Effects of the synthetic corticosteroid dexamethasone on bovine herpesvirus 1 productive infection

Virology 505 (2017) 71–79

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Effects of the synthetic corticosteroid dexamethasone on bovine herpesvirus 1 productive infection

MARK

⁎

Liqian Zhua,b, Jesse Thompsonc, Fangrui Mac, James Eudyd, Clinton Jonesa, a

Oklahoma State University, Center for Veterinary Health Sciences, Department of Veterinary Pathobiology, Stillwater, OK 74078, USA College of Veterinary Medicine and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, 48 Wenhui East Road, Yangzhou 225009, China c University of Nebraska, Nebraska Center for Virology, Morisson Life Science Center, Lincoln, NE 68583-09065, USA d Department of Genetics Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska, USA b

A BS T RAC T Sensory neurons are a primary site for life-long latency of bovine herpesvirus 1 (BoHV-1). The synthetic corticosteroid dexamethasone induces reactivation from latency and productive infection, in part because the BoHV-1 genome contains more than 100 glucocorticoid receptor (GR) responsive elements (GREs). Two GREs in the immediate early transcription unit 1 promoter are required for dexamethasone induction. Recent studies also demonstrated that the serum and glucocorticoid receptor protein kinase (SGK) family stimulated BoHV-1 replication. Consequently, we hypothesized that dexamethasone influences several aspects of productive infection. In this study, we demonstrated that dexamethasone increased expression of the immediate early protein bICP4, certain late transcripts, and UL23 (thymidine kinase) by four hours after infection. SGK1 expression and Akt phosphorylation were also stimulated during early stages of infection and dexamethasone treatment further increased this effect. These studies suggest that stress, as mimicked by dexamethasone treatment, has the potential to stimulate productive infection by multiple pathways.

1. Introduction Bovine herpesvirus 1 (BoHV-1) is a significant risk factor for bovine respiratory disease complex (BRDC), the most important disease in cattle. Infection of cattle with BoHV-1 frequently results in upper respiratory tract disease, erosion of mucosal surfaces, and immune suppression thus promoting opportunistic bacterial infections (Hodgson et al., 2005; Jones and Chowdhury, 2010). Mannheimia haemolytica (MH) is a commensal bacterium normally found in the upper respiratory track of healthy cattle: however, it is frequently found in lungs of cattle with pneumonia indicating it is important for BRDC (Highlander, 2001; Highlander et al., 2000; Zecchinon et al., 2005). BoHV-1 enhances interactions between the MH leukotoxin and bovine peripheral blood mononuclear cells, including neutrophils, leading to cell death and acute inflammation in the lung (Leite et al., 2004; Rivera-Rivas et al., 2009). As a result, co-infection of calves with BoHV-1 and MH consistently leads to life-threatening pneumonia (Yates et al., 1983). A receptor for BoHV-1, poliovirus receptor related 1 (PVRL1), was recently identified as a BRDC susceptibility gene for Holstein calves (Neibergs et al., 2014) underscoring the importance of BoHV-1 in this poly-microbial disease. BoHV-1 genes are expressed in three distinct phases during ⁎

productive infection of cultured cells: immediate early (IE), early (E), or late (L) (Jones, 1998, 2003). IE gene expression is stimulated by VP16, a tegument protein (Misra et al., 1994, 1995). IE transcription unit 1 (IEtu1) encodes two transcriptional regulatory proteins, bICP0 and bICP4 (Wirth et al., 1992, 1989, 1991). The IEtu1 promoter drives expression of a single IE transcript that is differentially spliced and then translated into bICP0 and bICP4. The bICP0 protein is also translated from an E mRNA (E2.6) because a separate E promoter regulates the bICP0 E transcript (Fraefel et al., 1994; Wirth et al., 1989, 1991,1992). Expression of the bICP4 protein represses IEtu1 promoter activity whereas bICP0 activates its E promoter and other viral promoters. IEtu2 expresses a 1.7 kb IE and L transcript that encodes bICP22, which has been reported to repress viral promoters in transient transfection assays (Koppel et al., 1997; Schwyzer et al., 1994). Following acute infection, trigeminal ganglia (TG) are a primary site for life-long latency (Jones et al., 2011; Jones, 2013). Increased corticosteroid levels, due to food and water deprivation during shipping of cattle, weaning, and/or dramatic weather changes increase the incidence of BoHV-1 reactivation from latency, (Jones, 2013, 2014). The synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress, stimulates productive infection (Kook et al., 2015) and

Corresponding author.

http://dx.doi.org/10.1016/j.virol.2017.02.012 Received 11 November 2016; Received in revised form 6 February 2017; Accepted 14 February 2017 0042-6822/ © 2017 Published by Elsevier Inc.

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samples was assessed by BioAnalyzer analysis (Agilent Technologies, Santa Clara, CA) and only intact, non-degraded RNA samples were used for further analysis. RNAseq libraries were constructed beginning with 500 ng of total RNA of each sample and the procedure was performed using the TruSeq Stranded Total RNA kit (Illumina, San Diego; catalogue # 15031048) per suggestions of the manufacturer. The libraries were subjected to 100 base pair/pair-end sequencing on an Illumina HiSeq2500 sequencer to an average depth of ~40 million reads per sample.

consistently initiates BoHV-1 reactivation from latency (Inman et al., 2002; Jones, 1998, 2003; Jones et al., 2000, 2006; Rock et al., 1992; Sheffy and Davies, 1972; Shimeld et al., 1990). Increased stress also correlates with herpes simplex virus 1 (HSV-1) reactivation from latency (Cassidy et al., 1997; Du et al., 2012; Glaser et al., 1985; Halford et al., 1996; Padgett et al., 1998). Canine herpesvirus type 1, another α-herpesvirinae subfamily member, consistently reactivates from latently infected beagles following treatment with the synthetic corticosteroid prednisone (Ledbetter et al., 2009). Corticosteroids bind and activate the GR or mineralocorticoid receptor (MR), (Oakley and Cidlowski, 2013). Collectively, these studies suggest that corticosteroids activate the GR and MR: consequently the incidence of reactivation from latency increases. Based on published studies, we hypothesize that activation of the GR and/or MR by corticosteroids stimulates productive infection by several mechanisms, including activation of viral gene expression. We further predict that understanding how DEX influences productive infection in cultured cells would shed light on its ability to initiate reactivation from latency. Consequently, initial studies focused on examining the effects of DEX on BoHV-1 gene expression during productive infection. Expression of bICP4 RNA and protein, but not the other viral IE genes (bICP0 and bICP22), was increased in bovine cells following DEX treatment. Expression of UL10, UL16, and UL17 expression, all late genes, was also increased more than 4 fold at 4 h after infection when cells were treated with DEX. Additional studies demonstrated that viral infection and DEX treatment stimulated expression of the protein kinase serum glucocorticoid receptor kinase 1 (SGK1), which stimulates productive infection (Kook and Jones, 2016). Finally, a protein kinase that has a similar catalytic domain as SGK1 and phosphorylates similar substrates (Akt) was phosphorylated within 30 min after infection and DEX enhanced Akt phosphorylation. These studies demonstrated DEX regulates viral gene expression and cellular protein kinases that enhance productive infection.

2.4. Western blot analysis Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) with protease and phosphatase inhibitors (Thermo-Scientific). The respective samples were boiled in Laemmli sample buffer for 5 min and all samples separated on an 8% or 10% SDS–polyacrylamide gel. Western blots were performed as previously described (Liu et al., 2016). Antibodies that specifically recognize the following cellular proteins were used for these studies: phosphorylated Akt at serine residue 473 (Cell Signaling Technology; catalogue# 9271), total Akt (Cell Signaling Technology; catalogue# 9272), SGK1 (Abcam; catalogue# ab59337), or tubulin. A peptide-specific rabbit antibody directed against bICP0 (Affinity Bioreagents, Golden, CO) was affinity purified and specifically recognizes bICP0 in infected or transfected cells. Peptide specific bICP4 and bICP22 antiserum were generated in rabbits. 2.5. Quantification of mRNA by qRT-PCR Total RNA was extracted from infected cells using TRIzol LS Reagent (Ambion, Cat: 10296010) following the manufacturers instructions. Freshly prepared total RNA (1 μg) was used as a template for synthesis of first-strand cDNA with commercial random hexamer primers using Thermoscript™ RT-PCR system Kit (Invitrogen, catalogue #11146-024) following the manufacturers instructions. The cDNA products were used as templates for real-time quantitative PCR to measure levels of viral mRNA with gene-specific primers. For these studies, we analyzed UL10 (forward primer: 5’CTGGTGGAAGTAGTCGTG-3’ and reverse primer: 5’TTCGTCGGGCTCTTTTGC-3’), UL16 (forward primer: 5’CGAGTGCGGGCGGTCTTC-3’ and reverse primer: 5’AGCAGGGCTTCGTCCATC-3’), and UL17 (forward primer: 5’GACTTGCTCTTCGCCTAC-3’ and reverse primer: 5’GTCGTCAAAGCCGCCTTC-3’). Analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. GAPDH primers used for this study are, forward primer: 5’CCATGGAGAAGGCTGGGG-3’ and reverse primer: 5’AAGTTGTCATGGATGACC-3’). Real-time PCR was carried out using the ABI 7500 fast real-time system (Applied Biosystems, CA). Expression levels of UL10, UL16 and UL17 were normalized to the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The mRNA level of each viral gene was shown as fold of expression (2ΔΔCT ) in the graph as compared to control cells that were infected with BoHV-1 for 4 h without DEX treatment.

2. Materials and methods 2.1. Viruses & plasmids The Cooper strain of BoHV-1 (wt virus) was obtained from the National Veterinary Services Laboratory, Animal and Plant Health Inspection Services, Ames, IA. BoHV-1 stocks were prepared in CRIB (established bovine kidney) cells. Bovine turbinate cells from a newborn calf were obtained from ATCC (CRL-1390). These are normal cells that exhibit contact inhibited growth and are not immortal. For these studies, BT cells were passaged less than 10 times and thus are referred to as low-passage cells. All cells were grown in Eagle's minimal essential medium (EMEM) supplemented with. 10% Fetal bovine serum (FBS), penicillin (10 U/ml), and streptomycin (100 μg/ml). Where indicated, BT cells were incubated with 2% stripped fetal calf serum. 2.2. RNA preparation Confluent BT cells in 100 mm dishes were incubated with 2% stripped FBS for 12 h. Cultures were then treated with 100 μM of DEX for 1 h, and infected with BoHV-1 at an MOI of 1 for 4 h in the presence of 2% stripped FBS and DEX. As controls, certain cultures were not treated with DEX prior to and after infection. RNA from BT cells was collected at four hours after infection and total RNA was prepared using Trizol reagent (Life Technologies) as previously described (Workman et al., 2012, 2009).

3. Results 3.1. Effect of DEX on viral gene expression during productive infection To examine the effect that the synthetic corticosteroid dexamethasone has on viral RNA expression, low passage bovine turbinate (BT) cells were infected with BoHV-1 for four hours and “deep sequencing” performed. Cultures not treated with DEX served as a control. The genes that were stimulated more than 2-fold by DEX treatment included UL10, UL16, UL17, UL21, UL23, UL24, UL25, UL26, US2,

2.3. Next generation sequence library generation protocol Prior to the generation of the libraries, the integrity of the RNA 72

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Fig. 1. Effects of DEX on viral gene expression in BT cells. Confluent BT cells in 100 mm dishes were incubated with 2% stripped FBS for 12 h. Cultures were then treated with 100 μM DEX for 1 h, and then infected with BoHV-1 at an MOI of 1 for 4 h in the presence of 2% stripped FBS and 100 uM DEX. As a negative control, infected cells were treated with media containing stripped fetal bovine serum, but no DEX. At 4 h after infection (hours), total RNA was prepared and Next Generation Sequencing performed, as described in the materials and methods.

not bICP0 or bICP22, expression at 4 h after infection. RT-PCR studies were utilized to confirm whether DEX stimulated expression of certain late gene. We examined the three late genes that were stimulated the most by DEX, UL10, UL16, and UL17 (Fig. 1 and Table 1). The levels of UL10 was approximately 4-fold higher at 4 h after DEX treatment relative to samples not treated with DEX, but by 8 h after infection the effect of DEX was minimal (Fig. 3A). In BT cultures infected with BoHV-1 for four hours and treated with DEX, a 7-fold increase of UL16 cDNA levels was observed (Fig. 3B). A 16-fold increase for UL17 cDNA levels was also observed at four hours after infection and DEX treatment (Fig. 3C). Although these studies demonstrated that DEX stimulated expression of UL10, UL16, and UL17 gene expression, it was also clear that the effects of DEX were transient during productive infection.

Table 1 Summary of BoHV-1 genes that were differentially expressed at least 2 fold by DEX. Function of the respective genes was derived from HSV-1 functions or when known BoHV-1 studies. Circ is a novel gene found in BoHV-1, but not HSV-1. Gene

DEX affect

Expression class

Function

UL10 (gM) UL16

>4

Late

Interacts with UL49.5

>4

Late

UL17

>4

Late

UL21

>2

Late

UL23

>2

Early

UL24 UL25

>2 >2

Late Late

UL26 UL50 bICP4 US2

>2 >2 >2 >2

Late Late IE Late

Circ

<2

IE

UL4 UL5 UL8

<2 <2 <2

Late Early Early

Tegument protein that interacts with gE: mutant grows poorly DNA Capsid & Tegument, packaging Facilitates viral gene expression & promotes egress from nucleus thymidine kinase: DNA replication Virulence factor Required for packaging but not cleavage of replicated viral DNA Viral encoded protease Non-essential Stimulates viral transcription Membrane associated ubiquitininteracting protein Non-essential gene, myristoylated protein Non-essential gene DNA helicase/primase DNA helicase/primase

3.2. BoHV-1 and DEX stimulate SGK1 expression Since an overall goal is to understand the effect that stress has on viral replication, we tested whether infection and/or DEX regulated steady state protein levels of the serum glucocorticoid protein kinase 1 (SGK1). The rational for this study is based on recent studies demonstrating that SGK1 stimulates BoHV-1 and HSV-1 productive infection (Kook and Jones, 2016) and SGK1 protein expression is stimulated by growth factors and glucocorticoids (Webster et al., 1993). Steady state SGK1 protein levels increased more than 2 fold at 4 and 8 h after infection (Fig. 4A, left and right panels). At 4 and 8 h after infection, virus infection and DEX treatment increased SGK1 protein levels by more than 4 fold (Fig. 4B). DEX and infection increased SGK1 levels more than 5-fold at 16 h after infection. DEX treatment also increased steady state SGK1 levels in uninfected BT cells approximately 2 fold after 4 h treatment (Fig. 4C), which was expected because a previous report demonstrated that DEX stimulated SGK1 protein levels (Brenan et al., 2000). As shown in Fig. 4B, DEX+infection further increased steady state SGK1 levels at 4 h after infection (Fig. 4C). In summary, these studies demonstrated that BoHV-1 infection and DEX stimulated SGK1 steady state levels better than DEX or viral infection alone.

bICP4, and UL50 (Fig. 1 and Table 1). UL8, Circ, UL5, and UL4 RNA expression were reduced more than 2-fold by DEX treatment. Although most of the viral genes induced by DEX were late genes, bICP4 and UL23 are expressed as an IE and E gene respectively. Western blot analysis was performed as an independent method to confirm bICP4 expression was stimulated by DEX. As controls, we examined expression of the other IE proteins (bICP0 and bICP22) because bICP0 RNA expression was not affected by DEX treatment but bICP22 RNA expression was slightly inhibited (Fig. 1). At 4 h after infection, a bICP4 specific band was detected in BT cells treated with DEX (Fig. 2A), but DEX only had a slight stimulatory effect. bICP4 protein levels were approximately 2-fold higher at 8 h after infection in samples treated with DEX. Steady state levels of bICP22 protein expression were reduced slightly at 4 and 8 h after infection when cultures were treated with DEX (Fig. 2B). By 16 h after infection, bICP22 levels were slightly higher in cultures treated with DEX. Regardless of whether cultures were treated with DEX, bICP0 steady state protein levels were nearly identical at 4, 8, and 16 h after infection (Fig. 2C). As expected, antibodies that recognize the respective viral specific IE proteins do not recognize a protein in uninfected cells. In summary, this study confirmed that DEX had an effect on bICP4, but

3.3. Effects of DEX and infection on phosphorylation of Akt SGK1 and Akt protein kinases belong to the AGC (cAMP-dependent, cGMP, and protein kinase C) family of serine/threonine protein kinases because they have similar catalytic domains and phosphorylate some of the same substrates, (Pearce et al., 2010). Akt activation requires phosphorylation at threonine 308 and serine 473 by two cellular protein kinases: PDK1 (phosphoinositide-dependent kinase) and mTORC2 [mTOR (mammalian target of rapamycin) complex 2]. Akt phosphorylation occurs in cultured cells infected with HSV-1 and 73

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Fig. 2. DEX differentially regulates expression of viral IE proteins. Confluent low-passage BT cells in 100-mm dishes were incubated with 2% stripped fetal bovine serum for 12 h prior to infection. Cultures were then pretreated with 100 μM water soluble DEX in 2% stripped fetal bovine serum for 1 h, and then infected with BoHV-1 at a MOI of 1. Throughout infection, cultures were treated with DEX (100 μM) in 2% stripped fetal bovine serum. Cells not treated with DEX were treated with media containing 2% stripped fetal bovine serum. At the designated times after infection (hours), cells were lysed using RIPA buffer, 50 μg protein loaded in each lane, and proteins separated on 8 or 6.5% SDS-PAGE gels. Western blots were performed to detect bICP4 (Panel A), bICP22 (Panel B), and bICP0 (Panel C). A representative Western Blot from three independent studies is shown. Quantification of bands from Western blots in Panels A-C were collected using the software image J package. The band intensity of bICP4, bICP22, and bICP0 was initially normalized to tubulin. Samples from the respective time-point after infection that were not treated with DEX were normalized to 1 and then the fold change in DEX-treated cultures from that time-point calculated. The graphs showing the quantification are the average of three independent experiments and error bars denote the mean of the three experiments.

infection (Fig. 5B). Infection+DEX consistently increased p-Akt(S473) levels more at 4, 8, and 16 h after infection when compared to infection alone. DEX treatment of BT cells for 4 h stimulated p-Akt(S473) levels approximately 10 fold (Fig. 5C), and as shown in Fig. 5A and B infection enhanced p-Akt(S473) levels. In summary, these studies demonstrated that infection and DEX treatment dramatically increased p-Akt(S473) levels. Although p-Akt(S473) stimulation varied between the individual repeats, low levels of p-Akt(S473) in uninfected BT cultures made it difficult to precisely quantify differences after infection. In contrast to p-Akt(S473), total Akt steady protein levels were not stimulated dramatically by DEX and infection.

viral protein kinases are, in part, required for Akt phosphorylation (Easton et al., 2014), which suggested BoHV-1 infection and DEX may regulate Akt phosphorylation. By 30 min after infection, Akt phosphorylation at serine 473 [p-Akt(S473)] was readily detected in BT cells (Fig. 5A). Levels of p-Akt(S473) increased more than 50 fold at 8 or 16 h after infection (Fig. 5A, right panel). Total Akt levels decreased at 8 and 16 h after infection, which may be due to the host-shutoff function of BoHV-1. Additional studies examined the effect that infection, DEX treatment, or DEX+infection had on p-Akt(S473) and total Akt protein levels. Consistent with studies presented in Fig. 5A, BoHV-1 infection increased p-Akt(S473) levels between 25 and 60 fold from 4 to 8 h after

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stimulated by DEX and viral infection in an additive fashion. Finally, these studies provided evidence that DEX and viral infection activated Akt by stimulating phosphorylation of Akt at serine 473, which must be phosphorylated for activation (Pearce et al., 2010). The presence of multiple GREs across the UL region of the BoHV-1 genome (Kook et al., 2015) correlated with increased expression of 9 genes within the UL region. With the exception of UL23 (thymidine kinase), the other UL genes stimulated by DEX are L transcripts. Interestingly, HSV-2 proteins encoded by UL21 (Sage et al., 2013) and UL24 (Blakeny et al., 2005) facilitate IE gene expression and enhance virulence respectively. Many genes in the UL region were not affected by DEX suggesting that specific GREs may mediate this process during productive infection. It is difficult to predict where the important GREs are within the UL because a GRE can be located 5–19 Kb pairs upstream of a promoter and stimulate promoter activity (Polman et al., 2012). It is also noteworthy to point out that HSV-1 and likely BHV-1 genomes exist as “silent” chromatin during latency, (Knipe and Cliffe, 2008): conversely HSV-1 DNA is associated with unstable chromatin during productive infection (Lacasse and Schang, 2010) and (Lacasse and Schang, 2012). Thus, chromatin conformation of the BoHV-1 genome may affect GR interactions with the viral genome and what viral genes are activated by stress. The impact of VP16 and other tegument proteins on IE gene expression may have also reduced or altered the impact of DEX on viral gene expression. The effects of DEX on viral gene expression may have been transient because the ½ life of DEX is 4 h in rats (Perez et al., 1998). Studies designed to understand the effect of DEX on BoHV-1 gene expression in the absence of VP16 need to be compared to results obtained in this study. The BoHV-1 repeats contain several putative GREs, including two that are crucial for DEX mediated activation of the IEtu1 promoter in transfected cells (Kook et al., 2015). The IEtu1 promoter drives expression of a single transcript, which is differentially spliced and translated into bICP0 and bICP4 (Wirth et al., 1989, 1991, 1992). There are several reasons why bICP4, but not bICP0 expression, was induced by DEX in productively infected BT cells. First, bICP0 expression levels may be primarily regulated by the bICP0 E promoter (Fraefel et al., 1994; Wirth et al., 1992). Secondly, a recent report suggested that a separate bICP0 promoter and bICP4 promoter regulate IE expression of these regulatory proteins (Pokhrial et al., 2016). Finally, it is possible that splicing of the bICP4 transcript preferentially occurred following DEX treatment. The finding that bICP22 RNA and protein expression was slightly reduced by DEX is consistent with the finding that DEX treatment reduced bICP22 promoter activity in transfected Neuro-2A cells (Kook et al., 2015). The ability of DEX and/or BoHV-1 infection to stimulate SGK1 protein levels in BT cells is intriguing because a SGK-specific inhibitor significantly reduces viral replication (Kook and Jones, 2016). The SGK family contains three members (SGK1, SGK2, and SGK3), (Pearce et al., 2010). SGK1, but not SGK2, mRNA and protein levels are rapidly stimulated by corticosteroids, cellular stress signals, and growth factors (Bell et al., 2000; Brenan and Fuller, 2000; Buse et al., 1999; Imaizumi et al., 1994; Maiyar et al., 1996; Mizuno and Nishida, 2001; Webster et al., 1993). p-Akt(S473), the activated form of Akt, has many substrates and functions, including inhibiting apoptosis signaling pathways (Manning and Cantley, 2007). HSV-1 encoded protein kinases (US3 and UL13) stimulate Akt phosphorylation during productive infection (Easton et al., 2014). A recent study reported that inhibiting Akt activity or reducing Akt expression reduced HSV-1 productive infection, in part by restricting viral entry (Cheshenko et al., 2017). Regardless of whether BoHV-1 encoded protein kinases phosphorylate Akt, these studies clearly revealed that infection rapidly increased p-Akt(S473) levels and suggest activation promotes productive infection. DEX treatment alone also stimulated Akt phosphorylation, which is consistent with results found for podocytes (Shengou and Li, 2013). Conversely, DEX interferes with Akt phosphorylation in rat pheochromocytoma derived cells (PC12) (Terada et al., 2014) and in

Fig. 3. DEX regulates transcription of viral L proteins during productive infection. Confluent low-passage BT cells in 60-mm dishes were incubated with 2% stripped fetal bovine serum for 12 h prior to infection. Cultures were then pretreated with 100 μM water soluble DEX in 2% stripped fetal bovine serum for 1 h, and then infected with BoHV-1 at a MOI of 1. Throughout infection, cultures were treated with DEX (100 μM) in 2% stripped fetal bovine serum. Cells treated with media containing stripped 2% fetal bovine serum without DEX were used as controls. Total RNA was prepared at the indicated time points after infection (hours) and then analyzed by qRT-PCR to measure the mRNA levels of UL10 (Panel A), UL16 (Panel B) and UL17 (Panel C). These results are the average of three independent experiments and error bars denote the mean of the three experiments.

4. Discussion We previously demonstrated that DEX stimulated BoHV-1 replication, in part because there are more than 100 putative GREs in the viral genome (Kook et al., 2015). The present study suggested DEX has the potential to stimulate expression of several viral genes, including bICP4 and several late genes, during early stages of productive infection. Furthermore, expression of a cellular protein kinase (SGK1), which promotes BoHV-1 and HSV-1 replication (Kook and Jones, 2016), was

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Fig. 4. DEX treatment and BoHV-1 infection increase SGK1 protein levels. Panel A: Confluent BT cells in 100 mm dishes were incubated with 2% stripped FBS for 12 h. Cultures were then infected with BoHV-1 at an MOI of 1 for the designated time after infection (hours) in the presence of 2% stripped FBS. Uninfected BT cells incubated with 2% stripped FBS for 4 h are designated as controls. Cells were lysed with RIPA buffer, proteins separated by SDS-PAGE, and Western blot analysis performed to detect expression of SGK1 and Tubulin. For each lane, 50 μg of protein was loaded. Panel B: Confluent BT cells in 100 mm dishes were incubated with 2% stripped FBS for 12 h. Cultures were then treated with 100 μM DEX for 1 h, and subsequently infected with BoHV-1 at an MOI of 1 for the designated time after infection (hours) in the presence of 2% stripped FBS and 100 uM DEX where indicated. Uninfected BT cells incubated with 2% stripped FBS for 4 h are designated as controls. Cells were lysed with RIPA buffer and Western Blot analysis determined to measure the effects of DEX and infection on SGK1 protein levels. For each lane, 50 μg of protein was loaded. Panel C: Confluent BT cells in 100 mm dishes were incubated with 2% stripped FBS for 12 h. Cultures were then treated with 100 μM DEX for 1 h, and subsequently infected with BoHV-1 at an MOI of 1 for 4 h in the presence of 2% stripped FBS and 100 uM DEX. SGK1 and tubulin were detected by Western Blot analysis. For each lane, 50 μg of protein was loaded. Western Blots presented in Panels A-C are representative of three independent experiments. To the right of gels in each panel, a graph summarizing the results from 3 independent experiments is presented. Quantification of SGK1 and tubulin bands from Western blots in Panels A-C were collected using the software image J package. The band intensity of SGK1 was initially normalized to tubulin. Samples from the respective time-point after infection were normalized to the Control lane (O hours after infection) and then the fold change after infection was calculated. Error bars denote the variability between the three independent studies.

cattle.

myotubes (Tsuchida et al., 2016) indicating the effect of DEX on Akt phosphorylation is cell type dependent. The precise roles that Akt and SGK-1 play during productive infection require additional studies. In conclusion, these studies provide evidence that the synthetic corticosteroid dexamethasone stimulates BoHV-1 productive infection by more than one mechanism. Considering that increased corticosteroid levels, as a result of stress, also interfere with immune responses, (Barnes, 1998; Funder, 1997; Oakley and Cidlowski, 2013; Rhen and Cidlowski, 2005; Schonevild et al., 2004; Smoak and Cidlowski, 2004), stressful stimuli may have numerous effects on BoHV-1 growth in

Acknowledgements This research was supported by grants from the USDA-NIFA Competitive Grants Program (13–01041 and 16–09370), funds derived from the Sitlington Endowment, and support from the Oklahoma Center for Respiratory and Infectious Diseases (National Institutes of Health Centers for Biomedical Research Excellence Grant # P20GM103648). L.Z. was partially supported by the China 76

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Fig. 5. Analysis of Akt phosphorylation following BoHV-1 infection and DEX treatment. Panel A: Confluent BT cells in 100 mm dishes were incubated with 2% stripped FBS for 12 h. Cells were then infected with BoHV-1 at a MOI of 1 for the designated times after infection (hours) in the presence of 2% stripped FBS. Uninfected BT cells incubated with 2% stripped FBS for 4 h are designated as a Control. Cells were lysed with RIPA buffer at the designated times after infection (hours), proteins separated by SDS-PAGE, and Western blot analysis performed to detect expression of the p-Akt(S473), total Akt, and Tubulin. For each lane, 30 μg of protein was loaded. Panel B: Confluent BT cells in 100 mm dishes were incubated with 2% stripped FBS for 12 h. Certain cultures were then treated with 100 μM of DEX for 1 h, and infected with BoHV-1 at an MOI of 1 for the designated times after infection (hours) in the presence of 2% stripped FBS and DEX. Uninfected BT cells incubated with 2% stripped FBS for 4 h are designated as a Control. For each lane, 30 μg of protein was loaded. Panel C: Confluent BT cells in 100 mm dishes were incubated with 2% stripped FBS for 12 h. Certain cultures were then treated with 100 μM of DEX for 1 h, and infected with BoHV-1 at an MOI of 1 in the presence of 2% stripped FBS and DEX. As designated, certain uninfected cultures were treated with DEX for 4 h. As a comparison, infected cultures were treated with DEX for 4 h. Cells were lysed with RIPA buffer, proteins separated by SDS-PAGE, and Western blots performed to detect expression of the designated proteins. For each lane, 30 μg of protein was loaded. Western Blots presented in Panels A-C are representative of three independent experiments. To the right of the gels in each panel, a graph summarizing the results from 3 independent experiments is presented. Quantification of p-Akt(S473) and tubulin bands from Western blots in Panels A-C were measured using the software image J package. The band intensity of p-Akt(S473) was initially normalized to tubulin. Levels of p-Akt(S473) from the respective time-point after infection were then normalized to the Control lane (0 h after infection) and the fold change after infection calculated. Error bars denote the variability between three independent studies.

References

Scholarship Council, Chinese National Science Foundation Grant (No. 31472172) and National Key Research Program (No. 2016YFD0500704). The University of Nebraska DNA Sequencing Core receives partial support from the National Institute for General Medical Science (NIGMS) INBRE - P20GM103427-14 and COBRE – 1P30GM110768-01 grants as well as The Fred & Pamela Buffett Cancer Center Support Grant - P30CA036727. This publication's contents are the sole responsibility of the authors and do not necessarily represent the official views of the NIH or NIGMS.

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