The novel ORFV protein ORFV113 activates LPA-p38 signaling

Viruses have evolved mechanisms to subvert critical cellular signaling pathways that regulate a wide range of cellular functions, including cell differentiation, proliferation and chemotaxis, and innate immune responses. Here, we describe a novel ORFV protein, ORFV113, that interacts with the G protein-coupled receptor Lysophosphatidic acid receptor 1 (LPA1). Consistent with its interaction with LPA1, ORFV113 enhances p38 kinase phosphorylation in ORFV infected cells in vitro and in vivo, and in cells transiently expressing ORFV113 or treated with soluble ORFV113. Infection of cells with virus lacking ORFV113 (OV-IA82Δ113) significantly decreased p38 phosphorylation and viral plaque size. Infection of cells with ORFV in the presence of a p38 kinase inhibitor markedly diminished ORFV replication, highlighting importance of p38 signaling during ORFV infection. ORFV113 enhancement of p38 activation was prevented in cells in which LPA1 expression was knocked down and in cells treated with LPA1 inhibitor. Infection of sheep with OV-IA82Δ113 led to a strikingly attenuated disease phenotype, indicating that ORFV113 is a major virulence determinant in the natural host. Notably, ORFV113 represents the first viral protein that modulates p38 signaling via interaction with LPA1 receptor.


Introduction
Orf or contagious ecthyma is a ubiquitous, highly contagious disease of sheep and goats caused by orf virus (ORFV). The disease is characterized by lesions at mucocutaneous margins in the mouth, reflecting virus tropism for keratinocytes [1]. Lesions progress from maculae to papulae, pustules and scabs, and usually resolve in 6-8 weeks [2]. The disease is usually mild and lesions remain confined to virus entry sites [3] and, although viremia has not been associated with ORFV infection, recently virus has been detected in blood of asymptomatic animals [4]. ORFV infection in sheep induces a potent early inflammatory response followed by a T-helper type 1 immune response at later stages. However, protective immunity against orf is short lived and virus can reinfect the host in both natural and experimental infections [5].
ORFV is the type member of the Parapoxvirus genus of Poxviridae, which also includes bovine pathogens pseudocowpox virus (PCPV) and Bovine papular stomatitis virus (BPSV), red deerpox virus, and Grey sealpox virus. The ORFV genome is approximately 138 kbp in length, and encodes for 132 genes [6]. The central genomic region contains genes highly conserved among the poxviruses that are essential for virus replication and structure, while the terminal genomic regions contains less conserved genes involved in virulence, and host range and immunomodulation [6]. The later genes are usually not essential for virus replication in cultured cells, but they affect virus virulence and pathogenesis in the host, and might play a role in viral persistence [7,8].
Many viruses hijack G-protein-coupled receptors to ensure viral replicative success and often contribute to their pathogenesis. However, no direct role for LPA signaling in the context of virus infection has been reported. A recent study identified a pro-viral role of LPA signaling during Hepatitis C virus infection [38]. Hepatitis C virus replication was shown to induce expression of autotaxin (ATX) which activates LPA signaling by hydrolyzing lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). Inhibition of ATX-LPA signaling decreased hepatitis C virus replication in hepatocytes.
While modulation of LPA signaling by viral factors has not been described, a few modulators of p38 signaling have been reported. For example, vaccinia virus protein A52R, known to inhibit IL-1-and Toll-like receptor-stimulated NF-κB activation, was found to activate p38 MAPK in a TRAF6 dependent manner [39,40]. E3, a vaccinia virus double-stranded RNA binding protein that inhibits cytokine expression, suppressed p38 phosphorylation in vaccinia virus-infected cells via an unknown mechanism [41].
Here, we show that ORFV113, a protein unique to parapoxviruses, enhances p38 phosphorylation in ORFV-infected cells in vitro and keratinocytes in vivo, and in cells transiently expressing ORFV113 or treated with soluble ORFV113. p38 signaling markedly affects ORFV plaque formation and replication in infected cells. We found that ORFV113 interacts with LPA 1 receptor, which is involved in ORFV113-mediated enhancement of p38 activation. When ORFV113 is deleted from the viral genome, the resulting virus loses the ability to replicate to high titers and to induce gross lesions in the skin of sheep. Notably, ORFV113 represents the first viral protein that modulates p38 signaling by interacting with LPA 1 receptor.

Ethics statement
All animal procedures were approved by University of Nebraska-Lincoln Institutional Animal Care and Use Committee (IACUC; protocol 1796) and were performed in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching.

Construction of ORFV113 gene deletion mutant and revertant viruses
To generate gene deletion mutant virus OV-IA82-Δ113, a recombination cassette (pΔ113-GFP) containing vaccinia virus 7.5 early-late promoter (VV7.5)-driven green fluorescent protein (GFP) gene flanked by ORFV113 left and right flanking regions was constructed as previously described [42]. To generate revertant viruses OV-IA82-RV113 Flag , sequences were synthesized containing C-terminally Flag tagged ORFV113 and a red fluorescent protein (RFP) reporter gene, all flanked by approximately 600 bp of homologous sequence on either side to mediate recombination (Genscript, Piscataway, NJ). To generate revertant viruses OV-IA82-RV113 HA (ORFV113 with HA tag) and OV-IA82-RV113 (untagged ORFV113), recombination plasmids containing ORFV113 sequence containing C-terminal HA tag or untagged ORFV113 and VV7.5-driven RFP reporter gene flanked by ORFV113 left and right flanking regions were constructed.
To obtain OV-IA82-Δ113, OFTu cells were infected with OV-IA82 using a multiplicity of infection (MOI) of 1 and transfected with recombination plasmid pΔ113-GFP. To obtain the revertant viruses OV-IA82-RV113 Flag , OV-IA82-RV113 HA and OV-IA82-RV113, OFTu cells were infected with OV-IA82-Δ113 (MOI, 1) and transfected with the respective recombination plasmids. Recombinant viruses were isolated by limiting dilution and plaque assay using fluorescence microscopy as previously described [16]. Identity and integrity of viral DNA sequences were confirmed by PCR and DNA sequencing.

Virus purification and virion protein characterization
OFTu cells infected with OV-IA82, OV-IA82-Δ113, OV-IA82-RV113 Flag , OV-IA82-RV113 HA or OV-IA82-RV113 (MOI, 0.1) were harvested at three days post infection (p.i.) and disrupted by three cycles of freeze and thaw. Cellular debris were removed by centrifugation at 1500 rpm for 5 min, and virus pelleted by ultracentrifugation at 25,000 rpm for 1 h. Pellets were resuspended in MEM, and pelleted again by ultracentrifugation, resuspended in MEM, aliquoted and frozen at -80˚C. Viral titers were obtained in OFTu cells by the Spearman-Karber's 50% tissue culture infectious dose (TCID 50 ) method.

ORFV113 protein expression and subcellular localization
To evaluate ORFV113 expression during virus infection, OFTu cells were mock infected or infected with OV-IA82-RV113 Flag (MOI, 10) for 1, 2, 4, 6, 8, 12 or 24 h p.i. Total protein extracts (50 μg) were resolved by SDS-PAGE or native PAGE, blotted and transferred to nitrocellulose membranes, and probed with primary antibody against Flag and GAPDH. Blots were then incubated with appropriate HRP-labeled secondary antibodies and developed using chemiluminescent reagents.

Detection of ORFV113 in culture supernatants
To check for the presence of ORFV113 in supernatant of ORFV infected cultures, OFTu cells were mock infected or infected with OV-IA82-Δ113 or OV-IA82-RV113 HA (MOI, 10) and culture supernatants were harvested at 24 h p.i. Cellular debris and virions were removed from supernatants by centrifugation at 2000 rpm for 10 min and of the virus by ultracentrifugation at 25,000 rpm for 1 h, respectively. Immunoprecipitation (see below) was performed on clarified supernatants using HA antibody (Cell signaling; 3724) and immunoprecipitation products were resolved by SDS-PAGE, blotted and incubated with antibody against HA (Cell signaling; 2367). Blots were processed as described above.
The presence of ORFV113 was examined in supernatants of OFTu cells transfected with pCMV-HA (Empty HA plasmid), pCMV-ORFV113 HA (ORFV113 HA plasmid) or pCMV-ORFV113 (Untagged ORFV113 plasmid) (2 μg). Culture supernatants were harvested 24 h post transfection and clarified by centrifugation at 2000 rpm for 10 min. Fifty μl of clarified culture supernatants were resolved by SDS-PAGE, blotted and incubated with antibodies against HA.

Growth curves
The replication characteristics of OV-IA82Δ113 was assessed in OFTu cells. Cells cultured in 12-well plates were infected with OV-IA82, OV-IA82Δ113 or OV-IA82-RV113 Flag using MOI 0.1 and 0.01 (multi-step growth curves) or 10 (single-step growth curve) and harvested at 6,

Plaque assay
To assess the effect of ORFV113 on plaque formation, OFTu cells in 6-well plates were infected with OV-IA82Δ113 (GFP reporter) or OV-IA82-RV113 Flag (RFP reporter) (10 −5 dilution of virus stock) for 1 h and cultures were washed three times with MEM media. Cultures were then overlaid with 2X MEM (MEM, 10% FBS, sodium bicarbonate, penicillin-streptomycin, Gentamycin and L-Glutamine) mixed with equal volume of 1% agarose. At 5 days post infection, random plaques (n =~100) were imaged for each treatment and plaque area measured using Nikon A1 software.

Co-immunoprecipitation
To prepare ORFV113-immunoprecipitation sample for mass spectrometry analysis of cellular interacting partners, OFTu cells was transfected with plasmid pCMV-ORFV113 Flag for 24 h and total cell protein was extracted as described above. Co-immunoprecipitation was performed using Active Motif Co-IP Kit (Active Motif, Carslbad, CA) following manufacturer's protocols. Extracts were co-immunoprecipitated with anti-Flag antibody overnight at 4˚C, and then incubated with 50 μl of pre-washed protein G agarose beads (Cat. #16-266; Millipore) at 4˚C for 2 h. Beads were washed four times with high stringency buffer and bound proteins eluted in 1X Laemmli buffer. LC-MS Mass Spectrometry and data analysis were performed by Protein Sciences Facility, Roy J. Carver Biotechnology Center, UIUC. For data analysis, Xcalibur raw files were converted by Mascot Distiller into peak lists submitted to an in-house Mascot Server and searched against specific NCBI-NR protein databases, Thermo Proteome Discover, MaxQuant and GeneOntology modules.
To investigate the potential interaction of ORFV113 with LPA 1 , OFTu cells co-transfected with pcDNA3.1 His and pCMV Flag or pCMV HA (control plasmids) or pcDNA3.1-LPA 1 His (pLPA 1 His ) and pCMV-ORFV113 Flag (p113 Flag ) or pCMV-CD2v HA (pCD2v HA ) (plasmid with African swine fever virus membrane protein CD2v used as a specificity control) (1 μg) were harvested 24 h post transfection and membrane extracts were prepared using membrane protein extraction reagent (Thermo Fisher, Waltham, MA). Co-immunoprecipitation was performed on membrane protein extracts with antibodies against His (Genscript; A00186), Flag and HA (Cell Signaling Technology; 3724) as described above. Bound proteins were eluted from beads in 2X Laemmli buffer, resolved by SDS-PAGE, blotted to nitrocellulose membranes, probed with antibodies against His, Flag and HA (Cell Signaling Technology; 2367) and developed as described above.

Co-localization of ORFV113 and LPA 1
To investigate the cellular localization of ORFV113 and LPA 1 , OFTU cells mock infected or infected with OV-IA82RV113 HA for 24 h were fixed with 4% paraformaldehyde, sequentially incubated with primary antibodies against HA and LPA 1 (Novus Biologicals; NBP1-03363), and secondary antibodies labeled with Alexa fluor 488 and 647, counterstained with DAPI, and examined by confocal microscopy.

Effect of p38 inhibitor (SB203580) on ORFV replication and plaque formation
SB203580, a widely used p38 inhibitor, was selected to investigate the effect of p38 inhibition on ORFV replication and plaque size formation [44][45][46]. OFTu cells were pretreated with vehicle control (DMSO) or inhibitor SB203580 (10 μM) for 1 h and then infected with OV-IA82Δ113 (GFP reporter) or OV-IA82RV113 (RFP reporter). Plaque assays were performed in presence of DMSO or SB203580, and fluorescent plaques imaged at 5 days p.i.
To assess the effect of p38 inhibition on ORFV replication, OFTU cell pretreated with vehicle control (DMSO) or inhibitor SB203580 for 1 h were infected with OV-IA82 or OV-IA82Δ113 (MOI:0.1) in presence of DMSO or SB203580, and harvested at various time points, and virus titers determined in OFTu cells and expressed as TCID 50 /ml.

Effect of ORFV113 expression on p38 phosphorylation
To investigate the effect of ORFV113 on p38 phosphorylation, HFF1 cells untreated, UV treated or transfected with pCMV-HA (Empty HA plasmid), or pCMV-ORFV113 HA (ORFV113 HA plasmid) (2 μg) were harvested at 2, 4 and 6 h post transfection. Total cell protein extracts (50 μg) were resolved by SDS-PAGE, blotted and incubated with antibodies against phos-p38, total p38, HA and GAPDH. Blots were processed and protein bands were quantified as described above.
To investigate the potential effect of ORFV113 present in cell culture supernatant on p38 signaling, OFTu cells were treated with clarified culture supernatants harvested from OFTu cells transfected with pCMV-HA (Empty HA plasmid), pCMV-ORFV113 HA (ORFV113 HA plasmid) or pCMV-ORFV113 (Untagged ORFV113 plasmid) for 3 h. Total cell extracts were prepared and p38 phosphorylation was assessed by Western blot and analysed as described above.

Real-time PCR
To investigate the effect of ORFV113 on p38-regulated gene expression, OFTU and HFF1 cells were transfected with pCMV-HA (Empty HA plasmid) or pCMV-ORFV113 HA (ORFV113 HA plasmid) (2 μg) for 1, 2 and 3h and for 6h. RNA was extracted using Zymo Direct-Zol RNA extraction kit (Zymo, Cat # 74104) and reverse transcribed as previously described [15]. The expression of interleukin-8 (IL-8) gene was assessed using Custom Plus TaqMan Gene Expression Assays (Applied Biosystems) based on ovine gene sequences in GenBank. Real-time PCR were performed as previously described [15]. Experiments were conducted with biological and technical triplicates. Results were expressed as fold changes relative to empty HA plasmid treatment.

Treatment of cells with immunoprecipitated ORFV113
OFTu cells transfected with pCMV-HA (Empty HA plasmid) or pCMV-ORFV113 HA (ORFV113 HA plasmid) (2 μg) were harvested 24 h post transfection and whole cell proteins were extracted using mammalian protein extraction reagent (Thermo Fisher, Waltham, MA). Immunoprecipitation was performed following manufacturer's protocols. Whole cell extracts were added to 40 μl pre-washed anti-HA antibody conjugated agarose (Thermo Fisher; PI26181) and incubated overnight at 4˚C. Beads were washed four times with 1X Tris buffered saline containing 0.05% Tween and protein was eluted with 130 μl anti-HA peptide (Thermo Fisher; PI26184). Presence of ORFV113 HA in immunoprecipitated product was assessed by western blot using antibody against HA as described above.
To investigate the effect of the ORFV113 protein on p38 phosphorylation, OFTu cells were treated with 60 μl of empty HA or ORFV113 HA immunoprecipitation product for 1 h and 3 h. Total cell protein extracts (50 μg) were resolved by SDS-PAGE, blotted and incubated with antibodies against phos-p38 and total p38. Blots were processed and protein bands were quantified as described above.

Effect of siRNA LPA 1 knockdown on ORFV113-induced p38 activation
To examine the role of LPA 1 on ORFV113-induced phosphorylation, LPA 1 expression was downregulated using siRNAs. OFTu cells were transfected with universal negative control siRNA (SIC001, Sigma Aldrich) or with three pooled siRNAs directed against sheep LPA 1  reagent (S1452, Sigma Aldrich) and then either harvested for assessing LPA 1 knockdown or treated with immunoprecipitation products from pCMV-HA or pORFV113 HA expressing cells and harvested at 1 h and 3 h post treatment. To measure the LPA 1 knockdown, total cell protein extracts were resolved by SDS-PAGE, blotted and incubated with antibodies against LPA 1 and GAPDH. Densitometry of LPA 1 bands were normalized to the loading control GAPDH. Fold changes were calculated relative to universal negative control siRNA treatment. To assess the effect of LPA 1 knockdown on ORFV113 induced p38 activation, total cell protein extracts were resolved by SDS-PAGE, blotted and incubated with antibodies against phos-p38 and total p38. Blots were processed and protein bands were quantified as described above.

Effect of CRISPR LPA 1 knockdown on ORFV113-induced p38 activation
To construct CRISPR LPA 1 knockdown cells, OFTu cells were co-transfected with LPA 1 CRISPR knock out plasmid and homology directed repair plasmid consisting of Puromycin drug resistance and RFP cassette (Santa Cruz) using Lipofectamine 2000 for 24h. Selection, amplification and maintenance of the culture were performed in the presence of Puromycin dihydrochloride (3μg/ml; Santa Cruz) and monitored by fluorescence microscopy. LPA 1 knockdown was measured by western blot using LPA 1 antibody (Santa Cruz; B-10).
To examine the effect of LPA 1 knock down on ORFV113 induced p38 activation, wildtype and CRISPR knockdown OFTu cells cultured for 16 h were either harvested for assessing LPA 1 knockdown or treated with immunoprecipitation products from pCMV-HA or pORFV113 HA expressing cells and harvested at 1 and 3 h post treatment. Level of LPA 1 knockdown and effect of LPA 1 knockdown on ORFV113 induced p38 activation were examined using western blot and densitometric analysis of bands as described above.

Effect of antibody against LPA 1 and LPA 1 inhibitor (KI16425) on ORFV113 induced p38 signaling
To assess the effect of antibody against LPA 1 on ORFV113 induced p38 phosphorylation, OFTu cells pretreated with IgG 2a mouse isotype control (Thermo Fisher; 026200) or LPA 1 mouse monoclonal antibody (sc-515665;Santa cruz) (10 μg/ml) for 45 min were treated with immunoprecipitation products from pCMV-HA or pORFV113 HA expressing cells in presence of isotype control or LPA 1 monoclonal antibody (10 μg/ml) and harvested at 1 h and 3 h post treatment.
KI16425, a widely used LPA 1 inhibitor was used to investigate the role of LPA 1 on ORF-V113-induced p38 phosphorylation [47][48][49]. OFTu cells were pretreated with DMSO (vehicle control) or KI16425 (10 μM) for 45 min and were then treated with immunoprecipitation products from pCMV-HA or pORFV113 HA expressing cells in the presence of DMSO or KI16425 and harvested at 1 h and 3 h post treatment. Total cell protein extracts were resolved by SDS-PAGE, blotted and incubated with antibodies against phos-p38 and total p38. Blots were processed and protein bands were quantified as described above.

Effect of LPA on ORFV113-induced p38 signaling
To investigate the effect of LPA on ORFV113-induced p38 signaling, phosphorylation of p38 was assessed by Western Blot. OFTu cells transfected with pEmpty HA or pORFV113 HA plasmids for 5 h, were induced with LPA for 5, 10 and 20 min, and harvested. Total cell protein extracts (50 μg) were resolved by SDS-PAGE, and bands were blotted as above. Blots were incubated with antibodies against phos-p38, total p38 and HA. Blots were processed and protein bands were quantified as described above.

Animal inoculations
To evaluate the effect of ORFV113 on ORFV virulence in the natural host, 66-80 lb. lambs were randomly allocated to three experimental groups, deletion mutant virus OV-IA82Δ113 (n = 4; sheep 5, 11, 55 and 130), revertant virus OV-IA82RV113 HA (n = 4; sheep 15, 40, 93 and 430) and mock (n = 3; sheep 25, 38 and 81). Following general anesthesia with ketamine/midazolam, the mucocutaneous junction of the lip near the right labial commissure and the inner sides of the hind limbs were scarified along 2 cm and 5 cm-long lines, respectively. Five hundred μl of virus inoculum (10 7.5 TCID 50 /ml; virus groups) or PBS (control group) were then applied topically to each site using cotton swabs. The scarified areas of the lips were monitored for 20 days for the presence of characteristic orf lesions, including erythema, papules, pustules, and attached scab. Skin biopsy specimens were collected from hind limb inoculation sites at 1 day (d), 2 d, 3 d, and 7 d. p.i., fixed in 10% buffered formalin, and processed following standard histology procedures. To quantify the viral load, skin biopsies were frozen and thawed three times, sonicated, and viral titers determined as described above.

Statistical analysis
Data were presented as mean±standard deviation. Statistical analysis was performed using GraphPad Prism software Version 9.2.0. Statistical significance was determined by Student's ttest for paired comparisons and by one-way ANOVA with post hoc Tukey test for multiple comparisons. A p< 0.05 was considered statistically significant.

ORFV113 is a novel ORFV virion protein that is present at early and late times during infection and localizes in the cytoplasm and plasma membrane
Parapoxvirus 113 (PPV113) are conserved proteins not found outside the Parapoxvirus genus of the Poxviridae. Proteins are 186 to 238 amino acids in length, with % amino acid identities ranging from 37% to 97%. Comparison with available PPV113 sequences reveals that ORFV strain OV-IA82 ORFV113 shares 81%-97% amino acid identity with ORFV homologues isolated from sheep and goat, 64%-69% with PCPV, 36%-42% with BPSV, 47% with sealpox virus, and 38% with red deerpox virus. OV-IA82 ORFV113 encodes for a protein of 211 amino acids, with predicted molecular weight of 22.2 kDa. ORFV113 lacks homology to known proteins outside the PPV genus, and domains suggestive of protein function. A N terminal transmembrane domain (IA82 positions 6-31) and two predicted N-linked glycosylation sites (IA82 positions 53 and 119) are present in the protein (S1 Fig).
The kinetics of ORFV113 expression was assessed during ORFV replication in primary ovine fetal turbinate cells (OFTu) by Western blot using a recombinant virus expressing C-terminally Flag-tagged ORFV113 (OV-IA82-RV113 Flag ). A protein of approximately 35 kDa was detected starting at 2 h p.i. with increasing protein levels and higher molecular weight species between 35 and 55 kDa observed at later time points (Fig 1A). The observed protein molecular weight was approximately 10 to 25 kDa higher than predicted, suggesting that the protein is post-translationally modified. Under non-reducing PAGE, ORFV113 species greater than 250 kDa were observed, suggesting non-covalent association with itself or other proteins under native conformation (S2 The presence of ORFV113 in virions was investigated in intracellular mature virions (IMV) purified from OFTu cells infected with an ORFV113 gene deletion virus (OV-IA82-Δ113) or revertant virus expressing Flag tagged ORFV113 (OV-IA82-RV113 Flag ). Western blot analysis showed a predominant protein band (~35 kDa) and higher molecular weight species (~35 to 55 kDa) corresponding to ORFV113 Flag in OV-IA82-RV113 Flag virions but not in OV-IA82-

PLOS PATHOGENS
ORFV113 activates LPA-p38 signaling Δ113 virions (Fig 1B). The virion core protein ORFV086 was used as a positive control and GAPDH was used as a control for potential cellular contamination. Data indicate that ORFV113 is a virion protein.
The presence of ORFV113 in supernatant of ORFV infected cells was investigated. OFTu cells were mock-infected or infected with OV-IA82-Δ113 or OV-IA82-RV113 HA for 24 h p.i., supernatants harvested, and immunoprecipitation and western blot performed using anti-HA antibody. ORFV113 HA was detected in the supernatant of OV-IA82-RV113 HA -infected cells (Fig 1C). To check for the presence of ORFV113 in supernatant of cells transiently expressing ORFV113, OFTu cells were transfected with pCMV-HA (Empty HA plasmid) or pCMV-ORFV113 HA (ORFV113 HA plasmid) for 24 h, and supernatants harvested, and examined by western blot. Similar to ORFV infected cells supernatant, ORFV113 HA was detected in culture supernatant of cells transiently expressing ORFV113 (Fig 1D). Data indicate that ORFV113 is present in supernatant of ORFV infected cells and in cells transiently expressing ORFV113.
To examine the subcellular localization of ORFV113, OFTu cells were infected with OV-IA82-RV113 HA and examined by confocal microscopy at various times post-infection. ORFV113 staining was observed in paranuclear cytoplasmic region and plasma membrane starting at 3 h p. i. (Fig 1E). Thus, ORFV113 is present at early and late times in infected cells localizing in the cytoplasm and plasma membrane.

ORFV113 is nonessential for ORFV replication in primary OFTu cells but affects ORFV plaque formation
Replication kinetics of ORFV113 gene deletion virus (OV-IA82Δ113) was compared with that of revertant virus (OV-IA82-RV113 Flag ) in OFTu cells. No differences in replication kinetics and viral yields were observed in multi-step and single-step growth curves between the two viruses (Figs 2A, 2B and S4). Plaque assay showed OV-IA82Δ113 plaques were significantly reduced in size (approximately 45% smaller) when compared to OV-IA82-RV113 Flag plaques (Fig 2C and 2D). These data suggest that ORFV113 is nonessential for ORFV replication in OFTu cell cultures but affects ORFV plaque formation.
To confirm the interaction between ORFV113 and LPA 1 , reciprocal co-immunoprecipitation experiments were performed. OFTu cells co-transfected with control plasmid or pORFV113 Flag together with plasmid expressing His tagged LPA 1 (pLPA 1 His ) were harvested at 24 h post-transfection and membrane proteins extracted. Reciprocal co-immunoprecipitation was observed following either anti-Flag or anti-His antibody pull downs (Fig 3A). As a control for specificity of ORFV113 and LPA 1 interaction, CD2v HA was used. No interaction was observed between CD2v HA and LPA 1 His in the membrane fraction of the cells expressing both CD2v HA and LPA 1 His (S6 Fig).
Colocalization of ORFV113 and endogenous LPA 1 was assessed in ORFV infected cells. OFTu cells were mock infected or infected with OV-IA82-RV113 HA and fixed at 24h, immunofluorescence was performed using unpermeabilized cells. Colocalization of ORFV113 and LPA 1 was observed in the membrane and paranuclear region of ORFV-infected cells (Fig 3B). Colocalization of ORFV113 and LPA 1 also was examined in skin biopsies of ORFV infected and mock infected sheep. Colocalization of ORFV113 and LPA 1 was observed in areas of ballooned degenerated keratinocytes in the outermost half of epidermis (Fig 3C). Together, data indicate that ORFV113 interacts with LPA 1 in vitro and in vivo.
To investigate the effect of ORFV113 on p38 activation during ORFV infection, OFTu cells were mock-infected or infected with OV-IA82, OV-IA82-Δ113, OV-IA82-RV113 HA (HAtagged revertant virus) or OV-IA82-RV113 (Untagged revertant virus) for 1, 3, 5, 12 and 24 h p.i. and phosphorylation of p38 was assessed by Western blot. Limited p38 phosphorylation was observed at 1 h p.i. and differences between the treatments were not evident (Fig 4A). Increased phosphorylation of p38 was observed at 3 and 5 h p.i. in virus infected cells

PLOS PATHOGENS
ORFV113 activates LPA-p38 signaling compared to mock infected cells (Fig 4A and 4B). Notably, infection with OV-IA82-Δ113 led to significantly decreased p38 phosphorylation compared to OV-IA82 infected cells at 3 h (40% reduction) and 5 h (10% reduction) post infection and no significant difference was observed between OV-IA82 and both revertant viruses (Fig 4A and 4B). At 12 h p.i., significantly increased p38 phosphorylation was observed in virus infected cells compared to mock infected cells (S7A and S7B Fig). Although data trended toward reduced p38 phosphorylation in OV-IA82-Δ113 cells compared to OV-IA82 infected cells, significant differences were not observed (S7B  (Figs  4A, 4B, S7A and S7B). This shows that p38 signaling regulation during ORFV infection is complex, with ORFV113 possibly being one of several viral factors involved in temporal regulation of p38 signaling. Data indicate that ORFV113 enhances p38 phosphorylation at early times post ORFV infection.

p38 signaling affects ORFV replication in infected OFTu cells
To assess the importance of p38 signaling during ORFV infection, the effect of p38 inhibitor SB203580 on plaque size and ORFV replication was examined. OFTU cells were pretreated with vehicle control (DMSO) or SB203580 for 1 h and infected with OV-IA82-RV113 Flag or OV-IA82Δ113, and plaque assay performed in presence of DMSO or SB203580. Cells were fixed at 5 days post infection and fluorescent plaques were imaged (Fig 5A). OV-IA82-RV113-Flag plaques size was markedly reduced (82% area reduction) in presence of p38 inhibitor compared with plaques in presence of vehicle control. Significant reduction (45% reduction) was observed in OV-IA82Δ113 plaque size in presence of p38 inhibitor compared to vehicle control, however the effect was less remarkable than that observed for OV-IA82-RV113 Flag (Fig 5B).
To examine the effect of p38 inhibitor SB203580 on ORFV replication, OFTU cells pretreated with vehicle control (DMSO) or SB203580 for 1 h were infected with OV-IA82 or OV-IA82Δ113 in presence of DMSO or SB203580, harvested at various time points, and titrated. Wildtype virus titers were significantly reduced in presence of p38 inhibitor at 36h (1.5 log), 48h (1.8 log) and 72h (1.7 log) (Fig 5C). Significant reduction was also observed in OV-IA82Δ113 titers in presence of p38 inhibitor at 36h (0.5 log), 48h (1 log) and 72h (1 log), however, the effect was less dramatic compared to OV-IA82 (Fig 5C). Together the plaque assay and growth curve data in the presence of p38 inhibitor indicate that 1-p38 signaling increases ORFV yields, and 2-presence of ORFV113 enhances susceptibility of ORFV to p38 inhibitor effects.

Membrane lysates (left) and extracts immunoprecipitated with anti-His (upper panel) or anti-Flag (lower panel)
antibodies were analyzed by SDS-PAGE-Western blotting with antibodies directed against proteins indicated on the right. � and �� denote light and heavy chain of the IgG antibody, respectively. (B) OFTU cells mock infected or infected with OV-IA82RV113 HA for 24 h were fixed and left unpermeabilized, and incubated with primary antibody against HA and LPA 1 , and Alexa Fluor 488 and 647 labeled secondary antibodies, counterstained with DAPI and examined by confocal microscopy. Green; ORFV113 HA , Violet; LPA 1 , Blue; DAPI. (C) Skin sections from animals mock infected or infected with OV-IA82RV113 HA were processed for immunofluorescence and confocal microscopy as described above. Green; ORFV113 HA , Violet; LPA 1 , Blue; DAPI. https://doi.org/10.1371/journal.ppat.1009971.g003

ORFV113 induces p38 phosphorylation in cell transiently expressing ORFV113 and in cells treated with soluble ORFV113 protein
To examine the possible effect of ORFV113 alone on p38 phosphorylation, human foreskin fibroblast (HFF-1) cells were untreated, treated with UV for 1 h (positive control), transfected with pCMV-HA (Empty HA plasmid) or plasmid pCMV-ORFV113 HA for 2, 4 and 6 h and p38 phosphorylation assessed by western blot. Significantly increased p38 phosphorylation
To examine downstream gene expression regulated by p38 phosphorylation, OFTu and HFF-1 cells were transfected with pCMV-HA or pCMV-ORFV113 HA , and IL-8 transcript levels assessed by RT-PCR. Significantly increased IL-8 transcription was observed in OFTu cells expressing ORFV113 compared to cells transfected with empty plasmid at 2h (4.9-fold) and 3h
To investigate the potential effect of ORFV113 present in cell culture supernatant (Fig 1C  and 1D) on p38 signaling, OFTu cells were treated with clarified culture supernatants harvested from OFTu cells transfected with pCMV-HA (Empty HA plasmid), pCMV-ORFV113 HA (ORFV113 HA plasmid) or pCMV-ORFV113 (Untagged ORFV113 plasmid) and phosphorylation of p38 was assessed by Western blot at 3 h post treatment. Treatment of cells with culture supernatants from cells expressing ORFV113 HA and Untagged ORFV113 induced significantly higher p38 phosphorylation (3.1 and 5.4 -fold, respectively) compared to control treatment (S8 Fig). To rule out the effect of potential confounding cellular factors present in the culture supernatant on p38 signaling, OFTu cells were treated with immunoprecipitation product from cells expressing pCMV-HA (Empty HA plasmid) or pCMV-ORFV113 HA (ORFV113 HA plasmid) and phosphorylation of p38 assessed by Western blot at 1 and 3h post treatment. Treatment of cells with ORFV113 immunoprecipitation product induced significantly higher p38 phosphorylation (2.8 and 1.9-fold) compared to control treatment at 1 and 3 h, respectively (Fig 6E and 6F). Together, data indicate that both expression of ORFV113 in cells and treatment of cells with soluble ORFV113 protein induces p38 activation.

LPA 1 is involved in ORFV113 induced p38 phosphorylation in OFTu cells
To examine the role of LPA 1 on ORFV113 induced p38 activation, OFTu cells pretreated with DMSO (vehicle control) or LPA 1 inhibitor KI16425 (10 μM) for 45 min were treated with immunoprecipitation products from pCMV-HA or pORFV113 HA expressing cells in presence of DMSO or KI16425 (10 μM), harvested at 1 h and 3 h post treatment and processed for Western blot. Significantly increased p38 phosphorylation was observed in vehicle controltreated cells in response to ORFV113 treatment compared to empty vector-treated cells, 1.7 fold at both 1 h and 3 h post treatment. However, no significant difference was observed between ORFV113 treatment compared to empty vector treated cells in presence of KI16425 (Fig 7A and 7B). Data indicate KI16425 inhibits ORFV113 induced p38 phosphorylation.
To assess the effect of LPA 1 knockdown on ORFV113 induced p38 phosphorylation, OFTu cells were transfected with negative control siRNA or pooled LPA 1 siRNA for 24 h. Cells were either harvested or treated with immunoprecipitation products from pCMV-HA or pORFV113 HA expressing cells, and p38 phosphorylation was assessed at 1 and 3 h post treatment by Western blot. Approximately 45% reduction in LPA 1 routinely was observed in knock down cells (Fig 7C). Significantly increased p38 phosphorylation was observed in negative control siRNA transfected cells in response to ORFV113 treatment compared to empty vector treated cells (1.4 and 1.3-fold at 1 h and 3 h post treatment, respectively). However, no significant difference was observed between ORFV113 treatment compared to empty vector treated cells in LPA 1 siRNA transfected cells (Fig 7D and 7E).
To further examine the effect of LPA 1 knockdown on ORFV113 induced p38 phosphorylation, LPA 1 knock down OFTu cells were obtained using CRISPR. Wild-type and CRISPR LPA 1 knockdown OFTu cells were treated with immunoprecipitation products from pCMV-HA or pORFV113 HA expressing cells, harvested at 1 h and 3 h post treatment and the average of three independent experiments. Error bars represent mean±SD. Statistical analysis was performed as described in B ( � , P<0.05; �� , P<0.01). https://doi.org/10.1371/journal.ppat.1009971.g006

PLOS PATHOGENS
ORFV113 activates LPA-p38 signaling assessed for p38 phosphorylation by Western blot. Approximately 50% reduction in LPA 1 was routinely observed in knockdown cells (Fig 7F). Significantly increased p38 phosphorylation was observed in wild-type OFTu cells in response to ORFV113 treatment compared to empty vector treated cells (2 and 1.5-fold at 1 h and 3 h post treatment, respectively). However, no significant difference was observed between ORFV113 and empty vector treatment in LPA 1 knockdown cells (Fig 7G and 7H). Together, these data suggest that LPA 1 in cells inhibits p38 phosphorylation and binding of ORFV113 to LPA 1 neutralizes its inhibitory function leading to increased p38 phosphorylation in cells mimicking that observed with KI16425 inhibition.

Treatment with LPA and antibody against LPA 1 enhances ORFV113 induced p38 phosphorylation in OFTu cells
Results above indicate that LPA 1 is involved in ORFV113 induced p38 phosphorylation. Several studies have shown that LPA induces p38 phosphorylation in different cell types [32][33][34][35]. In the skin, LPA levels increase in response to tissue injury and induces multiple signaling pathways including the p38 pathway [50]. Thus, a potential role of LPA in ORFV113-induced p38 signaling in natural infection can be hypothesized. To examine the effect of LPA on ORF-V113-induced p38 signaling, OFTu cells transfected with plasmids pCMV-HA or pCMV-ORFV113 HA for 5 h were treated with LPA for 5, 10 or 20 min, harvested and processed for western blot. In cells transfected with control plasmids, significantly increased p38 phosphorylation was observed in cells treated with LPA for 5 min (1.8-fold) compared to untreated cells. Significantly increased p38 phosphorylation was observed in ORFV113 expressing cells treated with LPA for 20 min (1.6 -fold) compared to untreated cells (Fig 8A  and 8B). Data indicate that LPA enhances ORFV113-induced p38 signaling.
To assess the involvement of LPA 1 on ORFV113 induced p38 phosphorylation, OFTu cells pretreated with isotype control or LPA 1 monoclonal antibody for 45 min were treated with immunoprecipitation products from pCMV-HA or pORFV113 HA expressing cells in presence of antibodies and harvested at 1 h and 3 h, and processed for Western blot. Treatment with antibody against LPA 1 and with ORFV113 significantly increased p38 phosphorylation compared to isotype control/ORFV113 treated cells at 1 h post treatment (1.5-fold) (Fig 8C and 8D). Data suggests that treatment with antibody against LPA 1 enhances ORFV113 induced p38 phosphorylation.

ORFV113 is a major virulence determinant in the natural host
The effect of ORFV113 in disease outcome was investigated in sheep, a natural ORFV host. Groups of animals were inoculated with deletion mutant virus OV-IA82Δ113 (n = 4), revertant virus OV-IA82RV113 HA (n = 4) or PBS (control group, n = 3) in the right labial commissure and the inner side of the thighs, and disease course was monitored for 20 days. Sheep in the revertant virus group exhibited gross changes in the lip compatible with orf as early as day 2 p. i., with mild erythema (#40), or erythema, papulae, and pustules (#15, #93 and # 430) at inoculation sites. By day 4 p.i., lesions exacerbated in all sheep in the group except sheep #40, in which erythema regressed (Fig 9A, RV113). Lesions in sheep #15, #93 and # 430 continued to expand and by day 8 p.i. showed marked scabby tissue deposition in the upper lip, while sheep #40 showed a single small papule in the lower lip. On day 16 p.i., lesions in sheep #40 and #93 regressed, while those in sheep #15 and #430 continued to enlarge by further scabby tissue deposition (Fig 9A). By day 20 p.i. (end point of the experiment), lesions in sheep #15 and #430 abated. Similar to control sheep, and in marked contrast with revertant virus-infected sheep, the four animals infected with OV-IA82Δ113 did not exhibit any sign of ORFV infection during the course of the experiment (Fig 9A, Δ113). This indicates that ORFV113 is a significant ORFV virulence determinant.
Punch biopsies were collected from inoculation sites in the thighs at 1, 2, 3 and 7 days p.i. and processed for histology. By day 2 p.i., samples from all animals showed changes associated with skin scarification, including epidermal hyperplasia, active re-epithelialization, and various degrees of neutrophil infiltration. Zero of three, 3 of four, and 2 of four sheep in control, revertant virus, and deletion mutant virus groups, respectively, exhibited small foci of keratinocytes with ballooning degeneration (BD) in the stratum spinosum of the epidermis, which represent dying cells containing progeny virions as previously described [51]. By day 3 p.i., multiple expanding BD sites were observed in all four sheep infected with revertant virus (Fig 9B, left  panel, RV113). In contrast, BD sites in the deletion mutant group were not seen in two sheep (#11 and 55), and remained restricted in size in sheep #5 and #130 (as an example for restricted BD, see arrowheads in sheep #130). In addition, skin infiltrates of inflammatory cells were more populated in the revertant group than in the deletion mutant group (Fig 9B, compare the two columns in left panel). By day 7 p.i., three of the sheep in the revertant virus group (# 15, # 40, and # 430) and none of the sheep in the deletion mutant virus group exhibited epidermal areas of BD and significant inflammatory cell infiltration (Fig 9B, right panel). No ORFVassociated changes were observed in control group samples during the experimental time. Together, data indicate that infection of sheep with OV-IA82Δ113, a virus lacking ORFV113, is controlled by the host before gross lesions and significant histopathological changes take place. Consistent with this, OV-IA82Δ113 titers in skin biopsy samples were approximately, 10-100 fold lower than revertant virus titers in the first week post infection, and 1,000-10,000 fold lower by day 12 p.i. (Fig 10).

ORFV113 enhances p38 signaling in ORFV-infected keratinocytes in vivo
To investigate the effect of ORFV113 on p38 signaling in the skin, skin sections from ORFVinfected animals were examined by immunofluorescence and confocal microscopy. We first determined that cells infected with OV-IA82RV113 HA or OV-IA82Δ113 (a virus expressing GFP) can be detected in the most apical layer of the hair follicle infundibulum as early as 24 h p.i (Fig 11). Next, expression of LPA 1 , phos-p38 and IL-8 was examined on skin sections from control and infected animals at 24, 36, 48 and 72 h. Increased LPA 1 expression was observed in keratinocytes infected with OV-IA82RV113 HA at 24, 36 and 48 h p.i., suggesting that ORFV113 induces expression of LPA 1 (Fig 12A and 12B). Likewise, keratinocytes infected with OV-IA82RV113 HA but not with OV-IA82Δ113 showed increased levels of phos-p38 and IL-8 compared to non-infected cells, indicating that ORFV113 induces p38 phosphorylation and IL-8 expression (Fig 12C-12F). Together, data suggest that ORV113 enhances p38 signaling in the epidermis of ORFV infected animals.

Discussion
LPA signaling in the skin is known to affect a wide range of cellular functions, including cell differentiation, migration, proliferation and chemotaxis, and it has been implicated in innate immune responses, inflammation and wound healing [21][22][23][24][25]. Here, we describe a novel ORFV protein, ORFV113, which interacts with the GPCR LPA 1 receptor (Fig 3) and enhances kinase p38 phosphorylation and signaling in ORFV-infected cells, cells transiently expressing ORFV113 and cells treated with soluble ORFV113 (Figs 4 and 6).
Some viruses have evolved mechanisms to hijack host GPCR signaling. Several herpes viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV) [52], human cytomegalovirus (HCMV) [53], and Epstein-Barr virus (EBV) [54], encode constitutively active GPCRs homologs (vGPCR) that allow viruses to activate multiple cellular signaling pathways akin to host GPCR. Other viruses use GPCRs as co-receptors for cell entry. For example, GPCRs CXCR4 and CCR5 are essential for HIV fusion and entry into target cells [55].

PLOS PATHOGENS
ORFV113 activates LPA-p38 signaling Viral modulators of p38 signaling have been described but none have been linked to LPA-p38 signaling pathway. For example vaccinia virus protein A52R, was found to activate p38 MAPK in a TRAF6 dependent manner and vaccinia virus E3 suppressed p38 phosphorylation in infected cells via an unknown mechanism [39][40][41]. Additional viral proteins modulating p38 signaling have been described. Chikungunya virus protein nsP2 interacted with phos-p38 and phos-JNK protein [56]. Herpes simplex virus type-1 (HSV-1) virion transactivator protein VP16 stimulated p38/JNK activation, however the mechanism of action is not known [57]. Expression of HSV-1 protein ICP27 alone directly activated p38 signaling, leading to stimulation of the host cell apoptotic pathways [58]. Signaling by KSHV-encoded GPCR resulted in p38 activation, leading to phosphorylation of hypoxia-inducible factor 1 alpha and resulting in increased expression of vascular endothelial growth factor [59].
We observed significantly decreased levels of p38 phosphorylation when cells were infected with OV-IA82Δ113 suggesting that ORFV113 enhances p38 signaling in infected cells (Fig 4). Consistent with this observation, decreased levels of phos-p38 and IL-8 at early times p.i. were observed in animals infected with OV-IA82Δ113 (Fig 12C-12F). These data indicate that ORFV113 expression enhances phosphorylation of p38 and subsequent signaling events (i.e increased IL-8 transcription) both in vitro and in vivo (Figs 6C, 6D, 12E and 12F).
Data show that ORFV113 is nonessential for ORFV replication in OFTu cells in vitro (Fig  2A and 2B) but is important for virus replication in keratinocytes in vivo (Fig 10). Notably, ORFV113 is important for ORFV plaque formation in cultured cells (Fig 2C and 2D), suggesting that expression of ORFV113 in infected cells and possibly signaling induced by ORFV113 in neighboring cells are important during ORFV cell-to-cell spread (Fig 13). It is tempting to speculate that the restricted size and small number of infection foci seen at 2 and 3 days p.i. in sheep infected with OV-IA82Δ113 (Fig 9B) reflect this same limited ability of virus to spread.
Consistent with the observation above, plaque assay and virus growth data in presence of a p38 kinase inhibitor (Fig 5) indicate that p38 signaling is essential for ORFV replication and normal plaque formation. As expected, the effect of the p38 inhibitor was not as pronounced on plaque size and viral yields of OV-IA82Δ113, a virus deletion mutant lacking ORFV113, compared to the wildtype virus, suggesting that ORFV113 enhances ORFV susceptibility to the effect of p38 inhibitor and may be involved in regulating p38 signaling during ORFV infection.
It is generally accepted that ORFV infection requires a severed skin or mucosa that expose activated keratinocytes to inoculum virus [2,60,61]. Prickly plants or stubble contaminated with shed scabs, and animals with active lesions are the usual sources of virus. The notion of epidermal damage as a prerequisite for infection is reinforced by studies showing that typical orf lesions developed after hair plucking or skin scarification followed by topical virus inoculation, but not after intradermal, subcutaneous or intravenous virus injection, or topical virus inoculation of intact skin [2]. The strategies of ORFV to ensure proper virus replication in the context of active host inflammatory and repair responses at virus entry sites are unknown. LPA levels significantly increase in areas with skin damage [50] and animal studies have shown that LPA reduces the duration of skin wound healing [62][63][64]. LPA has been shown to induce the migration, proliferation and differentiation of keratinocytes and dermal fibroblasts, which are essential processes underlying skin tissue repair and reconstruction [65][66][67]. We can speculate an interplay between ORFV113 and LPA 1 affecting downstream events of LPA signaling. phos-p38 (C) RV113 and Δ113 (D), Green: ORFV113 HA or GFP, Violet: phos-p38, Blue: DAPI.; (E and F) IL-8 expression (E) RV113 and Δ113 (F), Green: ORFV113 HA or GFP, Violet: IL-8, Blue: DAPI. https://doi.org/10.1371/journal.ppat.1009971.g012

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ORFV113 activates LPA-p38 signaling Infection of sheep with deletion mutant virus OV-IA82Δ113 resulted in striking disease attenuation compared with wildtype virus infection (Fig 9). This was reflected by the absence of gross lesions, the temporally and spatially restricted histopathological changes, and the markedly reduced viral load, most likely a consequence of the poor OV-IA82Δ113 replication, spread, or both (Figs 9, 10 and 12). Further studies will be required to investigate the interplay of LPA signaling and ORFV113 in the context of ORFV pathogenesis in vivo.
Previously, ORFV antigen has not been detected in vivo before 2 days post-infection using the scarification model [68]. Here, we detected viral antigen at 24 h p.i., with signal consistently associated with the apical layer of the outer root sheath (ORS) in the infundibular region of hair follicles (HF) (Figs 11 and 12), suggesting that keratinocytes in this layer are more sensitive to virus infection than keratinocytes in the interfollicular (superficial) epidermis (IFE). This was unexpected since ORFV is thought to target activated keratinocytes associated with repair responses in the IFE and HF's ORS [2,69]. In contrast, the apical layer of the HF infundibulum is thought to be fully differentiated and as such it represents a very different cell environment for virus replication. The infundibular region of HFs is a major site of chemokine and antimicrobial peptide production, and expresses high levels of toll-like receptors [70]. Our in vivo data showed transient p38 activation early in infection (Fig 12C and 12D), suggesting that this may be a requirement for early viral replication in these differentiated infundibular The fact that ORFV113 is a virion component suggests it performs an important function (s) in infected cells, which likely involves early targeting of LPA 1 and/or other cellular proteins (Fig 1B). ORFV113 expressed in the membrane of infected cells might be interacting with LPA 1 on adjacent cells in a juxtacrine manner, leading to p38 phosphorylation in neighboring cells (Figs 1E, 3B and 3C). Alternatively, soluble ORFV113 may perform the same function in a paracrine manner as ORFV113 is present in the supernatant of infected cells (Fig 1C). The mechanism by which ORFV113 is present in the supernatant is yet to be determined. Thus, LPA-p38 signaling targeted by ORFV113 affects ORFV replication and spread both in vitro and in vivo (Fig 13). Rock.