The 135 Gene of Goatpox Virus Encodes an Inhibitor of NF-κB and Apoptosis and May Serve as an Improved Insertion Site To Generate Vectored Live Vaccine

Abstract

Goatpox virus (GTPV) is an important member of the Capripoxvirus genus of the Poxviridae Capripoxviruses have large and complex DNA genomes encoding many unknown proteins that may contribute to virulence. We identified that the 135 open reading frame of GTPV is an early gene that encodes an ∼18-kDa protein that is nonessential for viral replication in cells. This protein functioned as an inhibitor of NF-κB activation and apoptosis and is similar to the N1L protein of vaccinia virus. In the natural host, sheep, deletion of the 135 gene from the GTPV live vaccine strain AV41 resulted in less attenuation than that induced by deletion of the tk gene, a well-defined nonessential gene in the poxvirus genome. Using the 135 gene as the insertion site, a recombinant AV41 strain expressing hemagglutinin of peste des petits ruminants virus (PPRV) was generated and elicited stronger neutralization antibody responses than those obtained using the traditional tk gene as the insertion site. These results suggest that the 135 gene of GTPV encodes an immunomodulatory protein to suppress host innate immunity and may serve as an optimized insertion site to generate capripoxvirus-vectored live dual vaccines.IMPORTANCE Capripoxviruses are etiological agents of important diseases in sheep, goats, and cattle. There are rare reports about viral protein function related to capripoxviruses. In the present study, we found that the 135 protein of GTPV plays an important role in inhibition of innate immunity and apoptosis in host cells. Use of the 135 gene as the insertion site to generate a vectored vaccine resulted in stronger adaptive immune responses than those obtained using the tk locus as the insertion site. As capripoxviruses are promising virus-vectored vaccines against many important diseases in small ruminants and cattle, the 135 gene may serve as an improved insertion site to generate recombinant capripoxvirus-vectored live dual vaccines.

Keywords: GTPV 135; NF-κB pathway; apoptosis; viral vector.

FIG 1

135 is an early gene and is nonessential for replication of GTPV in vitro. (A) Alignment of amino acid sequences of the 135 protein of GTPV (accession number AGZ95457.1) and the N1L proteins of VACV (accession number 2I39_A), myxoma virus (accession number NP_051859.1), and ectromelia virus (accession number NP_671537.1). Amino acids identical across all four viruses and three of the four viruses are highlighted in dark blue and light blue, respectively. (B) Schematic showing the genomes of wild-type and recombinant GTPV AV41 strains. Western blotting confirmed expression of the GTPV 135 protein (∼18 kDa) (C), and immunofluorescence assay indicated the subcellular localization of the 135 protein in LT cells 48 h after infection with AV41 and its r135-Flag recombinant (D). (E) The GTPV 135 gene is an early gene. LT cells were infected with r135Flag-GFP/tk at an MOI of 0.05 in the presence or absence of AraC, and expression of the 135 protein, GFP, and β-actin was detected by Western blotting. (F) The 135 gene is nonessential for GTPV replication. LT cells were infected with AV41, r135-Flag, or the Δ135 virus at an MOI of 0.05. Cells and medium were harvested at different times by freeze-thawing three times. After low-speed centrifugation, the supernatants were collected for viral titration in LT cells. Data are representative of independent experiments performed in triplicate.

FIG 2

Deletion of the 135 gene results in less attenuation than that with deletion of the tk gene for GTPV in sheep. Sheep were inoculated intradermally with the AV41, Δ135, or Δtk virus. (A) Diameters of lesions at inoculation sites in sheep that received 10^5.0 TCID₅₀ (I) and 10^6.0 TCID₅₀ (II) were measured and recorded every day. (B) Representative lesions are shown for day 6 after inoculation (I), and the significance of differences in diameters between viruses was determined using the t test (II). *, P < 0.05; **, P < 0.01.

FIG 3

Expression of the 135 protein inhibits NF-κB signaling pathway activation. HEK293T cells were cotransfected with p135, pN1L, and pCAGGS together with pNFκB-luc and pTK-Renilla. At 24 h, cells were treated with 100 ng/ml of TNF-α (AI) or IL-1β (BI) or left untreated for 8 h. The cell lysates were prepared for assays of NF-κB-inducible luciferase activity (AI and BI), and mRNAs were extracted for determination of TNF-α and IL-1β mRNA levels by qPCR (AIII and BIII). (AII and BII) Expression of the GTPV 135 protein and the VACV N1L protein was measured by Western blotting. Data are representative of independent experiments performed in triplicate. The significance of differences was determined by the t test. *, P < 0.05; **, P < 0.01.

FIG 4

GTPVΔ135 is attenuated in inhibition of NF-κB activation. HEK293T cells were infected with AV41 or the Δ135 virus at MOIs of 0.01, 0.1, and 1. Twenty-four hours after infection, cells were cotransfected with pNFκB-luc and pTK-Renilla. At 24 h posttransfection, cells were treated with 100 ng/ml of TNF-α or left untreated for 8 h. Cell lysates were prepared for assays of NF-κB-inducible luciferase activity (A), and mRNA was extracted for determination of the IL-1β mRNA level by qPCR (B). Data are representative of independent experiments performed in triplicate. The significance of differences was determined by the t test. *, P < 0.05; **, P < 0.01.

FIG 5

Expression of the 135 protein inhibits apoptotic death of HeLa cells after treatment with CHX and TNF-α. HeLa cells were transfected with p135, pN1L, and pCAGGS and treated with 2 μM CHX and 5 ng/ml of TNF-α for 12 h. CHX-treated cells were harvested for annexin V/PI staining (A) or treatment with caspase 3/7 detection reagents and Sytox AADvanced Ready flow reagent (C), examined by flow cytometry, and analyzed with FlowJo, using biexponential scaling. The horizontal axis shows the quantity of cells stained with annexin V dye (A) or caspase 3/7 green dye (C). The vertical axis represents the quantity of cells stained by PI (A) or Sytox AADvanced (C). B2 represents the percentage of late apoptotic cells, and B4 represents the percentage of early apoptotic cells. (B and D) Total numbers of apoptotic cells were calculated by adding late and early apoptotic counts. Expression of the GTPV 135 and VACV N1L proteins was confirmed by Western blotting. The significance of differences was determined by the t test. *, P < 0.05; **, P < 0.01.

FIG 6

GTPV-H/135 is more immunogenic than GTPV-H/tk for eliciting PPRV and GTPV NAs. Groups of six sheep were vaccinated with GTPV-H/135 or GTPV-H/tk at 10^3.5 TCID₅₀ (A) and 10^5.5 TCID₅₀ (B) and were boosted 4 weeks after priming. Sera were collected to determine the titers of NAs to PPRV (AI, AII, BI, and BII) and GTPV (AIII and BIII). The titers are presented as the mean and standard error for each group. Dotted lines indicate the thresholds of seroconversion of PPRV NAs (AI and BI). The significance of differences was determined by the t test (*, P < 0.05; **, P < 0.01).