expression of a human cytomegalovirus latency-associated homolog of interleukin-10 during the...

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Virology 370 (200

Expression of a human cytomegalovirus latency-associated homolog ofinterleukin-10 during the productive phase of infection

Christina Jenkins a, Winnie Garcia a, Allison Abendroth a,b, Barry Slobedman a,⁎

a Centre for Virus Research, Westmead Millennium Institute and University of Sydney, New South Wales, Australiab Department of Infectious Diseases and Immunology, University of Sydney, New South Wales, Australia

Received 6 June 2007; returned to author for revision 26 June 2007; accepted 5 September 2007Available online 17 October 2007

Abstract

The human cytomegalovirus UL111A region is active during both productive and latent phases of infection. During productive infection, the virusexpresses ORF79, a protein with oncogenic properties, and cmvIL-10, a functional homolog of human IL-10. During latent infection of myeloidprogenitor cells, an alternately spliced variant of cmvIL-10, termed latency-associated (LA) cmvIL-10 has previously been identified. To determinewhether LAcmvIL-10 transcription occurs during productive infection, we performed 5′ and 3′ RACE to map UL111A-region transcripts inproductively infected human foreskin fibroblasts (HFFs). This analysis revealed the presence of a singly spliced UL111A-region transcript predictedto encode LAcmvIL-10. This transcript was expressed in HFFs with early (β) kinetics, a temporal class that differs from that of ORF79 (α kinetics)and cmvIL-10 (γ kinetics). These data identify and map a transcript encoding a latency-associated homolog of IL-10 which is expressed by the virusduring the productive phase of infection.© 2007 Elsevier Inc. All rights reserved.

Keywords: Human cytomegalovirus; Latency-associated transcription; Productive infection; Rapid amplification of cDNA ends

Introduction

Human cytomegalovirus (HCMV) is the prototypic β-herpesvirus and causes serious disease in neonates andimmunocompromised individuals such as allograft recipientsand those with HIV AIDS. The virus is able to undergoproductive viral replication in a variety of differentiated celltypes including fibroblasts, endothelial cells, dendritic cells andmacrophages, and establish a latent infection in hematopoieticcells such as monocytes and their progenitors (Mocarski et al.,2007). Two open reading frames (ORF) expressed from theUL111A region of the HCMV genome have previously beenidentified during productive infection. Initial studies examiningthe morphological transforming region II (mtrII) identified a 79aa ORF with oncogenic properties (Kouzarides et al., 1983;

⁎ Corresponding author. Centre for Virus Research, Westmead MillenniumInstitute, PO Box 412, Westmead, NSW 2145, Australia. Fax: +61 2 98459100.

E-mail address: [email protected] (B. Slobedman).

0042-6822/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2007.09.002

Razzaque et al., 1988; Thompson et al., 1994). Chee et al. (1990)designated the gene encoding ORF79 as UL111A. Furtheranalysis of this region by Kotenko et al. (2000) and Lockridge etal. (2000) identified a splicing event that bypassed the UL111Astop codon and resulted in the expression of an HCMV homologto the potent immunosuppressive cytokine human IL-10(denoted cmvIL-10). Lockridge et al. (2000) extended thiswork using rapid amplification of cDNA ends (RACE)methodology and demonstrated that the UL111A transcriptthat encodes cmvIL-10 is a doubly spliced message that initiatesat position 159642 (HCMV strain AD169 coordinates, GenBankaccession number 38 X17403) and terminates at position160430, coding for a 175 aa protein. Like human IL-10,cmvIL-10 protein exhibits a range of immunosuppressiveproperties, including an ability to downregulate major histo-compatibility complex (MHC) class I and MHC class II onmonocytes, inhibit PBMC proliferation and secretion of pro-inflammatory cytokines, decrease matrix metalloproteinaseactivity, alter cell–cell and cell–matrix interactions of cyto-trophoblasts and endothelial cells at the uterine–placental

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http://dx.doi.org/10.1016/j.virol.2007.09.002

286 C. Jenkins et al. / Virology 370 (2008) 285–294

interface, and interfere with the maturation of dendritic cells(Spencer et al., 2002; Chang et al., 2004; Raftery et al., 2004;Yamamoto-Tabata et al., 2004).

In addition to expression during productive infection, asingly spliced UL111A-region transcript encoding a latency-associated (LA)-cmvIL-10 has been identified in latentlyinfected granulocyte–macrophage progenitor cells (GM-Ps)(Jenkins et al., 2004). In contrast to the transcript encodingcmvIL-10, the transcript encoding LAcmvIL-10 was shownto initiate within a 38 bp region (between nucleotide position159577 and 159615) upstream of the UL111A start site andencompasses 2 exons and 1 intron with the stop site oftranscription co-terminal with cmvIL-10 at position 160430.Both transcripts, however, encode ORFs predicted tooriginate from the same methionine at position 159678.Consequently, LAcmvIL-10 and cmvIL-10 are predicted tobe co-linear for the first 127 aa, but with divergentsequences for the remaining C-terminal portions due toretention of the second intron containing a stop codon inLAcmvIL-10, giving rise to 139 aa protein (Jenkins et al.,2004). In the present study, we sought to determine whetherthe transcript encoding LAcmvIL-10 detected during latentinfection of GM-Ps was expressed during the productivephase of HCMV infection.

Fig. 1. Mapping UL111A-region transcripts expressed during productive HCMV infencoding UL111A transcript region expanded to show the position of UL111A gene-s(B) Ethidium bromide-stained agarose gel of 5′ RACE PCR products derived fromArrows indicate 5′ RACE PCR products amplified at 769 bp, 693 bp, and 610 bp aterminus of the human transferrin receptor (TFR) (2.6 kb fragment), a negative conindicated. (C) Ethidium bromide-stained agarose gel of 3′ RACE PCR products derToledo. Arrows indicate 3′ RACE PCR products amplified at 736 bp, 660 bp, and 577of the human transferrin receptor (TFR) (3.5 kb fragment), a negative control conta

Results and discussion

5′ RACE mapping of UL111A-region transcripts duringproductive HCMV infection

The 5′ ends of transcripts expressed from the UL111Aregion during productive HCMV infection were examinedusing 5′ RACE mapping (SMART RACE cDNA Amplifica-tion Kit, BD Biosciences, USA). Experiments were performedusing RQ1 RNase-free DNase (Promega Corporation, USA)-treated total RNA extracted from human foreskin fibroblasts(HFFs) 3 days after infection with HCMV strain Toledo(MOI=3). 5′ RACE-Ready cDNA was PCR amplified with anabridged universal primer mix (UPM) and a gene-specificprimer (JAS-R5) designed to amplify across the two introns ofthe previously characterised UL111A transcript, which encodescmvIL-10 (Fig. 1A). 5′ RACE PCR products were detectedmigrating at sizes consistent with the predicted productsamplified from an unspliced UL111A transcript (769 bp), asingly spliced UL111A transcript (693 bp), and a doublyspliced UL111A transcript (610 bp) (Fig. 1B). A controlreaction using a human transferrin receptor (TFR)-specificreverse primer (BD Biosciences, USA) in conjunction withUPM yielded a 5′ RACE product of approximately 2.6 kb

ection. (A) Schematic representation of the HCMV genome with the cmvIL-10pecific (JAS-F1, JAS-R5 and JAS-2U) and generic (UPM) primers (arrowheads).RNA extracted from HFFs productively infected with HCMV strain Toledo.mplified with primers JAS-R5 and UPM. A positive control amplifying the 5′trol containing no RNA template (No RNA), and 100 bp DNA ladders (M) areived from RNA extracted from HFFs productively infected with HCMV strainbp with primers JAS-F1 and UPM. A positive control amplifying the 3′ terminusining no RNA template (No RNA) and 100 bp DNA ladders (M) are indicated.

287C. Jenkins et al. / Virology 370 (2008) 285–294

confirming RNA integrity. No products were detected whentemplate RNA was omitted from the reaction. Each UL111A-region 5′ RACE PCR product was excised from the gel, UA-cloned and their nucleotide sequence determined. Sequencingof 30 clones confirmed the intron/exon structure of each 5′RACE PCR product with the 769 bp product originating froman unspliced UL111A transcript (predicted to encode ORF79),the 693 bp product originating from a singly spliced UL111Atranscript (predicted to encode LAcmvIL-10), and the 610 bptranscript arising from a doubly spliced UL111A transcript(predicted to encode cmvIL-10) (Table 1).

Analysis of the 5′ ends of the 30 sequenced clonesoriginating from the three different sized transcripts revealedthat all mapped at or near to the previously characterised startsite of the cmvIL-10 transcript (Lockridge et al., 2000) with 27clones mapping within 2 bp of this start site at position 159642,and a single clone from each of the three detected transcripttypes mapping 5 bp downstream of the UL111A start site(Table 1). The same three transcript sizes, start sites oftranscription and splicing patterns were observed in HFFsinfected with two additional HCMV strains; TownevarRIT3 andAD169 (data not shown).

3′ RACE mapping of UL111A-region transcripts duringproductive HCMV infection

Poly dT primed cDNA was subjected to a single round ofPCR amplification with primers designed to both amplify the 3′end and to determine the splicing pattern of UL111A-regiontranscripts expressed during productive infection. Thus, primersspanned across both intron 1 and intron 2 of the previouslycharacterised cmvIL-10 transcript (gene-specific primer JAS-F1and generic primer UPM).

Table 1DNA sequences of start sites of 5′ RACE UL111A-region PCR clones derived from

aPosition coordinates according to HCMV strain AD169 (accession number X17403bStart site of transcription of UL111A is indicated by an arrow at nucleotide positiocDNA sequence of the HCMV UL111A gene region from nucleotide position 1596dUnspliced transcript predicted to encode the ORF79 protein.eSingly spliced transcript predicted to encode the 139 aa LAcmvIL-10 protein.fDoubly spliced transcript predicted to encode the 175 aa cmvIL-10 protein.

This analysis revealed three 3′ RACE PCR products mi-grating at the predicted sizes of products amplified from theunspliced (736 bp), the singly spliced (660 bp), and the doublyspliced UL111A transcripts (577 bp) (Fig. 1C). An additionalsmaller band was detected at approximately 430 bp. In contrastto the three larger PCR products, southern blotting did not resultin specific hybridization of an internal UL111A-regionoligonucleotide probe to this smaller PCR product indicatingthat it was likely a result of non-specific amplification (data notshown). As described for 5′ RACE reactions, a control reactionconfirmed full-length amplification of TFR (∼3.5 kb) using aTFR-specific forward primer (BD Biosciences, USA) in con-junction with UPM. No products were detected when templateRNA was omitted from the reaction. Each 3′ UL111A-regionRACE PCR product was excised, UA-cloned and sequenced todetermine their nucleotide sequence. Data obtained from 26clones demonstrated that 20 of these exhibited sequencesconsistent with a single polyadenylation site 14 bp downstreamof a consensus polyadenylation signal (AATAAA), indicatingthe same termination site as described for the cmvIL-10transcript during productive infection (AD169 nucleotide posi-tion 160430; Kotenko et al., 2000; Lockridge et al., 2000)(Table 2). No other clone terminated more than six nucleotidesfrom this site. Sequencing of fifteen clones arising from the660 bp product, revealed a processed transcript with a singlespliced region of 76 bp that was identical to intron 1 of thecmvIL-10 transcript, but unlike the cmvIL-10 transcript nosecond intron was observed. This transcript was predicted toencode LAcmvIL-10. Clones arising from the 736 bp and577 bp 3′RACE PCR products were confirmed to be generatedfrom an unspliced UL111A-region transcript (predicted toencode ORF79), and a doubly spliced transcript (predicted toencode cmvIL-10), respectively. The same 3′ end and splicing

productively infected HFFs

; Chee et al., 1990).n 159642 (Lockridge et al., 2000).30 to 159660.

Table 2DNA sequences of termination sites of 3′ RACE UL111A-region PCR clones derived from productively infected HFFs

aPosition coordinates according to HCMV strain AD169 (accession number X17403; Chee et al., 1990).bStop site of transcription of UL111A is indicated by an arrow at nucleotide position 160430 (Lockridge et al., 2000).cDNA sequence of the HCMV UL111A gene region from nucleotide position 160420 to 160450.dN=A, C, G, or T; V=A, G, or C.eUnspliced transcript predicted to encode the ORF79 protein.fSingly spliced transcript predicted to encode the 139 aa LAcmvIL-10 protein.gDoubly spliced transcript predicted to encode the 175 aa cmvIL-10 protein.


patterns were observed in a further two replicate experiments,using RNA from HFF cultures productively infected witheither HCMV strain TownevarRIT3 or strain AD169 (data notshown).

The results of the 5′ and 3′ RACE analyses confirmed thepresence of no fewer than three transcripts expressed from theUL111A region during productive infection of HFFs (Fig. 2).The first, a doubly spliced transcript, with two introns 76 bp and83 bp in size, initiates at nucleotide position 159642 and ter-minates at position 160430, correlating with the structure of thecmvIL-10 transcript previously characterized during productiveinfection (Kotenko et al., 2000; Lockridge et al., 2000). Asecond transcript extends from the same start site of transcriptionand is co-terminal with the UL111A transcript but remains

Fig. 2. Schematic depicting three UL111A-region transcripts detected during procorresponding 175 aa ORF that encodes cmvIL-10, the singly spliced UL111A-regionunspliced UL111A transcript and its corresponding 79 aa ORF encoding the mtrII orespectively. Nucleotide position numbers (HCMV strain AD169 genome) of the stindicated by a right-angled arrow and the signal peptide is indicated by the grey bo

unspliced. This transcript likely represents the HCMV geneoriginally designated UL111A that encodes ORF79 (Chee et al.,1990) which is thought to play a role in tumorigenic trans-formation, and as such has been designated the mtrII oncogene(Kouzarides et al., 1983; el-Beik et al., 1986; Razzaque et al.,1988; Jahan et al., 1989; Jariwalla et al., 1989; Chee et al., 1990;Thompson et al., 1994; Muralidhar et al., 1996). The third, atranscript containing a single intron of 76 bp and two exons, wasalso detected. This transcript initiates at the same start site and isco-terminal with the doubly spliced UL111A transcript encodingcmvIL-10, but differed with respect to intron 2, which remainedunspliced. This transcript is predicted to encode LAcmvIL-10,which is a 139 aa ORF derived from a singly spliced UL111A-region transcript expressed during the latent phase of infection

ductive infection. Shown are the doubly spliced UL111A transcript and thetranscript and its corresponding ORF that encodes 139 aa LAcmvIL-10, and thencoprotein (ORF79). Black boxes and open boxes depict transcripts and ORFs,art and stop sites of transcription are indicated. The first methionine residue isx.


(Jenkins et al., 2004). Consistent with our identification andmapping of IL-10 homolog transcripts expressed during pro-ductive infection, western blot analysis of productively infectedMRC-5 cells reacted with anti-cmvIL-10 antibody identified notonly cmvIL-10-like protein, but also a smaller protein productwhich the authors suggested may be LAcmvIL-10 protein(Chang et al., 2004). Thus, UL111A-region transcripts encodingLAcmvIL-10 protein are expressed during both latent and pro-ductive phases of infection.

The detection of HCMV latency-associated transcriptsduring both the latent and productive infection is not withoutprecedent. Indeed, the expression of LAcmvIL-10 transcriptsduring productive infection correlates with the detection ofHCMV sense major immediate early (MIE)-region CLTs duringproductive HCMV infection (Lunetta and Wiedeman, 2000),and also with observations that the major 2.0 kb latency-associated transcript (LAT) expressed by HSV-1 during latentinfection is also present in mice ganglia during productiveinfection (Spivack and Fraser, 1987). Expression of latency-associated transcripts during lytic viral infection has also beendemonstrated with the EBV LAT, EBNA-1, the latentlyexpressed VZV genes ORF21, ORF29, ORF62 and ORF63,and the pseudorabies virus LAT gene (Felser et al., 1988;Jackers et al., 1992; Lear et al., 1992; Debrus et al., 1995;Schaefer et al., 1995; Nonkwelo et al., 1996; Jin and Scherba,1999; Xia and Straus, 1999; Cohrs et al., 2002; Xia et al., 2003).

Surprisingly, the results from our study suggest that the virusmay utilise a different start site for LAcmvIL-10 transcriptionduring productive HCMV infection when compared to LAcm-vIL-10 transcription during latent infection. 5′ RACE mappingduring productive infection indicated that LAcmvIL-10 tran-scripts started at nucleotide position 159642 of the HCMVgenome, whereas 5′ primer walking analysis during latentinfection indicated that transcription initiated within a 38 bpregion (between nucleotide position 159577 and 159615) up-stream of this site (Jenkins et al., 2004). Thus, the initial 5′ regionof the LAcmvIL-10 coding transcripts expressed during pro-ductive and latent infections appear to differ, although bothtranscripts are predicted to use the same translational start siteand hence code for the same 139 aa protein.

Differences in the transcription start sites of viral genesexpressed during productive and latent phases of infection arenot without precedent among herpesviruses. EBV EBNA-1 hasbeen shown to be expressed during lytic infection under thecontrol of a promoter distinct from those used during latent EBVinfection (Lear et al., 1992; Schaefer et al., 1995; Nonkwelo etal., 1996). Transcription from the Cp and Wp promoters, whichare active during latency, are silenced during productiveinfection, and the downstream Fp promoter becomes activated(Nonkwelo et al., 1996). In addition, the RNAs expressed fromthe pseudorabies virus LAT gene during productive infectionhave been found to be distinct from the largest latency transcript(LLT), with a 2.0 kb productive infection transcript originatingabout 243 bp downstream of the transcriptional start site of theLLT (Cheung, 1990; Jin and Scherba, 1999). In HCMV, MIEregion transcription start site usage is differentially regulatedduring productive and latent infections. During latency, the

productive infection transcription start site (PSS) used to driveexpression of the major immediate MIE proteins IE1 and IE2 isrepressed, with latency-associated transcripts (sense CLTs)originating from two start sites which lie 292 bp and 356 bpupstream of PSS (Kondo et al., 1996).

Kinetics of LAcmvIL-10 transcription during productiveHCMV infection

During productive infection, HCMV genes are expressed inan ordered cascade. Viral genes are assigned to 1 of 3 classesbased upon their expression kinetics: immediate-early (IE), early(E), or late (L) (also designated α, β, or γ, respectively). Thekinetic class of singly spliced (LAcmvIL-10) and unspliced(ORF79) transcripts expressed during productive infection wasassessed using RT-PCR. HFFs were infected with HCMV strainToledo (MOI=3) and total RNAwas harvested at 12 h p.i. (afterincubation) in the presence or absence 100 μg/ml cyclohexamide(CHX; Sigma, USA), at 24 h and 48 h, and also at 72 h p.i. in thepresence or absence of 300 μg/ml phosphonoacetic acid (PAA;Sigma, USA) CHX inhibits both viral and cellular proteintranslation and PAA inhibits viral replication, a requirement forγ2 gene expression (Honess and Roizman, 1974; Honess andWatson, 1977).

Random primed cDNA was amplified with primers JAS-F1and JAS-2U. Primer JAS-2U amplifies from within the secondintron of the UL111A transcript and therefore allows amplifi-cation of only unspliced and singly-spliced (intron 1) UL111A-region transcripts. This analysis revealed a RT-dependent 421 bpproduct consistent with amplification of the unspliced UL111A-region transcript which was detected from 12 p.i. and remaineddetectable at all time points and in cells treated with either CHXor PAA (Fig. 3A). In addition, a RT-dependent 345 bp productconsistent with amplification of the singly spliced transcript (i.e.coding for LAcmvIL-10) was also detected from 12 h p.i.However, in contrast to the unspliced transcript, expression ofthe singly spliced transcript was sensitive to CHX as treatmentwith this inhibitor of protein synthesis abolished detectableexpression. On the other hand, expression remained detectablein the presence of PAA. No amplification was detected in RNAfrom uninfected HFFs or in reactions where the reversetranscriptase enzyme (RT) or RNAwas omitted. The specificityof these RT–PCR products was confirmed by DNA sequencing(data not shown). These RT-PCR data indicate that the unsplicedtranscript (ORF79) is expressed with immediate early (α)kinetics, a finding supported by the detection of the ORF79protein by immunohistochemistry as early as 12 h followinginfection of HEL 299 cells with HCMV (Muralidhar et al.,1996). In contrast, the singly spliced LAcmvIL-10 coding tran-script is expressed with early (β) kinetics. Although we did notspecifically examine the kinetics of expression of the doublyspliced (i.e. cmvIL-10) transcript, studies by Chang et al. (2004)previously demonstrated that this transcript was expressed as alate (γ) gene, detecting cmvIL-10 mRNA from 24 h post-inoculation using real-time PCR. Also, cmvIL-10 transcriptcopy number was profoundly reduced in the presence of twoviral replication inhibitors, foscarnet and ganciclovir. These

Fig. 3. Determination of kinetic class of the unspliced (ORF79) and singly spliced (LAcmvIL-10) transcripts during productive HCMV infection of HFFs. (A) RT–PCR analysis of expression of singly spliced UL111A transcripts in HFFs productively infected with HCMV strain Toledo. Arrows adjacent to the lanes indicate theexpected genomic-sized (i.e. unspliced RNA) product of 421 bp and spliced transcript (i.e. LAcmvIL-10)-sized product of 345 bp amplified using primers JAS-F1 andJAS-2U. Presence (+) or absence (−) of the reverse transcriptase (RT) in the reaction mixture and the presence (+) or absence (−) of cyclohexamide (CHX) orphosphonoacetic acid (PAA) are indicated. Mock-infected HFFs (Uninfected), a sample without RNA (No RNA) and DNA extracted from HCMV-infected HFFs(DNA) were included as controls and molecular weight size markers (M) are also shown. (B–D) Efficiency of CHX and PAA drug blocks during productive HCMVinfection of HFFs. RNA extracted from cells at 12 p.i. in the presence (+) or absence (−) of CHX, at 24, 48, and 72 h p.i. in the presence (+) or absence (−) of PAAanalysed by northern blot Hybridization with α-32P-labelled probes specific to either TRL4 (B), UL115 (C) or GAPDH (D). Hybridization of the probes to RNAsmigrating at the expected sizes of 2.7 kb for TRL4, 0.9 kb for UL115 and 1.3 kb for GAPDH are indicated. RNA from mock-infected HFFs (Uninfected) was includedas a control. Numbers to the left of the images indicate the size in nucleotides of an adjacent RNA marker.


differences in temporal expression observed between the threeUL111A-region transcripts imply that each may play differentroles at different stages of the replicative cycle during productiveHCMV infection.

In order to determine the efficacy of drug blocks, duplicateRNA samples were subjected to northern blotting with HCMVTRL4-specific, HCMV UL115-specific or GAPDH-specificα-32P-labelled double-stranded DNA probes. Hybridization ofblots with the TRL4 probe detected the ∼2.7 kb TRL4 majorearly (β) transcript at all time points, with the exception of 12 hp.i. in the presence of CHX (Fig. 3B). This is consistent withearly gene kinetics, as transcription of early genes is reliant ontranslation of transactivating immediate early (α) gene productsand treatment with CHX, a protein synthesis inhibitor, preventsthis from occurring (McDonough et al., 1985). Hybridizationwith the UL115 probe detected these 0.9 kb transcripts from48 h p.i. in the absence of PAA (Fig. 3C). In contrast, expressionat 72 h p.i. in the presence of PAA was abolished, a resultconsistent with the characterization of UL115 as a late (γ) gene(Leatham et al., 1991). These data confirmed the efficacy ofdrug treatments employed to delineate temporal gene expres-sion during productive infection. In addition, the quality of eachRNA sample was determined by probing with a 32P-labelled

DNA probe specific to the housekeeping gene GAPDH.GAPDH transcripts (1.3 kb) were readily detected in allsamples (Fig. 3D), confirming the integrity of the RNA used inthis study.

The mechanisms that control the temporal patterns of ex-pression of the different UL111A-region RNAs have not yetbeen defined. The fundamental difference between these RNAsis alternate splicing, which results in different protein products.Differentially spliced RNAs originating from the same promoterhave been shown to have very different expression patternsduring HCMV infection. For example, despite being initiated atthe same IE promoter, the UL37x1 unspliced RNA is expressedat high levels at IE times and remains abundant until late times ofinfection. In contrast, the UL37 spliced RNAs are expressed atlow abundance during IE times (Kouzarides et al., 1988; Tenneyand Colberg-Poley, 1991a,b; Goldmacher et al., 1999). Whilst anumber of regions of the genome, including the UL36–38 locusand the MIE region produce products from different temporalclasses as a consequence of different promoter usage and mRNAsplicing signals (Stenberg, 1996; Mocarski and Courcelle, 2001;Su et al., 2003), we hypothesize that in the case of UL111A-region RNAs, regulatory factors linked to the different temporalclasses control alternate splicing of transcripts originating from a


single UL111A promoter. This will be an important consider-ation for future work aimed at determining how the expression ofthese gene products is regulated.

Expression of LAcmvIL-10 protein from the singly splicedUL111A-region transcript

To determine whether the singly spliced transcript couldexpress LAcmvIL-10 protein, the cDNA was cloned and theconstruct transfected into HEK293 cells using Nucleofectortechnology (Amaxa Biosystems, Germany). The cDNA from thedoubly spliced cmvIL-10 transcript was similarly cloned andexpressed in HEK293 cells, as was a mock control generated bytransfecting HEK293 cells with the empty parental pcDNA3.1-mycHis(B) vector (which encodes resistance to the cytocidaldrug Geneticin). Forty-eight hours post transfection, success-fully transfected cells were selected with 400 μg/ml Geneticin,and stable transfectants were maintained in culture with passageinto fresh DMEM supplemented with 10% FCS and 400 μg/mlGeneticin every 3 days.

Conditioned culture media were collected and clarified bylow-speed centrifugation and total protein was concentratedfrom 1 ml aliquots using trichloroacetic acid (TCA) precipita-tion, before SDS–PAGE and western blotting with an anti-cmvIL-10 antibody (Fig. 4). Conditioned media from cellstransfected with the LAcmvIL-10 plasmid were found to expressa protein of 15.9 kDa, the predicted size of the epitope-taggedLAcmvIL-10, and epitope-tagged cmvIL-10 was detected at thepredicted size of 20.1 kDa in conditioned media from cellstransfectedwith the cmvIL-10 construct. An additional bandwasdetected in conditioned media from cmvIL-10 plasmid-trans-fected cells of∼25 kDa, consistent with the predicted size of thisprotein with glycosylation (Kotenko et al., 2000). A larger bandwas not detected in the LAcmvIL-10 sample, suggesting that thisprotein may not be glycosylated. No proteins were detected by

Fig. 4. Expression of recombinant LAcmvIL-10 and cmvIL-10 frommammaliancells. Western blot of total protein precipitates of conditioned media fromHEK293 cells transfected with LAcmvIL-10 and cmvIL-10 cDNA-derivedexpression vectors, or the empty parental vector (mock), following incubation ofthe membrane with a polyclonal anti-cmvIL-10 antibody. Arrows indicatecmvIL-10 at the predicted size of 20.1 kDa (with a mycHis fusion) and at a sizeconsistent with glycosylation of the protein (∼25 kDa) and LAcmvIL-10 (with amycHis fusion) at 15.9 kDa. Sizes of corresponding molecular weight markerare indicated with molecular masses in kilodaltons (kDa).

the anti-cmvIL-10 antibody in conditioned media from parentalvector (mock)-transfected cells. These data show that the singlyspliced transcript codes for LAcmvIL-10 and is secreted fromcells into culture supernatant. In addition, an antibody to cmvIL-10 exhibited sufficient cross-reactivity with LAcmvIL-10 suchthat it could be used to detect both proteins.

In conclusion, we have identified and mapped a UL111A-region transcript encoding the latency-associated cmvIL-10homolog, LAcmvIL-10, which was expressed during productiveHCMV infection in vitro, and defined the temporal class towhich this transcript belongs. Thus, transcripts encodingLAcmvIL-10 are expressed during both the latent andproductive phases of infection. The initiation and terminationsites of the singly spliced LAcmvIL-10 coding transcriptsexpressed during productive infection were identical to the startand stop sites of transcription of the doubly spliced cmvIL-10transcript, and the unspliced transcript coding for ORF79,indicating that no fewer than three different UL111A-regiontranscripts are expressed during productive infection. TheUL111A region has been shown to be dispensable for productiveinfection of permissive cultured cells (Chang et al., 2004; Yu etal., 2003; Pepperl-Klindworth et al., 2006). However, recombi-nant cmvIL-10 exhibits potent immunosuppressive properties(Spencer et al., 2002; Chang et al., 2004; Raftery et al., 2004)and LAcmvIL-10 exhibits some, but not all of the immunosup-pressive properties of cmvIL-10 in so far as it is able todownregulate MHC class II expression by monocytes and GM-Ps but does not inhibit dendritic cell maturation (C. Jenkins andB. Slobedman, unpublished data). Thus, this protein may act toenhance the capacity of the virus to limit the CD4+ T cellresponse during the productive phase of infection. The relativecontributions of cmvIL-10 and LAcmvIL-10 to the immuno-evasive capabilities of the virus remains to be determined, buttheir expression implies at least a partial redundancy in functionsthat are likely to enhance the capacity of the virus to transientlylimit the host immune response during the initial stages ofinfection.

Materials and methods

Cells and virus

Primary human foreskin fibroblasts (HFFs) were used toculture HCMV for both production of viral stocks and pro-ductive HCMV RNA for later molecular analysis. HFFs werederived from normal foreskin tissue and cultured in DMEM+10% fetal calf serum (FCS). Cells were not used after theyreached the 30th passage. Infections were performed usingHCMV strain Toledo, TownevarRIT3 or AD169.

RNA isolation

Total RNAwas extracted from cells using a phenol-free, filterbased RNA extraction system, RNAqueous™ (Ambion Inc.,USA). Cells were first pelleted (270×g, 10 min) and washedonce in 1× PBS. The cells were then disrupted using a Lysis/Binding solution containing guanidinium thiocyanate. The


lysate was then diluted with an equal volume of 64% ethanol andpassed through a silica-based filter cartridge by centrifugation at20,800×g (30 s) and the flow-through was discarded. The filtercartridge was then washed three times with ethanol to removeresidual DNA, proteins, and other contaminants. Followingwashing, the RNA was eluted by applying hot (85 °C) elutionsolution to the filter cartridge. Following isolation, the RNAwasconcentrated for later DNase treatment and/or subsequent use inRT–PCR.

DNase treatment

Total RNA was treated with RQ1 RNase-Free DNase (Pro-mega Corporation, USA) in order to remove contaminatinggenomic DNA. Reactions were performed in a total volume of10 μl containing 1× RQ1 RNase-Free DNase Reaction Buffer,1 U per 1 μg RNA of RQ1 RNase-Free DNase, and the sampleRNA. Reactions were incubated at 37 °C for 30min. To terminatethe reaction and inactivate the DNase, 1 μl of RQ1 DNase StopSolution was added to the reactions which were then incubated at65 °C for 10min. DNase-treated RNAwas precipitated in order toprevent excess magnesium present in the RQ1 DNase digestionfrom inhibiting subsequent PCR amplification.

RT–PCR

DNase-treated RNA samples (0.5 μg) were reverse tran-scribed in a 20 μl volume in the presence of: 1× first-strandbuffer (Tris–HCl [pH 8.3], 75 mM KCl, 3 mM MgCl2), 20 mMdithiothreitol, 40 U RNaseOUT recombinant ribonucleaseinhibitor (Invitrogen, USA), 0.5 mM concentrations of deox-ynucleoside triphosphates, 90 ng random primers, 200 U ofSuperscript II (Invitrogen, USA). Samples were then incubatedat room temperature for 10 min, followed by 1.5 h at 42 °C.Reactions were terminated by heating at 70 °C for 15 min. Analiquot of the resultant cDNA (5 μl) was amplified in a 50 μlreaction containing 50 mM KCl, 20 mM Tris–HCl (pH 8.4),1.5 mM MgCl2, 0.1 mM concentrations of deoxynucleosidetriphosphates, 0.2 μM concentrations of forward (JAS-F1; 5′-TACAAAGCCGCAGTGTCGTCCAGAGGATTACG-3′) andreverse (JAS-2U; 5′-CAGCGTGCTATGAACACGTTGTTAC-CT-3′) primers, 2.5 U Platinum Taq DNA polymerase (Invitro-gen, USA) for 40 cycles of 94 °C for 30 s, 68 °C for 30 s and72 °C for 2 min.

Northern blot hybridization

Total RNAwas analysed by northern blot hybridization usingthe NorthernMax kit (Ambion Inc., USA). Total RNA samples(20 μg) were separated under denaturing conditions using 1.5%(w/v) agarose gels containing 2.2 M formaldehyde (Sigma,USA) in 1× MOPS running buffer. RNAwas then transferred tonylon membrane and UV cross-linked to the membrane (120 mJof UV light applied twice; Stratalinker; Stratagene, USA). DNAprobes for detection of RNA by northern blot hybridization weregenerated by PCR amplification with the following primers:HCMV TRL4 (forward; 5′-ATGCAGCATGCACGCGTGT-3′,

reverse; 5′-CTATACGGAGATCGCGGTCCT-3′), HCMVUL115 (forward; 5′-CGTTTTAGGGATCGAAGACCTG-3′,reverse; 5′-TTAGCGAGCATCCACTGCTT-3′) and GAPDH(forward; 5′-CGAGATCCCTCCAAAATCAA-3′, reverse; 5′-TGTGGTCATGAGTCCTTCCA-3′). PCR products were puri-fied using Nucleotrap Gel Extraction Kit (BD Biosciences,USA) and labelled with α-32P-dCTP using DECAprime II™(Ambion Inc., USA). Nylonmembranes were first prehybridizedat 65 °C in preheated ULTRAhyb solution (Ambion Inc., USA)for 6 h. Labelled probe DNA mixture was first denatured byheating at 100 °C for 10 min and then added to the prehybridizedmembrane and incubated overnight at 65 °C.

Following hybridization, membranes were washed in 2×SSC, 0.1% SDS at 65 °C for 3×30 s and 2 subsequent 30 minwashes at 65 °C. Bound radiolabelled probes were detected byautoradiography using BioMax X-ray film (Kodak, USA) anddeveloped using a CP1000 Automatic Developer (AGFA,Australia).

Rapid amplification of cDNA ends (RACE)

The 5′ and 3′ ends of UL111A-region transcripts weremapped using the SMART RACE cDNA Amplification Kit(BD Biosciences, USA). Aliquots (1 μg) of DNase-treated totalRNA from HCMV-infected HFFs were treated as permanufacturer′s recommendations. 3′ RACE products were ge-nerated by PCR amplification of poly dT-generated cDNAswith primers JAS-F1 and UPM (5′-CTAATACGACTCACTA-TAGGGC-3′) (BD Biosciences, USA) for 25 cycles using of94 °C for 30 s, 68 °C for 30 s and 72 °C for 2 min. 5′ RACE-Ready cDNA was synthesized using a modified lock-dockingoligo (dT) primer and PowerScript reverse transcriptase (RT) inthe presence of the SMART oligonucleotide (BD Biosciences,USA). PowerScript RT exhibits terminal transferase activityupon reaching the end of an RNA template, adding 3–5 residues(predominately dC) to the 3′ end of the first strand cDNA. TheSMART oligo, whose terminal consists of a stretch of dGresidues, can subsequently anneal to the dC-rich cDNA tail andact as an extended template for RT. 5′ RACE products weregenerated by PCR amplification of 5′ RACE-Ready cDNAwithprimers JAS-R5 (5′-TCTCGAGTGCAGATACTCTTCGA-GACGG-3′) and UPM (BD Biosciences, USA) for 25 cyclesusing of 94 °C for 30 s, 68 °C for 30 s, 72 °C for 2 min. All 5′and 3′ RACE PCR products resolved by agarose gel electro-phoresis were purified using the Nucleotrap Gel Extraction Kit(BD Biosciences, USA) and then UA-cloned into the pDriveCloning Vector (QIAGEN Pty Ltd, Australia) for subsequentsequence analysis.

Construction of LAcmvIL-10 and cmvIL-10 mammalian cellexpression vectors

The cDNA sequences of cmvIL-10 and LAcmvIL-10 derivedfrom cells infected with HCMV strain Toledo were amplifiedusing primers pcB1.5ABamHI (5′-ACTGGGATCCCATGCTG-CCACCATGCTG-3′) andpcX1.5ABsp (5′-GGACTGCAAATCG-CATTCGAACTTTCTCG-3′) for cmvIL-10 or pcB1.5ABamHI


and pcXLAcmvBsp (5′-AGCGTGCTATGAACACGTTCGA-ACCTCTGC-3′) for LAcmvIL-10. These primers weredesigned to both amplify these viral IL-10 sequences as wellas introduce BamHI and Bsp119I restriction endonuclease sitesinto the amplified products. The resultant PCR products werethen cloned into pcDNA3.1mycHis(B) (Invitrogen, USA) usingBamHI and Bsp119I. This construction inserted the cmvIL-10and LAcmvIL-10 cDNA sequences, beginning 17 nucleotidesupstream of the translation start site and extending to thetranslational stop codon of both cmvIL-10 and LAcmvIL-10, in-frame with mycHis epitope tags. In both cmvIL-10 andLAcmvIL-10 these nucleotide changes resulted in the elimina-tion of the native translational stop codon and the addition of asingle phenylalanine residue before the start of the mycHisepitope tag.

Acknowledgments

C.J. was the holder of an Australian Postgraduate Award anda Westmead Millennium Foundation Stipend EnhancementAward. This work was supported by Australian National Healthand Medical Research Council grant #301942 awarded to B.S.and A.A. B.S. and A.A. contributed equally to this work.

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