Purinergic signaling during Marek’s disease in chickens

Abstract Purinergic receptors (PRs) have been reported as potential therapeutic targets for many viral infections including herpesviruses, which urges the investigation into their role in Marek’s disease (MD), a herpesvirus induced cancer in chickens that is an important pathogen for the poultry industry. MD is caused by MD virus (MDV) that has a similar viral life cycle as human varicella zoster virus in that it is shed from infected epithelial skin cells and enters the host through the respiratory route. In this report, PR responses during natural MDV infection and disease progression was examined in MD-resistant white Leghorns (WL) and MD-susceptible Pure Columbian (PC) chickens during natural infection. Whole lung lavage cells (WLLC) and liver tissue samples were collected from chickens infected but showing no clinical signs of MD (Infected) or presenting with clinical disease (Diseased). RNA was extracted followed by RT-qPCR analysis with gene specific primers against members of the P1, P2X, and P2Y PR families. Differential expression (p < 0.05) was observed in breed and disease conditions. Some PRs showed tissue specific expression (P1A1, P2X1, and P2X6 in WLLC) whereas others responded to MDV infection only in MD-susceptible (PC) chickens (P1A2A, P2X1, P2X5, P2X7). P2Y PRs had differential expression in both chicken lines in response to MDV infection and MD progression. This study is the first to our knowledge to examine PR responses during MDV infection and disease progression. These results suggest PR signaling may an important area of research for MDV replication and MD

Effect of ORFV119 on IKKα/β, IκBα and NF-κB-p65 phosphorylation.

<p>(A) OFTu cells were mock infected or infected with OV-IA82, OV-IA82-Δ119, OV-IA82-RV119^{Lx<u>G</u>xE-Flag} or OV-IA82-RV119^Flag viruses (MOI, 10) and harvested at the indicated times p.i. Total cell protein extracts (50 μg) were resolved by SDS-PAGE, blotted, and probed with specific antibodies shown on the right. Results are representative of three independent experiments. (B and C) Densitometric analysis of bands corresponding to phosphorylated IKKα/β, IκBα and NF-κB-p65 at 30 min (B) and 1 h p.i. (C). Densitometry of p-IKKα/β, p-IκBα and p-NF-κB-p65 bands were normalized to the loading control GAPDH. Fold changes are shown relative to OV-IA82 treatment and results are mean values of three independent experiments. (*<i>P < 0</i>.<i>05</i>) (OV-IA82 vs OV-IA82-Δ119 and OV-IA82 vs OV-IA82-RV119^{Lx<u>G</u>xE-Flag}).</p

Co-immunoprecipitation of pRb with TRAF2 in 293T cells and ORFV119 with TRAF2 in Saos-2 cells.

<p>(A-C) Co-immunoprecipitation of pRb with TRAF2 in 293T cells. 293T cells were transfected with control plasmid (pFlag) or pORFV119^Flag. Total cell lysate and proteins extracts immunoprecipitated with antibodies against TRAF2 (A), pRb (B) or Flag (C), were examined by Western blot (WB) using anti-pRb, anti-TRAF2 or anti-Flag antibodies. (D and E) Co-immunoprecipitation of ORFV119 with TRAF2 in Saos-2 cells. Saos-2 cells were transfected with control plasmid (pFlag) or pORFV119^Flag and harvested at 12 h post transfection. Total cell lysate and protein extracts immunoprecipitated with anti-TRAF2 (D) and anti-Flag (E) antibodies were examined by Western blot using anti-Flag and anti-TRAF2 antibodies. (F-H) Co-immunoprecipitation of ORFV119 with TRAF2 in Saos-2 cells transiently expressing ORFV119^Flag and pRb. Saos-2 cells were co-transfected with control plasmid (pFlag) or pORFV119^Flag and pRb plasmids and harvested at 12 h post transfection. Total cell lysate and protein extracts immunoprecipitated with anti-TRAF2 (F), anti-Flag (G) and anti-pRb (H) antibodies were examined by Western blot using anti-Flag, anti-TRAF2 and anti-pRb antibodies. Results are representative of two independent experiments. Percentage of (A) pRb co-immunoprecipitated by TRAF2 in pFlag transfected cells: 5.1±0.58%; pORFV119^Flag transfected cells: 28.4±2.73%; (B) TRAF2 co-immunoprecipitated by pRb in pFlag transfected cells: 3.1±0.53%; pORFV119^Flag transfected cells: 10.3±1.64%. (F) ORFV119^Flag co-immunoprecipitated by TRAF2 in pORFV119^Flag and pRb transfected cells: 15.2±1.26%; (G) TRAF2 co-immunoprecipitated by ORFV119^Flag in pORFV119^Flag and pRb transfected cells: 18.7±0.96%.</p

A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-κB signaling - Fig 6

<p>(A) Detection of ORFV119 or ORFV119^Lx<u>G</u>xE in virions. OFTu cells were infected with OV-IA82, OV-IA82-Δ119 (Δ119), OV-IA82-RV119^{Lx<u>G</u>xE-Flag} (RV119^Lx<u>G</u>xE) or OV-IA82-RV119^Flag (RV119^Flag) and IMV and EEV virions were purified as described in Materials and Methods. Whole cell lysate (W.C.L) (60 μg) from OV-IA82-RV119^Flag infected OFTu cells, and purified virions (10 μg) were resolved by SDS-PAGE and analyzed by Western blot using antibodies against Flag, ORFV086 structural core protein (positive control) or GAPDH. Results are representative of three independent experiments. (B, C) Effect of translation inhibition on ORFV119 and control cellular protein p53 expression. OFTu cells mock treated or pre-treated with CHX (50 μg/ml) for 30 min were mock infected or infected with OV-IA82-RV119^Flag (MOI, 10) in presence of the drug and harvested at 0.5 h, 1 h, 1.5 h, and 2 h p.i. Cell extracts (50 μg) were resolved by SDS-PAGE, blotted, and probed with anti-Flag, anti-p53 and anti-GAPDH antibodies. (D, E) OFTu cells were drug treated and infected as in B with OV-IA82 or OV-IA82-Δ119 viruses, fixed at 30 min (6D) or 1 h p.i (6E), incubated with anti-NF-κB-p65 antibody, and examined by confocal microscopy. Cells were counted (n = 300 cells/slide) and results are shown as percentage of cells expressing nuclear NF-κB-p65. Results are mean values from two independent experiments. NS, non-significant by Student t test. [OV-IA82 (CHX+) vs OV-IA82 (CHX-), OV-IA82-Δ119 (CHX+) vs OV-IA82-Δ119 (CHX-)].</p

ORFV119 interacts with pRb.

<p>(A and B). 293T cells were transfected with plasmids pORFV119^Flag (p119^Flag), pORFV119^{Lx<u>G</u>xE-Flag} (p119^{Lx<u>G</u>xE-Flag}) or control plasmid (pFlag) and harvested at 12 h post transfection. Total cell lysate (CL) and protein extracts immunoprecipitated (IP) with anti-Flag (A) or anti-pRb (B) antibodies were examined by Western blot (WB) with antibodies against Flag or pRb. Results are representative of four independent experiments. (C-F) OFTu cells were mock infected or infected with OV-IA82-RV119^Flag (C and D), OV-IA82-RV119^{Lx<u>G</u>xE-Flag} (E and F) (MOI, 10) and harvested at 12 h p.i. Total cell lysate and protein extracts immunoprecipitated with anti-Flag (C and E) or anti-pRb (D and F) antibodies were examined by Western blot with antibodies against Flag or pRb. Results are representative of three independent experiments. Percentage of (A) pRb co-immunoprecipitated by ORFV119^Flag: 16.7±1.2%; (B) ORFV119^Flag co-immunoprecipitaed by pRb: 28.4±2.3%; (C) pRb co-immunoprecipitated by ORFV119^Flag in OV-IA82-RV119^Flag infected cells: 8.7±1.4%; (D) ORFV119^Flag co-immunoprecipitated by pRb in OV-IA82-RV119^Flag infected cells: 18.5±1.91%.</p

Regulation of NF-κB signaling pathway by ORFV virion associated NF-κB inhibitors.

<p>ORFV073 inhibits NF-κB signaling by preventing activation of IKK complex through interaction with NEMO, the regulatory subunit of the IKK complex. ORFV119 interacts with pRb and TRAF2 inhibiting the activity of IKK complex and downstream NF-κB signaling early in infection. Localization of pRb in virions remains to be demonstrated.</p

Effect of ORFV119 on TNFα-induced NF-κB-p65 nuclear translocation.

<p>(A) HeLa cells were transfected with control plasmid (pEGFP-N1) or pORFV119-GFP, which expresses ORFV119 fused to GFP. At 12 h post-transfection, cells were treated with TNFα (20ng/ml) for 30 min and processed for immunofluorescence. Green, GFP; Red, NF-κB-p65; Blue, DAPI. (B) Cells were counted (n = 500 cells/slide) and results are shown as percentage of GFP or ORFV119GFP expressing cells with nuclear NF-κB-p65. Results are mean values of three independent experiments (**<i>P<0</i>.<i>01</i>, GFP vs GFP + TNFα and GFP + TNFα vs ORFV119-GFP + TNFα). White arrows indicate ORFV119GFP expressing cells with reduced nuclear NF-κB-p65.</p

Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host.

Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to a lack of natural virus-host model systems. Marek's disease is a devastating herpesviral disease of chickens caused by Marek's disease virus (MDV) and an excellent natural model to study skin-tropic herpesviruses and transmission. Like varicella zoster virus that causes chicken pox in humans, the only site where infectious cell-free MD virions are efficiently produced is in epithelial skin cells, a requirement for host-to-host transmission. Here, we enriched for heavily infected feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined short- and long-read RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning sequencing reads, and a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5' ends of two core herpesviral genes, pUL47 and ICP4, along with strong evidence of independent transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems

Clustal W alignment of PPV119 amino acid sequences.

<p>Aligned sequences are ORFV strains OV-IA82, NZ2, OV-SJ1, OV-XY, OV-SA00, OV-GO, D1701, NA1/11, ORF11, and B029, PCPV strains F00.120R and It1303/05, and BPSV strains BV-AR02 and BV-TX09c5 (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006779#sec002" target="_blank">Materials and Methods</a> for accession numbers). Numbers on the right indicate amino acid positions for OV-IA82 strain ORFV119. Shaded columns, boxed sequences and the underlined sequence correspond to the conserved LxCxE motif, the acidic-rich region, and the predicted mitochondrial localization signal, respectively. Asterisks [*], colons [:], and periods [.] below the alignment indicate fully, strongly, and weakly conserved, residues, respectively.</p