A parapoxviral virion protein inhibits NF-κB signaling early in infection - Fig 7

<p>(A) Detection of ORFV073 in virions. OFTu cells were infected with OV-IA82Δ073 or OV-IA82RV073^Flag and harvested at 90–95% cytopathic effect. Supernatants and pellets from infected cultures were purified for extracellular enveloped virus (EEV) and intracellular mature virus (IMV) as described in Material and Methods. Whole cell lysates (10 μg) from mock and OV-IA82RV073^Flag infected cells (MOI = 10) (24 h p.i) and purified OV-IA82Δ073 and OV-IA82RV073^Flag virion proteins (10 μg) were resolved by SDS-PAGE, blotted to the nitrocellulose membrane and probed with antibodies against flag or structural protein ORFV086. # and ## denote 30 kDa and a doublet 40 kDa ORFV073 bands in EEV fraction, respectively. (B) Effect of translation inhibition on ORFV073-mediated block of NF-κB-p65 nuclear translocation. OFTu cells were pre-treated with the protein synthesis inhibitor cycloheximide (CHX) (50 μg/ml) for 30 min and then mock infected or infected with OV-IA82, OV-IA82Δ073 or OV-IA82RV073^Flag (MOI = 10) in presence of CHX (50 μg/ml). Cells were fixed at 1 h p.i., incubated with antibody against NF-κB-p65, stained with Alexa Fluor 488-labeled antibody and DAPI, and examined by confocal microscopy. Cells (n = approximately 300) expressing nuclear NF-κB-p65 in each group were counted and results depicted as mean percentage of cells containing nuclear NF-κB-p65 over two independent experiments (*, <i>P<0</i>.<i>05</i>). Result shown in (A) are representative of six independent experiments.</p

Clustal W alignment of amino acid sequences corresponding to parapoxvirus ORFV073 homologs, squirrellpox virus (SQPV)-084 protein, and murid betaherpesvirus m170 protein.

<p>The ORFV strain IA82 sequence is at the top of the alignment, with amino acid positions indicated by numbers on the right. The predicted nuclear localization signal is shown underlined. Sequences listed below strain IA82 are arranged in order of decreasing % amino acid identity relative to strain IA82. For clarity, only the murid betaherpesvirus m170 domain with homology to parapoxvirus ORFV073 proteins is shown (m170 positions 86–147). Asterisks [*], colons [:], and periods [.] below the alignment indicate fully, strongly, and weakly conserved, residues, respectively. Viruses, strains, and GenBank accession numbers (in parentheses) used for the alignment are as follows: ORFV strains OV-IA82 (AY386263.1), NZ2 (DQ184476.1), B029 (KF837136.1), NA1/11 (KF234407.1), D1701 (HM133903.1), NP (KP010355.1), SJ1 (KP010356.1), OV-SA00 (NC 005336.1), GO (KP010354.1), and YX (KP010353.1); Pseudocowpox virus strain F00.120R [PCPV-F00] (GQ329669.1); Bovine papular stomatitis virus strain BV-AR02 [BPSVar02] (NC 005337.1); Parapoxvirus of the red deer in New Zealand strain HL953 [PPV-RD] (NC 025963.1); Squirrellpox virus strain Red squirrel UK [SQPV-084] (YP_008658509); Murid betaherpesvirus 1 strain N1 [MHV1m170] (CCE57166).</p

Replication characteristics of ORFV073-gene deletion virus.

<p>Primary OFTu cells were infected with wild-type OV-IA82 or deletion mutant OV-IA82Δ073 viruses and virus titers determined at various times p.i. as described in Material and Methods. (A) Multi-step growth curve, MOI 0.1; (B) single-step growth curve, MOI 10. (C) CPE of wild-type OV-IA82 or deletion mutant OV-IA82Δ073 virus infected OFTu cells at 48 h p.i (MOI = 10) (magnification, X20).</p

Effect of ORFV073 expression on TNFα-induced phosphorylation of IKKα/β, IκBα and NF-κB-p65.

<p>(A) HeLa cells stably expressing GFP (GFP/HeLa) or ORFV073-GFP (073GFP/HeLa) were treated with TNFα (20 ng/ml) and harvested at 5, 10 and 15 min post treatment. Whole cell protein extracts (50 μg) were resolved by SDS-PAGE, blotted to nitrocellulose membranes and probed with antibodies against phospho or total IKKα/β, IκBα and NF-κB-p65, and GAPDH and GFP. (B-D) Densitometry of p-IKKα/β, p- IκBα and p-NF-κB-p65 bands, respectively are normalized to the loading control GAPDH. Relative fold inductions across treatments in B-D are shown relative to GFP/HELA (*, <i>P<0</i>.<i>05</i> and **, <i>P<0</i>.<i>01</i>). Results are mean values of three independent experiments.</p

Co-immunoprecipitation of ORFV073 with NEMO.

<p>OFTu cells co-transfected with plasmids pcDNA3.1-NEMO, pcDNA/V5-His (control), or pcDNA3.1-NEMO and pcDNA/V5-073His (ORFV073-His) were harvested at 24 h post transfection and nuclear extracts prepared as described in Material and Methods. Nuclear lysates (left) and extracts immunoprecipitated with anti-His (upper right) or anti-NEMO (lower right) antibodies were examined by SDS-PAGE-Western blotting with antibodies directed against proteins indicated on the right. # denote light chain of the IgG antibody. Results are representative of three independent experiments.</p

Effect of ORFV073 on NF-κB-p65 activation during ORFV infection.

<p>(A) OFTu cells were mock infected or infected with OV-IA82, OV-IA82Δ073 or OV-IA82RV073^Flag (MOI = 10) and transcription of selected NF-κB regulated genes was assessed by real-time PCR at 1 and 2 h p.i. (*, <i>P<0</i>.<i>05</i>, **, <i>P<0</i>.<i>01)</i>. Fold changes are relative to OV-IA82 treatment and data are mean mRNA levels from two independent experiments. (B) OFTu cells were mock infected or infected with OV-IA82, OV-IA82Δ073 or OV-IA82RV073^Flag (MOI = 10). Cells were fixed at 1 h p.i. and incubated with antibody against NF-κB-p65. Cells were then stained with Alexa Fluor 488-labeled secondary antibody and DAPI, and examined by confocal microscopy. Green, NF-κB-p65; blue, DAPI. (C) Cells (n = approximately 300) expressing nuclear NF-κB-p65 in each group were counted and results depicted as the mean percentage of cells containing nuclear NF-κB-p65 at the indicated time points post infection. Results are mean of two independent experiments (*, P<<i>0.05</i>). (D) OFTu cells were mock infected or infected with OV-IA82 or OV-IA82Δ073 (MOI = 10) and harvested at the indicated times p.i. Whole cell protein extracts (50 μg) were resolved by SDS-PAGE, blotted to nitrocellulose membranes and probed with antibodies against phospho or total NF-κB-p65, and GAPDH. Results in (B and D) are representative of two independent experiments.</p

Effect of ORFV073 on phosphorylation of IKKα/β, IκBα and NF-κB-p65 during ORFV infection.

<p>(A) OFTu cells were mock infected or infected with OV-IA82, OV-IA82Δ073 or OV-IA82RV073^Flag (MOI = 10) and harvested at 30 min and 1 h p.i. Whole cell protein extracts (50 μg) were resolved by SDS-PAGE, blotted to nitrocellulose membranes and probed with antibodies against phospho and total IKKα/β, IκBα and NF-κB-p65, and GAPDH. (B-D) Densitometry analysis of p-IKKα/β, p-IκBα and p-NF-κB-p65, respectively; bands were normalized to the loading control GAPDH. Fold changes are shown relative to OV-IA82 and data are mean values of three independent experiments (*, <i>P<0</i>.<i>05</i> and **, <i>P<0</i>.<i>01</i>).</p

Histological changes in the skin of the inner side of thighs following inoculation with OV-IA82Δ073 (sheep 17, 62, 122, and 609) and OV-IA82RV073 (sheep 21, 114, 124, and 656).

<p>Results are shown for days 2 and 5 p.i. Inset in top left panel shows normal skin at the inoculation site (uninfected control). HF, hair follicle. H&E, 2 dpi and ctrl, X200; 5 dpi, X100.</p

Effect of ORFV073 expression on TNFα-induced NF-κB-p65 nuclear translocation.

<p>(A) HeLa cells stably expressing GFP (GFP/HeLa) or ORFV073-GFP fusion protein (073GFP/HeLa) were treated with TNFα (20 ng/ml) and fixed 30 min or 1 h after treatment. Cells were sequentially incubated with anti-NF-κB-p65 and Alexa Fluor 594-labeled antibodies, and stained with DAPI. Shown are representative results of two independent experiments after 30 min TNFα treatment. GFP and 073GFP, Green; NF-κB-p65, red; DAPI, blue. Arrows indicate ORFV073-expressing cells with absence or reduced nuclear NF-κB-p65. (B) Cells (n = approximately 200) from randomly selected fields were scored for nuclear NF-κB-p65 and results are depicted as mean percentage of GFP/073GFP expressing cells containing nuclear NF-κB-p65 at 30 min and 1 h post TNFα induction over two independent experiments (*, P<0.05).</p

Spatial and space-time clustering of seroprevalence of SARS-CoV-2 antibodies in Illinois household cats during 2021–2023.

(A) Purely spatial clusters of SARS-CoV-2 antibodies in Illinois household cats. The relative risk (RR) of counties within a spatial cluster where cats had higher than expected SARS-CoV-2 antibodies is shown. The circle represents the location of the cluster. Within the circle, the color of each dot represents the value range of the RR rate in each county. (B) Space-time clusters of SARS-CoV-2 antibodies in Illinois household cats. Retrospective analysis, scanning for clusters with high seroprevalence, using 50% population at risk and 50% period scanning window, 999 Montecarlo permutation, and the Bernoulli model. The circle represents the location of the cluster, and the period of the cluster is also represented. Within the cluster, the color of each dot represents the value range of the RR in each county. Statistically significant at p ≤ 0.05.</p