The amino terminus of γ134.5 is required to facilitate neuroinvasion. Mice were mock infected or infected with HSV-1(F), R3616, H1001, or H1002 at 4 × 105 PFU through corneal scarification. At 3 days postinfection, eyes, trigeminal ganglia, and brain tissues were collected and virus yields were determined. Data are expressed as means ± standard deviations for six mice for each group. (B) Immunohistochemistry analysis. The sections from eye tissues described above were reacted with anti-HSV-1 antibody, and immunohistochemistry was performed. Specific HSV-1 staining is shown in brown. Representative images from each group were chosen for the panels.
Fig 9
Kinetics of viral replication in vivo. Mice were infected with HSV-1(F), R3616, H1001, or H1002 at 4 × 105 PFU via corneal scarification. Mice were sacrificed on days 1, 3, and 5. Tissue samples from the eye (A), trigeminal ganglia (B), or brain stem (C) were analyzed for viral yields on Vero cells. Data are expressed as means ± standard deviations for data from four mice for each group.
References
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