Viral Interferon Regulatory Factors Decrease the ... - Journal of Virology

Viral Interferon Regulatory Factors Decrease the Induction of Type I and Type II Interferon during Rhesus Macaque Rhadinovirus Infection Bridget A. Robinson,a,b Ryan D. Estep,b Ilhem Messaoudi,a,b,c Kelsey S. Rogers,b and Scott W. Wonga,b,c Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, Oregon, USAa; Vaccine and Gene Therapy Institute, Oregon Health & Science University, Beaverton, Oregon, USAb; and Division of Pathobiology and Immunology, Oregon National Primate Research Center, Beaverton, Oregon, USAc

Kaposi’s sarcoma-associated herpesvirus and rhesus macaque rhadinovirus (RRV), two closely related gammaherpesviruses, are unique in their expression of viral homologs of cellular interferon regulatory factors (IRFs), termed viral IRFs (vIRFs). To assess the role of vIRFs during de novo infection, we have utilized the bacterial artificial chromosome clone of wild-type RRV17577 (WTBAC RRV) to generate a recombinant virus with all 8 of the vIRFs deleted (vIRF-ko RRV). The infection of primary rhesus fibroblasts and peripheral blood mononuclear cells (PBMCs) with vIRF-ko RRV resulted in earlier and increased induction of type I interferon (IFN) (IFN-␣/␤) and type II IFN (IFN-␥). Additionally, plasmacytoid dendritic cells maintained higher levels of IFN-␣ production in PBMC cultures infected with vIRF-ko RRV than in cultures infected with WTBAC RRV. Moreover, the nuclear accumulation of phosphorylated IRF-3, which is necessary for the induction of type I IFN, was also inhibited following WTBAC RRV infection. These findings demonstrate that during de novo RRV infection, vIRFs are inhibiting the induction of IFN at the transcriptional level, and one potential mechanism for this is the disruption of the activation and localization of IRF-3.

T

he interferon (IFN) response is integral to a host’s antiviral defenses. IFNs are divided into three distinct types (types I to III), characterized by sequence, receptor usage, and biological activity (18). There are two well-studied type I IFNs (IFN-␣ and -␤). IFN-␣, which is expressed as multiple subtypes (13 in humans), is produced primarily by plasmacytoid dendritic cells (DCs) (pDCs) (19). IFN-␤, on the other hand, is produced by a wide variety of cell types, including fibroblasts and epithelial cells (18). One of the most important and earliest-studied functions of type I IFNs is the promotion of an antiviral environment within a virus-infected cell as well as surrounding cells (26). Additionally, type I IFN signaling also stimulates the adaptive immune response through the promotion of T cell survival, effector function, and proliferation (25, 46, 65); the activation of natural killer (NK) cells (50); and the maturation and activation of DCs (27, 44, 48). Type II IFN (IFN-␥) is produced by activated T cells and NK cells, promotes the activation of monocytes and macrophages (68), and is crucial for an effective Th1 adaptive response. Type III IFNs (IFN-␭1, -␭2, and -␭3) are the most recently identified IFNs. They exhibit innate and antiviral properties similar to those of type I IFNs, but they signal through a different receptor, so their biological impact is likely distinct (15). The expression of IFNs is governed by a family of transcription factors known as interferon regulatory factors (IRFs) (24, 64). In particular, the transcription of type I IFN (IFN-␣/␤) relies on the activation of IRF-3 and IRF-7, highly homologous proteins that become activated via C-terminal phosphorylation (58). IRF-3 is constitutively expressed in most cell types and becomes phosphorylated following the recognition of a variety of pathogenassociated molecular patterns (PAMPs) (33), including viral nucleic acids (74). Following the C-terminal phosphorylation of IRF-3, homodimeric complexes accumulate in the nucleus, where an interaction with the transcriptional cofactor p300/CBP potentiates the transcription of IFN-␤ (30, 40, 75), human IFN-␣1 (23,

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38), and murine IFN-␣4 (58). The activation of IRF-7 occurs in a similar manner, except that it has a more restricted expression profile: IRF-7 is constitutively expressed in some lymphoid cells and pDCs and is transcriptionally upregulated following type I IFN signaling in a variety of cell types (45, 57). Because of specific cell type expression and regulation, IRF-7 plays a vital role in the induction of IFN-␣ in pDCs (12) and is crucial for the induction of most IFN-␣ subtypes that constitute the second wave of type I IFN production (45, 57, 58). To efficiently withstand IRFdependent antiviral responses and establish an infection within the host, viruses have evolved a number of mechanisms to interfere with cellular IRFs, the induction of IFN, and subsequent IFNinduced signaling (3, 6). Kaposi’s sarcoma-associated herpesvirus (KSHV) and rhesus macaque (RM) rhadinovirus (RRV) are two highly related gammaherpesviruses and the only viruses known to carry genes with significant homology to cellular IRFs, aptly named viral interferon regulatory factors (vIRFs) (2, 54, 56, 60). Because of their unique homology to cellular IRFs, it has been hypothesized that the vIRFs employ novel mechanisms of immune evasion. Indeed, 3 of the 4 vIRFs encoded within KSHV can individually inhibit the induction of IFN and IFN-induced signaling by disrupting the functions of cellular IRF-1, IRF-3, IRF-5, and IRF-7 (9, 10, 20–22, 29, 43, 71, 76). The inhibitory mechanisms employed by each of the vIRFs are varied, suggesting that each vIRF plays a unique and significant role. For example, KSHV vIRF-1 binds to the transcriptional co-

Received 6 May 2011 Accepted 17 November 2011 Published ahead of print 7 December 2011 Address correspondence to Scott W. Wong, [email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.05047-11

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activator p300/CBP and blocks the necessary interaction between p300/CBP and cellular IRF-3, effectively inhibiting the transcription of type I IFNs and other p300-dependent cytokines (9, 36, 39, 61). KSHV vIRF-3 can interact directly with cellular IRF-7 to block IRF-7 binding at promoter regions, thus inhibiting the subsequent transcription of several subtypes of IFN-␣ (29). Moreover, KSHV vIRFs also interfere with cell cycle control proteins and apoptosis (reviewed in reference 52). The above-mentioned studies have provided insight into the molecular strategies employed by KSHV vIRFs to inhibit IFN induction and signaling but have not thoroughly defined their roles in the context of de novo KSHV infection due to inefficient lytic replication in vitro (reviewed in reference 51). In contrast, RRV displays robust lytic replication in vitro and encodes 8 vIRFs (open reading frames [ORFs] R6 to R13) with similarity to KSHV vIRF-1 (60), providing a unique opportunity to study the roles of vIRFs during the early phase of de novo infection. Sequence analysis suggests that RRV likely acquired the first 4 vIRFs (ORFs R6 to R9) initially and that these genes later underwent a duplication event to result in a total of 8 vIRFs within the RRV genome (60). Therefore, it is plausible that the duplicated vIRFs (R10 to R13) share redundant functions with their respective antecedents. Even in the absence of the duplication of these genes, the vIRFs potentially have overlapping functions, as is the case for the KSHV vIRFs. For example, KSHV vIRFs 1 to 3 have disparate molecular functions, but they collectively interfere with IFN induction and signaling (52). In this study, we tested the hypothesis that RRV vIRFs have antagonistic effects on cellular IRFs and the IRF-dependent induction of IFN. To do so, we have generated a recombinant RRV clone lacking all 8 vIRF genes (vIRF-ko RRV) utilizing a pathogenic molecular bacterial artificial chromosome clone of wildtype RRV17577 (WTBAC RRV17577) (16). The results presented herein demonstrate that the deletion of the vIRFs resulted in an increased induction of type I and type II IFN in RRV-infected rhesus macaque fibroblasts (RFs) and rhesus macaque peripheral blood mononuclear cells (PBMCs) along with an increased production of IFN-␣ within pDCs. The induction of type I IFN was preceded by increased IRF-3 phosphorylation and nuclear accumulation, which was notably inhibited during WTBAC RRV infection. The ectopic expression of a single vIRF, encoded by ORF R6, was able to significantly inhibit IRF-3-mediated transcription, as induced by poly(IC), and to specifically localize with endogenous IRF-3. These data suggest that vIRFs play a critical role in dampening antiviral responses early during RRV infection and demonstrate that at least one vIRF could be targeting IRF-3 to modulate the type I IFN response. MATERIALS AND METHODS Cells, virus, drugs, and cytokines. Primary rhesus fibroblasts (RFs), telomerized RFs (tRFs) (32), and HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Ogden, UT). Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of expanded-specific-pathogen-free (ESPF) rhesus macaques (RMs) by using Histopaque (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s guidelines. RM splenocytes were collected from spleens, and red blood cells (RBCs) were subsequently lysed with RBC lysis solution (Five Prime, Gaithersburg, MD) (10 min at room temperature [RT]). RRV infection of RM PBMCs and splenocytes was carried out with serum-free RPMI (Mediatech). ESPF RMs are serologically negative for RRV, simian immunodeficiency virus (SIV), type D simian retrovirus

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(SRV), herpesvirus simiae (B virus), simian T-lymphotropic virus 1 (STLV-1), rhesus cytomegalovirus (RCMV), and simian foamy virus (SFV). These studies utilized plaque-purified isolates of bacterial artificial chromosome (BAC)-derived RRV17577 (WTBAC RRV17577) (16) and WT RRV17577-GFP. WT RRV17577-GFP was generated via homologous recombination in RFs to express green fluorescent protein (GFP) via the EF-1␣ promoter and is located in the intergenic space between ORF 57 and ORF R6. All RRV stocks were purified through a 30% sorbitol cushion and resuspended in phosphate-buffered saline (PBS), and viral titers were determined by using a serial dilution plaque assay with RFs. Herpes simplex virus 1 (HSV-1) strain F was a generous gift from David Johnson (Oregon Health & Science University). Poly(IC) (Sigma) was resuspended in PBS and transfected into cells by using TransIT LT1 transfection reagent (Mirus, Madison, WI). Recombinant human IFN-␣2 (PBL, Piscataway, NJ) was resuspended in PBS with 0.1% bovine serum albumin (BSA) and used at a final concentration of 10 U/ml. Construction of a vIRF-ko RRV clone using the WT RRV17577 BAC. The construction of vIRF-ko RRV was performed by using WTBAC RRV17577 DNA as a template (16). First, an Flp recombination target (FRT)-flanked kanamycin resistance (Kanr) cassette was engineered with 40-bp arms of homologous RRV sequence to target the 10.9-kb region (nucleotides [nt] 78436 to 89386) encoding the 8 vIRFs (ORF R6 to ORF R13) within RRV. The Kanr cassette with RRV homology arms was then cloned into pSP73 and sequenced before the linearized cassette was electroporated into recombinogenic Escherichia coli strain EL250 (35), which contains the WT RRV17577 BAC. Recombinant EL250 clones were selected for kanamycin resistance, and BAC DNA was subsequently isolated by using a standard alkaline lysis method to identify potential clones containing the Kanr cassette in place of the vIRFs. EL250 clones with a successful recombination of the vIRF region were then treated with arabinose to induce Flp recombination within the EL250 system for the removal of the Kanr cassette within the BAC. The construction of vIRF-ko RRV-GFP was also done by using WTBAC RRV as a template but with the galK recombination system in E. coli SW105 cells, and vIRF deletion clones were identified via growth on plates with galactose as the sole carbon source (69). The galK cassette was subsequently removed via recombination with a GFP cassette and excised from pQ100 and consisted of the open reading frame of GFP driven by the constitutive promoter EF-1␣. GalK-negative recombinants were selected for resistance to 2-deoxygalactose in minimal medium with glycerol as the sole carbon source. Individual vIRF-ko RRV-GFP clones were analyzed via restriction digestion and Southern analysis and were sequenced across the vIRF region. For Southern blot analysis, BAC-derived DNA was isolated from EL250 clones, digested with BamHI overnight at 37°C, visualized on a 0.7% agarose gel, and subsequently transferred onto a Duralon-UV membrane (Stratagene, La Jolla, CA). Probes were labeled with digoxigenin (DIG) by using the DIG-High Prime kit (Roche, Indianapolis, IN), and hybridization, washing, and detection were done according to kit protocols. Probes were made for the vIRF deleted region (Kanr cassette), the intact vIRF region (ORF R9), and an additional RRV gene (ORF 4), as a control. Generation of infectious virus using vIRF-ko RRV BAC DNA. Infectious virus was isolated from RRV17577 BAC DNA as previously described (16). Briefly, vIRF-ko RRV17577 BAC DNA from EL250 clones was transfected into RFs by using TransIT LT1 reagent (Mirus, Madison, WI). After the development of an RRV-associated cytopathic effect (CPE) (73), the supernatant and cells were collected, freezethawed once, and sonicated to release any cell-associated virus. The virus collected after this transfection underwent two rounds of infection in RFs transiently transfected with Cre recombinase to remove the loxP-flanked BAC cassette. To confirm the removal of the BAC cassette, the BAC cassette insertion site between ORF57 and R6 was am-

Journal of Virology

vIRFs Inhibit IFN Production during RRV Infection

plified and sequenced by using purified viral DNA as a template. The recombinant virus subsequently went through two rounds of plaque purification in RFs to obtain a purified clone of vIRF-ko RRV. Viral DNA isolation and complete genome hybridization. As previously described (16), RFs were infected with BAC-derived WT or vIRF-ko RRV (multiplicity of infection [MOI] of 0.01) and allowed to progress to complete CPE before the supernatant and cells were collected and digested overnight with proteinase K. Viral DNA was then isolated via cesium chloride centrifugation (77,400 ⫻ g for 72 h). Fractions containing viral DNA were pooled and dialyzed against Tris-EDTA. Viral DNA from vIRF-ko RRV-infected cells was subsequently analyzed via comparative genome hybridization (CGH) at NimbleGen Systems, Inc. (Madison, WI), as described previously by Estep et al. (16). WTBAC RRV17577 DNA was used as a reference genome, and data were analyzed by using SignalMap software (NimbleGen Systems, Inc.) to identify potential nucleotide changes in vIRF-ko RRV. In vitro growth curves. Single-step (MOI ⫽ 2.5) and multistep (MOI ⫽ 0.1) growth curve analyses were carried out with RFs. Cells were seeded into culture tubes (Corning, Aston, MA) at a density of 2 ⫻ 105 cells per tube and infected in duplicate on the following day. After a 2-h adsorption period, the tubes were washed twice with phosphate-buffered saline to remove any unbound virus, and fresh medium was added. Cells and supernatants were collected at 2, 12, 24, 48, 72, and 96 h postinfection (hpi). Samples were freeze-thawed once and sonicated to release any remaining cell-associated virus, and titers were determined via a plaque assay with RFs. RNA isolation, RT-PCR, and real-time RT-PCR. RNA was isolated from tRFs and rhesus macaque PBMCs by using the RNeasy kit (Qiagen, Valencia, CA) and DNA endonuclease, RQ1, was used to remove DNA from RNA preperations (Promega, Madison, WI) according to kit protocols. Reverse transcription-PCR (RT-PCR) was performed by using Superscript III one-step RT-PCR with Platinum Taq (Invitrogen, Carlsbad, CA). Transcripts were detected with the specific oligonucleotide pairs listed in Table 1. First-strand cDNA synthesis was carried out by using the High Capacity cDNA RT kit (ABI, Foster City, CA), and cDNA was subsequently amplified by using TaqMan PreAmp master mix (ABI) and TaqMan gene expression assays specific for the following transcripts: IFN-␤ (ABI catalog no. Rh_03648734), IFN-␣1/13 (Rh_03456606), IFN-␥ (Rh_02621721), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Rh_02621745) (ABI). All data were normalized to the levels of GAPDH in each sample, and normalized levels of IFN transcripts were made relative to a standard, positive sample included on each plate: either poly(IC)stimulated tRFs or IFN-␣-stimulated PBMCs. 3= RACE. Primary rhesus fibroblasts were infected (MOI ⫽ 2) for 72 h, followed by RNA extraction and first-strand cDNA synthesis. Rapid amplification of 3= cDNA ends (3= RACE) was performed by using the GeneRacer kit (Invitrogen) with gene-specific primers upstream of the stop codon of ORF 57 (ORF-57gsp [5=-ACG CGC AAA AAC ACG CTA GCG3=]) or ORF 58 (ORF-58gsp [5=-GCT CCT CGG ACT TGT ACA CTA TT-3=]). 3= RACE products were analyzed on a 1% agarose gel, purified, and cloned into pCR4-TOPO (Invitrogen), and at least 3 clones of each product were subsequently sequenced. Immunoprecipitation (IP), PAGE analysis, and immunoblotting. Cell lysates were immunoprecipitated with an anti-hemagglutinin (HA) monoclonal antibody (MAb) or an anti-IRF-3 polyclonal antibody (pAb) (FL-425) in native lysis buffer (50 mM Tris-Cl [pH 8.0], 1% NP-40, and 150 mM NaCl supplemented with phosphatase inhibitors [100⫻ cocktail; Sigma] and protease inhibitors [100⫻ cocktail; Sigma]), followed by incubation with protein A/G Plus-agarose (Santa Cruz), and lysates were finally collected in radioimmunoprecipitation assay (RIPA) buffer (native lysis buffer with 0.1% SDS and 0.5% sodium deoxycholate). Whole-cell extracts were also collected in RIPA buffer, nuclear and cytoplasmic lysates were collected according to kit protocols (NE-PER; ThermoScientific), and all samples were analyzed by 10% SDS-PAGE.

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TABLE 1 Oligonucleotide sets used for RT-PCR ORF

Oligonucleotide

Oligonucleotide sequence (5=–3=)

R6

R6 for R6 rev

ATC GTT ACT CAC GCA AAT CTT TGA AGA CCA CGT TTG CAA ATG

R7

R7 for R7 rev

TGG AAC AAA GTA ACT GCA GAT AAC TCA CTG ATT AAA CCA AGG

R8

R8 for R8 rev

TGC TGC GAG ACA GGT CGT CAT CAG TAG TGC CGG AGG CCT GAT

R9

R9 for R9 rev

TCC GAC GAG ATC AGC GTA C CCC TCG TTA TAC GCG ACC A

R10

R10 for R10 rev

CGT TTC CCA ATT ATG ATT ATC CCG ATA CCG TCT CTC TTG ATC

R11

R11 for R11 rev

AAC CGG TGC ACC GAC AGT CGC CCG TGT CCT CTC GAA AAC ATC

R12

R12 for R12 rev

ATT GTT GCG ATA ATG ATA AGC CCG GTG GCA TCC GCT TCG TTA

R13

R13 for R13 rev

ATG CAA CCT GTG GTT GCG TAC CTG GCG GCC CTG GCA TAT A

R3 (vMIP)

R3 for R3 rev

CCT ATG GGC TCC ATG AGC ATC GTC AAT CAG GCT GCG

GAPDH

GAPDH for GAPDH rev

GTG GAT ATT GTT GCC ATC AAT ATA CTT CTC ATG GTT CAC ACC

Protocols for native PAGE analysis of dimeric forms of cellular IRF-3 were originally described elsewhere (28). Briefly, 7.5% Ready Gels (BioRad, Hercules, CA) were prerun (30 min at 40 mA at 4°C) with 25 mM Tris (pH 8.4) and 192 mM glycine with and without 1% sodium deoxycholate (Sigma) in the cathode and anode chambers, respectively. Lysates were resuspended in native sample buffer and electrophoresed for 60 min at 25 mA. Proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) via semidry transfer (60 min at 100 mA at RT). Membranes were probed with anti-human IRF-3 pAb (FL-425) (Santa Cruz), anti-human phospho-IRF-3 (Ser396) (4D4G) (Cell Signaling Technology), anti-human GADPH MAb (SC 51906) (Santa Cruz), and anti-human poly(ADP-ribose) polymerase 1/2 (PARP1/2) pAb (H-250) (Santa Cruz). Data were analyzed by using densitometry. Immunofluorescence. Cells were grown on glass coverslips in 12-well plates and fixed with 4% paraformaldehyde in PBS (20 min at RT). Cells were then permeabilized and blocked in 5% normal goat serum (NGS)– 0.1% Triton X in PBS (PBST) (1 h at RT) prior to staining, and all subsequent steps were performed with 1% NGS–PBST. Cells on coverslips were stained with anti-human IRF-3 MAb (clone SL012.1) (BD Pharmingen, San Diego, CA) overnight at 4°C and subsequently stained with antimouse IgG-Texas Red (Vector Labs, Burlingame, CA). Subsequently, cells were stained with anti-HA-fluorescein isothiocyanate (FITC) (HA-7) (Sigma) (2 h at RT), and nuclei and/or DNA was detected by using Hoechst 33258 dye. Cells on coverslips were mounted onto slides by using Vectashield (Vector Labs) and examined on a Zeiss Axio Imager.M1 microscope (Zeiss Imaging Solutions, Thornwood, NY). Images were acquired by using a Zeiss Axiocam camera (MRm) with Axiovision software (version 4.6) and subsequently processed by using Adobe Photoshop (Adobe Systems, San Jose, CA). Intracellular cytokine staining (ICCS). Freshly isolated PBMCs or splenocytes were maintained in serum-free medium. Under each condi-

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TABLE 2 Oligonucleotide pairs used to generate vIRF constructs vIRF

Oligonucleotide

Oligonucleotide sequence (5=–3=)a

R6

R6 for R6-HA rev

ctt ggc agt gcggccgc ATG GCT ACC TGG CGC CCA CCT cgt gaattc tca agc gta gtc tgg gac gtc gta tgg gta TTC AAA GTG CCG ATA TAT TTC

R7

R7 for R7-HA rev

ctt ggc agt gcggccgc ATG GCG GGC CGT GGA GTC GAT cgt gaattc tga agc gta gtc tgg gac gtc gta tgg gta CGA GCA GGC CAC CCC ATC ATC

R10

R10 for R10-HA rev

ctt ggc agt gcggccgc ATG GCC GCT GGG GAA TCG AGA agc ggatcc tta agc gta gtc tgg gac gtc gta tgg gta TTC AAA GTG CCT ATA GAT TTC

R11

R11 for R11-HA rev

ctt ggc agt gcggccgc ATG GCG GAA CGC GAT ATG GAT cgt gaattc tta agc gta gtc tgg gac gtc gta tgg gta CTT CCT CCC ATA CGG TGC GAG

K9

K9 for K9-HA rev

ctt ggc agt gcggccgc ATG GAC CCA GCC AAA GA cgt gaattc cta agc gta gtc tgg gac gtc gta tgg gta TTG CAT GGC ATC CCA TAA

a

Restriction enzyme sites are in boldface type; gene-specific sequences are in uppercase type.

tion, 4 million cells were infected (MOI ⫽ 1) and treated with brefeldin A (0.02 ␮g/␮l) (Sigma) during the final 6 h of each experiment to block cytokine secretion. PBMCs were then surface stained with fluorescently labeled antibodies, CD3 (SP34), CD20 (B9E9), CD14 (M5E2), HLA-DR (L243), CD11c (3.9), and CD123 (6H6), to delineate specific populations, including plasmacytoid DCs (CD3⫺, CD20⫺, CD14⫺, HLA-DR⫹, CD11c⫺, and CD123⫹). Cells were subsequently fixed in fixation buffer (BioLegend) for 20 min at 4°C, permeabilized, and stained for intracellular IFN-␣ (MMHA-2). IFN-␣ was labeled by using Zenon labeling technology according to kit protocols (Molecular Probes, Eugene, OR). Samples were acquired on an LSRII instrument (BD, San Jose, CA), and data were analyzed by using FlowJo software (TreeStar, Ashland, OR). Cloning of vIRFs for transient-expression assays. RRV vIRFs were amplified from purified WTBAC RRV17577 DNA, engineered with a C-terminal HA tag, and cloned into pcDNA3.1(⫺) (Invitrogen). Oligonucleotide primers utilized for amplification are listed in Table 2. Each vIRF clone was sequenced and analyzed via immunofluorescence and Western blot analysis to verify expression. Luciferase assays. IRF-3-mediated transcription was measured by using a reporter plasmid encoding firefly luciferase driven by 5 copies of the interferon-stimulated response element (ISRE) in the promoter (pISRE-

LUC) (Promega, Madison, WI). Firefly luciferase readings were normalized via the constitutive expression of Renilla luciferase (pRL-SV40) (Promega). HEK293 cells were transfected overnight with 500 ng total DNA [250 ng pISRE-LUC, 10 ng pRL-SV40, 50 ng vIRF-HA-pcDNA3.1, and 190 ng empty pcDNA3.1(⫺)]. As a negative control, HEK293 cells were transfected overnight with 250 ng pISRE-LUC, 10 ng pRL-SV40, and 240 ng empty pcDNA3.1(⫺). Cells were then transfected with poly(IC) (10 ␮g) for 6 h and analyzed with the Dual-Glo luciferase assay according to the manufacturer’s protocol (Promega). Data were made relative to luciferase units recorded for an empty vector stimulated with poly(IC). Statistical analysis. Data were analyzed by using GraphPad Instat (GraphPad Software, La Jolla, CA), and significant differences were determined by a paired t test, with P values of ⱕ0.05 being considered significant.

RESULTS

RRV vIRFs are expressed early during infection. RRV encodes 8 vIRFs (ORFs R6 to R13), encoded in a cluster between ORF 57 and ORF 58 (Fig. 1A). To analyze the temporal expression of the 8 RRV vIRFs, reverse transcription-PCR (RT-PCR) was performed to analyze the expression of the vIRF transcripts during the first 24

FIG 1 Expression of 8 vIRFs during rhesus rhadinovirus infection. (A) Schematic of the RRV genome showing the positions and orientations of the eight vIRFs (ORFs R6 to R13) encoded within a 10.95-kbp region (nucleotides 78436 to 89386) between ORF 57 and ORF 58. The arrows point toward the 3= end of each ORF. TR, terminal repeat. (B) tRFs were infected (MOI ⫽ 2), and RNA was extracted at 3, 6, 12, and 24 hpi and analyzed by RT-PCR using 8 vIRF-specific oligonucleotide pairs (Table 1). As controls for RNA input and purity, levels of GAPDH in each sample were analyzed, and a reaction was run without reverse transcriptase (⫺RT). ORF, open reading frame; hpi, hours postinfection; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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FIG 2 Construction and molecular characterization of vIRF-ko RRV using WTBAC RRV17577. (A) Schematic representation of the RRVBAC DNA highlighting the BamHI restriction sites (B1 to B4) used to monitor the recombination of the vIRF region and removal of the Kanr cassette. (B) WTBAC and vIRF-koBAC DNAs were digested with BamHI before and after Flp recombination. The 15.7-kbp band (⬍) in WTBAC DNA shifts upwards to 16.3 kbp (ⴱ) after the recombination of the vIRF region and migrates with a similar-sized fragment. After the Kanr cassette is removed via Flp recombinase, a new band appears at 14.7 kbp (°). (C) Southern blot analysis performed on BamHI-digested DNA using DIG-labeled probes against the vIRF region, the Kanr gene, and another RRV gene (ORF4), as a control. (D) Comparative genome hybridization used to directly compare viral DNA from the vIRF-ko RRV recombinant clone to that from WTBAC RRV. The schematic of the RRV genome shows the vIRFs (ORFs R6 to R13) shaded in gray. Any alterations within the vIRF-ko RRV genome resulted in incomplete hybridization to the array, depicted by the ratio of vIRF-ko to WT RRV, and signaled a potential nucleotide mismatch between the two viruses. This comparison identified mismatches only within the region of the vIRF deletion (nt 78436 to 89386). TR, terminal repeats; Flp, flippase.

h of WTBAC RRV infection. RT-PCR revealed an early and sustained expression of all eight vIRFs during the first 24 h of infection (Fig. 1B). Specifically, R10 was expressed as early as 3 hpi, and R6, R7, and R11 transcripts were all present by 6 hpi (Fig. 1B). Moreover, the expression pattern of the first four vIRFs (R6 to R9) closely paralleled the expression pattern of the duplicated vIRFs (R10 to R13) (60), demonstrating that the sequence-related vIRFs are similar in their temporal expressions. Overall, these data demonstrate the early expression of the vIRFs during RRV infection of RFs, which would position their respective proteins to be present during the virus-stimulated induction of IFN. Generation of vIRF-ko RRV using WTBAC RRV17577. To determine the collective role of the vIRFs during RRV infection, an infectious BAC-derived clone of RRV17577 (16) was utilized to generate a recombinant virus lacking all 8 vIRFs, designated vIRF-ko RRV. Because the vIRFs are encoded within a cluster in the RRV genome, we were able to delete all 8 vIRFs concurrently. To achieve this, we replaced the vIRFs with a single kanamycin resistance (Kanr) cassette via homologous recombination using the Red recombination system within E. coli strain EL250 (35). To verify the targeted recombination of the vIRFs, BAC-derived DNA


was digested with BamHI (Fig. 2A and B) and examined via Southern analysis (Fig. 2C) after the initial recombination of the vIRF region and again after the Flp removal of the Kanr cassette. The deletion of the vIRFs and the Flp removal of the Kanr cassette resulted in a 199-bp lesion, including a single FRT site (48 bp), in the genomic region where the vIRFs were deleted. To verify this, BAC DNA from the vIRF-ko RRV clone was isolated, and the genomic region containing the vIRF deletion was amplified and sequenced to confirm targeted deletion and that there were no changes in the surrounding genomic sequence (data not shown). BAC DNA from the vIRF-ko clone was subsequently transfected into RFs to produce infectious virus. This virus was then used to infect RFs transiently expressing Cre recombinase to remove the LoxP-flanked BAC cassette (16) and then subjected to two rounds of plaque purification in RFs to obtain a purified isolate of vIRF-ko RRV. Viral DNA was purified and analyzed via comparative genomic hybridization (CGH) to compare the complete genomic sequence of vIRF-ko RRV viral DNA to that of WTBAC RRV17577 (Fig. 2D). CGH is a sensitive, array-based analysis used to identify single-nucleotide changes, insertions/deletions, and rearrangements between two highly similar genomes.

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FIG 3 In vitro characterization of vIRF-ko RRV. (A and B) RFs were infected with recombinant RRV isolates in single-step (MOI ⫽ 2.5) (A) and multistep (MOI ⫽ 0.1) (B) growth curves. Infected RFs were harvested at the specified time points and subjected to a serial-dilution plaque assay on RFs to ascertain viral titers, displayed as PFU/ml. The data from 2 separate experiments were averaged (⫾ standard errors of the means [SEM]). (C) RFs infected with GFP-expressing clones of RRV were fixed at 48 hpi, and GFP expression was analyzed via immunofluorescence (green, RRV-GFP; blue, Hoechst 33342). This experiment was repeated three times, and a representative image is shown (magnification, ⫻200), with the percentage of GFP-expressing cells (⫾SEM) included within each image. (D to G) RFs were infected with WTBAC RRV or vIRF-ko RRV (MOI ⫽ 1), and RNA was harvested at 72 hpi and analyzed via 3= RACE to verify the transcription of ORF 57 and ORF 58. (D) Two ORF 57 products were detected (arrows labeled a and b), and a third transcript was also detected in vIRF-ko RRV infection but resulted in a truncated transcript (#). (E) ORF 57 3= RACE products were cloned and sequenced, and the full-length transcripts (a and b) utilized 2 distinct poly(A) sites (shaded in gray). The stop codon is underlined, and the addition site of the poly(A) tail is noted (⬎). (F and G) 3= RACE analysis of the ORF58 transcript. The most prevalent transcript is marked (arrow) (F) and was subsequently cloned and sequenced to identify a single poly(A) site (shaded in gray) (G). The stop codon (underlined) and the addition of the poly(A) tail (⬎) are noted.

The effectiveness and validity of CGH have been demonstrated by its use in comparisons of different strains of WT RRV and WTBAC RRV17577 isolates (16), in comparisons of coronavirus isolates (72), and in studies of bacterial evolution (1). The CGH analyses did not detect any changes in the vIRF-ko RRV isolate compared to WTBAC RRV viral DNA (Fig. 2D), indicating that WTBAC RRV and vIRF-ko RRV have identical sequences outside the deleted vIRF region. Deletion of vIRFs did not affect growth kinetics or inhibit transcription of neighboring ORFs during RRV infection of RFs. To evaluate viral replication in the absence of the vIRFs,

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single-step (MOI ⫽ 2.5) and multistep (MOI ⫽ 0.1) growth curves were performed to assess viral growth and spread, respectively. In both the single-step (Fig. 3A) and multistep (Fig. 3B) growth analyses, vIRF-ko RRV displayed growth kinetics and magnitude similar to those of WTBAC RRV. These data show that the vIRFs are not essential for RRV infection, replication, or spread during RRV infection of RFs in vitro. Additionally, the GFP-expressing clones of WT and vIRF-ko RRV were further analyzed to validate GFP expression as a suitable marker for RRV infection. WT RRV-GFP and vIRF-ko RRV-GFP infections resulted in 16.8% and 21.6% GFP-positive (GFP⫹) cells at 36 hpi,

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respectively (Fig. 3C). The similar expression intensities and percentages of GFP positivity suggest that GFP is an adequate marker for comparing the efficiency of RRV infection. To further characterize the vIRF-ko RRV recombinant clone, gene expression of the ORFs directly upstream (ORF 57) and downstream (ORF 58) of the deleted vIRFs during vIRF-ko RRV infection was verified. RRV ORF 57 encodes a protein with immediate-early expression and shares similarity with ORF 57 genes of other gammaherpesviruses, which function in the nuclear export of unspliced viral mRNA (7, 51, 63). RRV ORF 58 is also conserved among gammaherpesviruses (51) and encodes a protein with a putative epithelial binding function, as was shown previously for Epstein-Barr virus (67). The coding regions for ORF 57 and ORF 58 are oriented in opposing directions, both being transcribed toward the vIRF region (Fig. 1A), so we utilized 3= RACE with gene-specific primers within ORF 57 and ORF 58 to detect both transcripts during vIRF-ko RRV infection. We identified two poly(A) sites that yielded full-length ORF 57 transcripts in WTBAC RRV- and vIRF-ko RRV-infected tRFs (Fig. 3D, arrows a and b, and E). The poly(A) site further downstream of the stop codon is preferentially used during vIRF-ko RRV infection, while the poly(A) site that overlaps with the stop codon is preferentially used during WTBAC RRV infection (Fig. 3E). There was also a third, less abundant product identified for ORF 57 in vIRF-ko RRV-infected cells (Fig. 3D), which resulted in an abbreviated transcript. The transcription of ORF 58 also utilizes multiple poly(A) sites during WTBAC and vIRF-ko RRV infections, but the dominant transcript used the same poly(A) site (Fig. 3F) and resulted in a full-length transcript (Fig. 3G). Collectively, these data suggest that the removal of the vIRFs did not alter the growth of RRV in RFs or considerably impact the transcription of adjacent ORFs. vIRFs are necessary for efficient infection of rhesus macaque PBMCs. Although RRV infection of fibroblasts is easily studied, peripheral blood mononuclear cells (PBMCs), and B cells specifically, represent an important cellular target for RRV in the rhesus macaque (4). Because such a small percentage of PBMCs becomes infected with RRV in vitro, it is difficult to compare growth properties within PBMCs by using a standard plaque assay. Thus, RTPCR was initially utilized to verify the presence of RRV transcripts as a marker of RRV infection of rhesus macaque PBMCs. RRV ORF R3, which encodes viral macrophage inflammatory protein (vMIP), was examined due to the ease of detection (DNA and RNA), even at low levels of infection (data not shown). In fact, the vMIP transcript was present in both WTBAC RRV- and vIRF-ko RRV-infected PBMCs throughout the 48-h time course (Fig. 4A). Additionally, we looked for the presence of one of the vIRF transcripts to verify that the vIRFs are also expressed during RRV infection of PBMCs. We chose the vIRF encoded by ORF R10, because it was transcribed early and persisted during the first 24 h in RRV-infected RFs (Fig. 1B). As expected, the vIRF R10 transcript was present only in WTBAC RRV-infected PBMCs, and the transcript was present at 3 hpi and persisted through 48 hpi (Fig. 4A). To evaluate the infection efficiency within total PBMCs as well as within specific immune cell populations in the blood, PBMCs or splenocyte populations from 6 different RMs were infected with GFP-expressing clones of WT and vIRF-ko RRV. The frequency of GFP⫹ cells was significantly lower in vIRF-ko RRVinfected cultures. Approximately 5% of PBMCs were infected


with vIRF-ko RRV, compared to 15% and 19% of PBMCs in WT RRV-infected cultures at 24 and 48 hpi, respectively (Fig. 4B). To determine if the deletion of the vIRFs equally reduced infection in specific immune cell subsets, infected PBMCs were also surface stained to delineate T cells, B cells, monocytes, myeloid DCs (mDCs), and plasmacytoid DCs (pDCs) (8) (Fig. 4C). Similar to total PBMC cultures, these analyses revealed that vIRF-ko RRV infects T cells, B cells, monocytes, and mDCs at a reduced efficiency (Fig. 4D). Interestingly, we did not detect any difference in the frequencies of GFP⫹ pDCs between WT- and vIRF-ko RRVinfected cultures (Fig. 3D). Deletion of vIRFs results in increased induction of type I and type II IFNs during RRV infection. To examine whether the vIRFs collectively function to inhibit the induction of IFNs, we measured the induction of type I (IFN-␤ and IFN-␣) and type II (IFN-␥) IFN in WTBAC RRV- and vIRF-ko RRV-infected tRFs and rhesus macaque PBMCs using real-time RT-PCR. Gene expression was normalized to GAPDH expression and determined relative to the positive control. The induction of IFN-␤ occurs early during viral infection in a variety of cell types, including fibroblasts, but WTBAC RRV infection did not result in a robust induction of IFN-␤, especially within PBMCs (Fig. 5A). This is particularly evident compared to poly(IC), which induced 105 times more IFN-␤ transcript than did RRV infection (Fig. 5A). However, the deletion of the vIRFs resulted in an average 2-fold increase in IFN-␤ transcript levels at 72 hpi compared to those of WTBAC RRV, although these differences were not statistically significant (Fig. 5A). To further assess the type I IFN response during RRV infection, we also examined the induction of IFN-␣1, as it is the only IFN-␣ subtype that is immediately induced upon viral infection (23, 38). As measured by real-time RT-PCR, the induction of IFN-␣1 in WTBAC RRV-infected tRFs was unremarkable throughout the 72-h time course, with levels between 0.5 and 1% of the magnitude of IFN-␣1 induction after poly(IC) stimulation, the positive control (Fig. 5B). In contrast, vIRF-ko RRV infection of tRFs resulted in a progressive increase in IFN-␣1 transcript levels, which culminated in a statistically significant 3-fold-higher induction at 72 hpi than that with WTBAC infection (Fig. 5B). For rhesus macaque PBMCs, the levels of the IFN-␣1 transcript peaked much earlier, and the induction of IFN-␣1 at 6 hpi in vIRF-ko RRV-infected PBMCs was 12-fold higher than that during WTBAC RRV infection (Fig. 5B). These data demonstrate that the vIRFs inhibit the induction of type I IFNs during RRV infection in tRFs and PBMCs. Finally, we assessed the impact of vIRFs on the induction of type II IFN (IFN-␥) during RRV infection. In tRFs, vIRF-ko RRV infection induced IFN-␥ expression at 48 to 72 hpi (Fig. 5C), whereas WTBAC RRV infection failed to induce detectable levels of IFN-␥ (Fig. 5C). RRV infection of PBMCs induced IFN-␥ with kinetics similar to those of IFN-␣1 induction, peaking at 12 hpi (Fig. 5C), and vIRF-ko RRV infection induced 3-fold more IFN-␥ at 6 hpi (Fig. 5C). Collectively, these data demonstrate the vIRFs can significantly reduce and delay the induction of both type I and type II IFN during RRV infection. vIRFs limit IFN-␣ production by pDCs in RRV-infected PBMCs. To determine if the vIRF-dependent inhibition of IFN was also detectable at the protein level, IFN-␣ production in RRVinfected PBMCs was examined via intracellular cytokine staining (ICCS) (Fig. 6). Freshly isolated PBMCs from six expanded-

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FIG 4 WTBAC RRV and vIRF-ko RRV infect rhesus macaque PBMCs. (A) Rhesus macaque PBMCs were isolated from (RRV-seronegative) RMs and infected with WTBAC RRV or vIRF-ko RRV (MOI ⫽ 1). RNA was harvested at the indicated time points and analyzed by RT-PCR for RRV vMIP (ORF R3), RRV vIRF (ORF R10), and GAPDH, as a loading control. Oligonucleotide pairs used for each transcript are listed in Table 1; viral transcripts were detected by using 300 ng RNA, and GAPDH was detected by using 50 ng RNA per sample. Samples were run simultaneously with Taq polymerase only (⫺RT) to control for input and purity, and purified RRV DNA was used as a positive control (⫹). (B to D) Leukocytes were isolated from the spleens of six RMs, and GFP-expressing clones of WT RRV and vIRF-ko RRV were used to infect total leukocyte cultures. (B) GFP expression levels in WT- and vIRF-ko RRV-infected cultures were compared by flow cytometry at 24 and 48 hpi, and median responses are represented by a horizontal line. Data were compared via paired t tests. (C) Surface markers were used to delineate cellular populations within the infected cultures: T cells (CD3⫹), B cells (CD20⫹), monocytes (Lin⫺ MHC-II⫹ CD14⫹), total dendritic cells (DCs) (Lin⫺ MHC-II⫹ CD14⫺), myeloid DCs (CD11c⫹ CD123dim), and plasmacytoid DCs (CD11c⫺ CD123⫹). (D) GFP expression in each cellular subset was analyzed at 24 and 48 hpi, as was done for the total leukocyte population in panel B. RM, rhesus macaque; GFP, green fluorescent protein; hpi, hours postinfection; FSC, forward scatter; SSC, side scatter.

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FIG 5 Induction of type I and type II IFN during RRV infection. Telomerized RFs (tRFs) and ESPF rhesus macaque PBMCs were infected with WTBAC RRV or

vIRF-ko RRV (MOI ⫽ 1), and RNA was extracted at the indicated time points and used to prepare and amplify cDNA for semiquantitative real-time RT-PCR specific for IFN-␤ (A), IFN-␣1 (B), and IFN-␥ (C), using TaqMan PreAmp master mix and gene expression assays (ABI) specific for rhesus macaque transcripts. All transcripts were normalized to levels of GAPDH in each sample and made relative to a positive-control sample included on each plate. The positive control (⫹ctrl) for IFN-␣ and IFN-␤ transcripts was cDNA from tRFs transfected with poly(IC) (10 ␮g/ml for 6 h), and the positive control for IFN-␥ was cDNA from PBMCs treated with recombinant human IFN-␣2 (10 units/ml for 6 h). Data are average data (⫾SEM) from at least three independent experiments each with tRFs and PBMCs. N.S., not significant; ND, not detected; PI, postinfection.

specific-pathogen-free (ESPF) rhesus macaques were infected with WTBAC or vIRF-ko RRV (MOI ⫽ 1) for 12, 24, and 48 h. For all samples, brefeldin A was added during the last 6 h of incubation to inhibit cytokine secretion. Subsequently, PBMCs were surface stained to delineate pDCs (shown in Fig. 4C) (8), as they are the major producers of type I IFN in PBMCs (11, 62), and IFN-␣ production within pDCs was measured by ICCS (Fig. 6). HSV-1 stimulation of rhesus macaque PBMCs was used as a positive control and resulted in a marked production of IFN-␣ within the pDC population (Fig. 6A), as previously described (11, 62). Likewise, approximately 15% of the pDC population was producing IFN-␣ at 12 hpi in both WTBAC RRV- and vIRF-ko RRV-infected PBMC


cultures (Fig. 6A and B). Interestingly, IFN-␣ production was sustained in vIRF-ko RRV-infected cultures, with approximately 17% of the pDCs still producing IFN-␣ at 24 hpi (Fig. 6A and B). In contrast, only 6% of the pDCs were still producing IFN-␣ at 24 hpi in WTBAC RRV-infected cultures (Fig. 6A and B). Thus, the deletion of the vIRFs resulted in a sustained production of IFN-␣ following RRV infection. vIRFs inhibit nuclear accumulation of IRF-3 during RRV infection. IRF-3 is a vital component of the transcription machinery that drives the transcription of type I IFN following viral infection (23, 38, 58). Therefore, the activation of IRF-3 was examined during the initial 24 h of infection with RRV. The total levels of IRF-3

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FIG 6 Production of IFN-␣ by pDCs in RRV-infected PBMC cultures. Freshly collected PBMCs from ESPF RMs were mock infected or infected with WTBAC or vIRF-ko RRV (MOI ⫽ 1) in serum-free medium. During the last 6 h of infection, brefeldin A (0.02 ␮g/␮l) was added to inhibit cytokine secretion, and at 12, 24, and 48 hpi, cells were surface stained for CD3, CD20, CD14, HLA-DR (MHC-II), CD11c, and CD123. PBMCs were then fixed, IFN-␣ production was measured by intracellular cytokine staining, and cells were analyzed by flow cytometry. PBMCs were stimulated with HSV-1 for 12 h as a positive control for IFN-␣ production, and surface marker expression was utilized to delineate pDCs, as shown in Fig. 4C. (A) A representative experiment is shown, displaying the percentage of pDCs producing IFN-␣ in each histogram. (B) This experiment was repeated with PBMCs from 6 different RMs, and the percentages of pDCs producing IFN-␣ were compared via a paired t test.

present in whole-cell extracts remained unchanged after WTBAC and vIRF-ko RRV infections (Fig. 7A). Moreover, both WTBAC and vIRF-ko RRV induced similar levels of IRF-3 dimerization, which peaked at 6 to 12 hpi, as measured by native PAGE and Western analysis (Fig. 7B and C). However, vIRF-ko RRV infection did result in an increase in the nuclear accumulation of IRF-3 at 6 hpi (Fig. 7D and E). Indirect immunofluorescence of total IRF-3 showed that 61% of cells contained IRF-3 in the nucleus at 6 hpi in vIRF-ko RRV-infected cells, compared to only 30% in cultures infected with WTBAC RRV (Fig. 7D and E). Further characterization of nuclear IRF-3 revealed that vIRF-ko RRV infection specifically results in an earlier and sustained accumulation of hyperphosphorylated forms of IRF-3 within the nucleus from 2 to 8 hpi, as shown by Western blot analysis (Fig. 7F). In contrast, WTBAC RRV infection did not result in a similar nuclear accumulation of phosphorylated IRF-3 (Fig. 7F), nor was there direct evidence that phosphorylated forms of IRF-3 were simply retained in the cytoplasm (Fig. 7F). These data suggest that interference with the activation and/or nuclear accumulation of phosphorylated IRF-3 is a potential mechanism by which vIRFs inhibit the induction of type I IFN during RRV infection. R6 vIRF independently inhibits IRF-3-mediated transcription. To assess the role of individual RRV vIRFs in inhibiting the induction of type I IFN, vIRF expression clones were generated with a C-terminal HA tag for ectopic expression in cell cultures. RRV vIRFs R6 and R10 were of specific interest, since R10 shares the highest level of amino acid identity with KSHV vIRF-1 (ORF K9), which is known to inhibit IRF-3-induced gene expression (9, 39), and R6 is most similar to R10. The vIRFs encoded by ORFs R7 and R11 were also included for comparison. The expression of these vIRFs in tRFs and HEK293 cells was initially verified by utilizing the HA tag within each construct (Fig. 8A and data not shown). Individual RRV vIRFs were then expressed alongside an IRF-3-responsive reporter to determine if the expression of any of

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these RRV vIRFs can inhibit IRF-3-mediated transcription. HEK293 cells were transiently transfected with a firefly luciferase plasmid under the control of an IFN-stimulated response element (ISRE)-containing promoter, along with a single vIRF clone. Cells were stimulated 24 h later with poly(IC) to induce the activation of IRF-3. Relative luciferase units (RLU) were determined by defining 100% RLU as the output in poly(IC)-stimulated cells that had been transfected with the empty vector (Fig. 8B). Similar to previous findings, KSHV vIRF-1 was able to inhibit 80% of IRF3-mediated transcription, in comparison to the empty vector (Fig. 8B) (9, 39). Likewise, three of the four RRV vIRFs tested (R6, R10, and R11) resulted in a 40 to 60% inhibition of IRF-3-mediated transcription in these assays (Fig. 8B). The expression of R6 vIRF resulted in the most significant effect, inhibiting IRF-3-mediated transcription by at least 60% (Fig. 8B) in a dose-dependent manner (Fig. 8C). Interestingly, R7 vIRF further enhanced IRF-3mediated transcription by more than 2-fold in these assays (Fig. 8B), suggesting a potentially unique role for R7 vIRF in stimulating transcription. To further evaluate the R6 vIRF inhibition of IRF-3-mediated transcription, the HA-tagged expression clone of R6 was expressed in tRFs and immunoprecipitated with IRF-3 pAb or HA MAb, followed by Western blot analysis. The IP-Western data showed that R6 vIRF interacts with endogenous, cellular IRF-3 (Fig. 8D), and the specificity of this interaction is highlighted by the lack of an interaction between R7 vIRF and IRF-3 in these same analyses (Fig. 8D). The spatial relationship between R6 vIRF and IRF-3 within the cell was also examined by immunofluorescence analysis. In unstimulated cells, IRF-3 was detected mainly within the cytoplasm, as shown in Fig. 7D and 8E, and expression with a control plasmid, pEGFP, or either of the vIRF expression plasmids, did not increase the nuclear accumulation of IRF-3 (Fig. 8Ei to iii). R6 vIRF was expressed in both the cytoplasm and nucleus,

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FIG 7 Nuclear accumulation of IRF-3 is inhibited during WTBAC RRV infection of RFs. RFs were infected with WTBAC RRV or vIRF-ko RRV (MOI ⫽ 1). (A) Whole-cell extracts were analyzed for total IRF-3 (FL-425) and GAPDH by SDS-PAGE. (B and C) Native cellular lysates (40 ␮g per sample) were run on a 7.5% PAGE nondenaturing gel with 1% sodium deoxycholate in the cathode chamber. Native lysates were probed for IRF-3, and data were analyzed by using densitometry to quantify the amount of IRF-3 present in the dimeric form (slower-migrating band) in relation to total IRF-3 (the sum of IRF-3 present in the dimeric and monomeric forms). Data are expressed as a percentage based on this ratio (percentage of IRF3 present in the dimeric form) and are presented as averages (⫾SEM) of data from 3 independent experiments. (D) RFs were fixed and analyzed via indirect immunofluorescence for IRF-3 (SL012.1) (shown in red), and nuclei were detected by using Hoechst dye (blue). RFs were transfected with poly(IC) (10 ␮g for 6 h) as a positive control for IRF-3 activation/nuclear localization. A representative image at 6 hpi is shown (magnification, ⫻400). (E) The percentage of cells expressing IRF-3 in the nucleus was calculated by counting ⬃200 cells within 5 different fields under each condition. Data are represented as means (⫾SEM) of data from 5 separate experiments. (F) Cytosolic and nuclear lysates were analyzed by SDS-PAGE for total IRF-3 (FL-425) and phospho-Ser396 IRF-3 (IRF-3p). Full-length PARP and GAPDH were used as loading controls. Gel images are representative of 3 independent experiments. N.S., not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PARP, poly(ADP-ribose) polymerase.

with some colocalization with cellular IRF-3; however, the colocalization of R6 vIRF and IRF-3 appeared to be enhanced when cells were stimulated with poly(IC) (Fig. 8Eiv). The colocalization of R6 vIRF and IRF-3 does not seem to be restricted to either the cytoplasm or the nucleus, and there was no apparent colocalization between R7 vIRF and cellular IRF-3, even following poly(IC) stimulation (Fig. 8E). Likewise, vIRF expression did not inhibit the nuclear localization of IRF-3 following poly(IC) stimulation, nor did poly(IC) stimulation alter the expression of either vIRF (Fig. 8E). These results show that R6 vIRF interacts and colocalizes with cellular IRF-3, and


this interaction may be enhanced when the cells are stimulated with poly(IC) to induce IRF-3 activation. DISCUSSION

KSHV allocates a significant portion of its genome to encoding immunomodulatory proteins (3). Of particular interest are the viral interferon regulatory factors (vIRFs), unique to KSHV and RRV, a closely related simian gamma-2-herpesvirus (2, 54, 56, 60). KSHV vIRF-1 was initially identified and named because of its sequence similarities with cellular IRFs; specifically, it shares 13% amino acid (aa) identity with human IRF-8 and IRF-9 (49). More-

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FIG 8 R6 vIRF inhibits IRF-3-mediated transcription. RRV vIRFs (R6, R7, R10, and R11) were C-terminally tagged with HA, cloned into pcDNA3.1(⫺) using oligonucleotides listed in Table 2, and transiently transfected. (A) Whole-cell lysates were collected from RFs transfected with a single vIRF construct at 36 h posttransfection. Equal amounts of lysate were subjected to SDS-PAGE and probed for HA expression. (B) HEK293 cells were transfected overnight with 500 ng total DNA (vIRF-HA construct [50 ng or 0 ng], pISRE-LUC [250 ng], pRL_SV40 [10 ng], and empty pcDNA3.1 [190 ng or 240 ng]). Cells were then transfected with poly(IC) and assayed 6 h later. Firefly luciferase levels were normalized to constitutively expressed Renilla luciferase levels in each well, and all conditions were made relative to the positive control [empty vector plus poly(IC)]. KSHV vIRF-1 (K9) was included as a control. Data are average data (⫾SEM) from 4 independent experiments and are compared to the positive control via Student’s t test. (C) HEK293 cells were transfected as described above for panel B but with increasing amounts of R6-HA-pcDNA3.1 (5 ng, 25 ng, 50 ng, and 100 ng). Data are average data (⫾SEM) from 3 independent experiments. (D) RFs were nucleofected with R6-HA, R7-HA, or pEGFP or mock transfected (no DNA) for 48 h and immunoprecipitated with anti-HA or anti-IRF-3 antibody. Immunoprecipitated lysates were then analyzed by SDS-PAGE; half was used to probe for HA, and the other half was used to probe for IRF-3. The image is representative of 3 separate experiments. (E) At 40 h postnucleofection, RFs were transfected with poly(IC) for 6 h or mock transfected, fixed, and analyzed by immunofluorescence for the detection of vIRF (anti-HA) (green) and cellular IRF-3 (red) and stained with Hoechst (blue) for the detection of nuclei. I.P., immunoprecipitated; WB, Western blot.

over, in vitro studies have demonstrated that KSHV vIRF-1, -2, and -3 possess a variety of mechanisms to independently inhibit cellular IRFs (52). Similarly, RRV encodes eight vIRFs that also share homology with cellular IRFs, and R6, R7, R8, R10, and R11

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vIRFs share between 19 and 26% aa identity with KSHV vIRF-1 (60). However, the RRV vIRFs do not share any significant homology with the other 3 KSHV vIRFs (60). Due to these similarities, the RRV vIRFs are also proposed to interfere with

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the transcriptional functions of cellular IRFs. The deletion of the vIRFs using WTBAC RRV, presented herein, has finally allowed an evaluation of the impact of vIRFs during de novo RRV infection, something that has proven difficult with KSHV due to its poor lytic replication in vitro. These data are the first to demonstrate that the deletion of the vIRFs results in increased gene expression and production of IFN and an increased nuclear accumulation of phosphorylated IRF-3 during de novo RRV infection. The IFN response is an important component of a cell’s antiviral defenses. To effectively circumvent the induction of IFN, an invading pathogen must also act quickly. Accordingly, transcriptional analysis has demonstrated that all 8 RRV vIRFs are detected by 12 hpi by RT-PCR, and vIRF R10 was expressed as early as 3 hpi in RRV-infected tRFs and PBMCs (Fig. 1B and 4A). Previous transcriptional analyses of RRV26-95 also demonstrated that at least 2 vIRFs (corresponding to R6 and R10) were expressed as early as 6 to 12 hpi (14). Likewise, KSHV vIRF-1 is also transcriptionally upregulated upon the induction of lytic replication in latently infected B cells (13). Therefore, the early expression of the vIRFs during RRV infection suggests that they also play a role in inhibiting the innate antiviral response, including the induction of IFN. A comparison of WTBAC and vIRF-ko RRV de novo infections revealed no difference in growth kinetics in RFs, and there were minimal effects on the expression of ORFs adjacent to the deleted vIRF region. The detection of an additional, truncated ORF 57 transcript in vIRF-ko RRV infection did not affect viral growth in RFs, and because the full-length ORF 57 transcripts were still detected, it is unlikely that the less abundant, truncated transcript interferes with the efficient expression of ORF 57. Further analysis showed that vIRF-ko RRV can also infect PBMCs albeit at a lower infection efficiency. Despite a decrease in the infection efficiency, vIRF-ko RRV infection resulted in an earlier and more robust induction of type I and type II IFN during RRV infection in both tRFs and PBMCs. Not surprisingly, the IFN-␣ produced within RRV-infected PBMCs was restricted to pDCs (Fig. 6 and data not shown), which are known to produce large amounts of IFN-␣ in response to a number of different viruses (11, 17, 19). The decrease in IFN-␣ production following WT RRV infection cannot be attributed to reduced levels of infection, since WT RRV infected PBMCs at a higher frequency (Fig. 4B), and the infection efficiencies in pDCs were comparable for both WT RRV- and vIRF-ko RRV-infected PBMC cultures (Fig. 4D), as was the survival of pDCs (data not shown). The data presented here do not directly demonstrate that pDCs producing IFN-␣ are infected, and the utilization of GFP-expressing clones of WT and vIRF-ko RRV suggested that only a small percentage of IFN-␣-producing pDCs also express GFP (data not shown). However, it is likely that a maximal production of IFN-␣ precedes the detection of GFP expression in an RRV-infected cell, as the detection of a GFP signal is inconsistent before 24 h post-RRV infection (data not shown), and a more sensitive detection method is not currently available. It is important to consider, however, that pDCs are unique in their sole expression of Toll-like receptor 7 (TLR7) and TLR9, which specifically equips them to recognize bacterial and viral nucleic acids, including genomic DNAs from several herpesviruses (31, 53). Moreover, it was recently demonstrated that KSHV infection of pDCs induces the production of IFN-␣ in a TLR9-dependent manner (70). Therefore, the detection of RRV transcripts by 3 hpi in RRV-infected PBMC cultures (Fig. 3A) and the dramatic in-


crease in IFN transcript levels at 6 hpi imply that pDCs are likely responding to direct infection with RRV, potentially through the recognition of its genomic DNA. The induction of type I IFNs is orchestrated by IRF-3, constitutively expressed in most cell types, and IRF-7, constitutively expressed in pDCs. These IRFs are quickly activated following viral infection and subsequently accumulate in the nucleus, where they drive the transcription of type I IFNs. Data presented herein suggest that vIRFs inhibit the nuclear accumulation of phosphorylated IRF-3 within the first 8 h of RRV infection, preceding the inhibition of type I IFN induction during RRV infection, which peaked at around 12 hpi. Additionally, reporter assays were used to examine the role of individual RRV vIRFs in inhibiting the induction of IFN. These analyses showed that R6 vIRF and, to a lesser extent, R10 and R11 vIRFs significantly inhibit IRF-3mediated transcription. Further examination of the relationship between R6 vIRF and IRF-3 revealed that R6 vIRF can directly or indirectly interact with endogenous IRF-3, and R6 vIRF and IRF-3 also colocalize within the cell. Therefore, R6 vIRF could be sequestering IRF-3, disrupting IRF-3 associations with other transcriptional factors, or inhibiting IRF-3 from binding promoter regions in target genes, such as type I IFN. Indeed, KSHV vIRFs are known to bind cellular transcription factors and inhibit the induction of type I IFN; KSHV vIRF-1 can bind p300/CBP and inhibit its essential interaction with IRF-3 (9, 39), and KSHV vIRF-3 can bind to and retain NF-␬B in the cytoplasm (61). Interestingly, R7 vIRF enhanced IRF-3-mediated transcription in our reporter assays. This phenotype is potentially independent of a direct targeting of IRF-3, as a further characterization of the relationship between R7 vIRF and IRF-3 showed that R7 vIRF does not bind or colocalize with IRF-3 in the cell (Fig. 8). However, the R7 vIRF-mediated enhancement of transcription was not entirely surprising, since KSHV vIRF-1 and vIRF-3 have also been shown to enhance the transcription of cellular genes (41, 42, 55). Moreover, a role for vIRFs in the stimulation of IRF-mediated transcription (41) could promote the cytokine-enhanced reactivation of the virus (47), providing the necessary drive to maintain an adequate pool of infected cells within the host. In addition to establishing an antiviral state early during viral infection, type I IFNs (IFN-␣/␤) also have effects on NK cells (5, 50), T cells (25, 46, 65), and DCs (27, 44, 48), directly impacting the adaptive immune response. Therefore, the inhibition of type I IFNs by vIRFs could disrupt both the innate and adaptive immune responses during in vivo RRV infection. Additionally, our data indicate that RRV vIRFs also inhibit the induction of type II IFN (IFN-␥), which is essential for promoting an efficient Th1 immune response. In fact, previous in vitro analyses demonstrated that KSHV vIRF-1 and vIRF-3 can also interfere with type II IFN signaling. Specifically, KSHV vIRF-1 inhibits transcription induced by IFN-␥ (37, 76) and results in the decreased surface expression of major histocompatibility complex class I (MHC-I) (34). Likewise, KSHV vIRF-3 similarly inhibits IFN-␥-responsive promoters, resulting in a decreased expression of MHC-II on latently infected B cells (59). A preliminary examination of IFN-␥ production in RRV-infected PBMC cultures, and T cells and DCs specifically, did not show any measurable production over levels in mock-infected cultures in the first 24 hpi (data not shown), despite the significant induction at the transcript level in vIRF-ko RRV infection (Fig. 5). However, the vIRF-mediated inhibition of IFN-␥ may be important for other cell types that were not exam-

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ined here, such as natural killer cells (5, 66). Additionally, vIRFs may be essential for inhibiting IFN-␥ at later times during RRV infection, potentially in response to other cytokines, such as interleukin-12 (IL-12) (50), which would be important during the development of the adaptive immune response. Future studies will utilize vIRF-ko RRV to focus on the impact of vIRFs during in vivo RRV infection in the rhesus macaque to further our understanding of their role(s) in immune evasion and pathogenesis. REFERENCES 1. Albert TJ, et al. 2005. Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori. Nat. Methods2:951–953. 2. Alexander L, et al. 2000. The primary sequence of rhesus monkey rhadinovirus isolate 26-95: sequence similarities to Kaposi’s sarcomaassociated herpesvirus and rhesus monkey rhadinovirus isolate 17577. J. Virol. 74:3388 –3398. 3. Areste C, Blackbourn DJ. 2009. Modulation of the immune system by Kaposi’s sarcoma-associated herpesvirus. Trends Microbiol. 17:119 –129. 4. Bergquam EP, Avery N, Shiigi SM, Axthelm MK, Wong SW. 1999. Rhesus rhadinovirus establishes a latent infection in B lymphocytes in vivo. J. Virol. 73:7874 –7876. 5. Biron CA, Nguyen KB, Pien GC, Cousens LP, Salazar-Mather TP. 1999. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu. Rev. Immunol. 17:189 –220. 6. Bowie AG, Unterholzner L. 2008. Viral evasion and subversion of pattern-recognition receptor signalling. Nat. Rev. Immunol. 8:911–922. 7. Boyne JR, Whitehouse A. 2006. Gamma-2 herpes virus posttranscriptional gene regulation. Clin. Microbiol. Infect. 12:110 –117. 8. Brown KN, Barratt-Boyes SM. 2009. Surface phenotype and rapid quantification of blood dendritic cell subsets in the rhesus macaque. J. Med. Primatol. 38:272–278. 9. Burysek L, et al. 1999. Functional analysis of human herpesvirus 8-encoded viral interferon regulatory factor 1 and its association with cellular interferon regulatory factors and p300. J. Virol. 73:7334 –7342. 10. Burysek L, Yeow WS, Pitha PM. 1999. Unique properties of a second human herpesvirus 8-encoded interferon regulatory factor (vIRF-2). J. Hum. Virol. 2:19 –32. 11. Chung E, et al. 2005. Characterization of virus-responsive plasmacytoid dendritic cells in the rhesus macaque. Clin. Diagn. Lab. Immunol. 12:426 – 435. 12. Dai J, Megjugorac NJ, Amrute SB, Fitzgerald-Bocarsly P. 2004. Regulation of IFN regulatory factor-7 and IFN-alpha production by enveloped virus and lipopolysaccharide in human plasmacytoid dendritic cells. J. Immunol. 173:1535–1548. 13. Dittmer DP. 2003. Transcription profile of Kaposi’s sarcoma-associated herpesvirus in primary Kaposi’s sarcoma lesions as determined by realtime PCR arrays. Cancer Res. 63:2010 –2015. 14. Dittmer DP, et al. 2005. Whole-genome transcription profiling of rhesus monkey rhadinovirus. J. Virol. 79:8637– 8650. 15. Donnelly RP, Kotenko SV. 2010. Interferon-lambda: a new addition to an old family. J. Interferon Cytokine Res. 30:555–564. 16. Estep RD, Powers MF, Yen BK, Li H, Wong SW. 2007. Construction of an infectious rhesus rhadinovirus bacterial artificial chromosome for the analysis of Kaposi’s sarcoma-associated herpesvirus-related disease development. J. Virol. 81:2957–2969. 17. Feldman SB, et al. 1994. Viral induction of low frequency interferonalpha producing cells. Virology 204:1–7. 18. Fensterl V, Sen GC. 2009. Interferons and viral infections. Biofactors 35:14 –20. 19. Fitzgerald-Bocarsly P, Dai J, Singh S. 2008. Plasmacytoid dendritic cells and type I IFN: 50 years of convergent history. Cytokine Growth Factor Rev. 19:3–19. 20. Flowers CC, Flowers SP, Nabel GJ. 1998. Kaposi’s sarcoma-associated herpesvirus viral interferon regulatory factor confers resistance to the antiproliferative effect of interferon-alpha. Mol. Med. 4:402– 412. 21. Fuld S, Cunningham C, Klucher K, Davison AJ, Blackbourn DJ. 2006. Inhibition of interferon signaling by the Kaposi’s sarcoma-associated herpesvirus full-length viral interferon regulatory factor 2 protein. J. Virol. 80:3092–3097. 22. Gao SJ, et al. 1997. KSHV ORF K9 (vIRF) is an oncogene which inhibits the interferon signaling pathway. Oncogene 15:1979 –1985.

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