Human Cytomegalovirus Immediate-Early 2 Protein IE86 Blocks Virus-Induced Chemokine Expression

Cell culture and virus infections.Telomerase 12 human foreskin fibroblast (HFF) cells (8) and 293 cells were cultured in Dulbecco's modification of Eagle's medium supplemented with 10% (vol/vol) fetal calf serum (Gemini), 100 units/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% CO₂ at 37°C. HCMV stocks were purified by ultracentrifugation in an SW40 rotor for 1 hour at 20,000 rpm. Purified virus was resuspended in serum-free medium and used for infection. For HCMV infection, cells were infected at a multiplicity of 5 PFU/cell with either wild-type (WT) HCMV or UV-irradiated (360 mJ/cm² in a Stratalinker instrument) HCMV (UV-HCMV) or with the mutant viruses IE2ΔSX, ΔUL83, or UL83Stop. Sendai virus (Charles River labs) infections were performed using 100 hemagglutinin (HA) units/ml as previously described (50).

Oligonucleotides.Primers for shuttle vector construction were as follows: 83RF F, 5′-GGATCCGATATCATTTCGGGACAACGGCG-3′; 83RF R, 5′-AGATCTACACTCGCGGTCCACATCCC-3′; 83LF F, 5′-AGATCTCCACGCAGCGGCCCTTGATG-3′; 83LF R, 5′-GGATCCCATGCATCGCCTCGACGCCC; 83Stop F, 5′-GATATCCtagactaGTCtagTTTCGGGGCACGTGCTGAAAGC-3′; and 83Stop R, 5′-GCAGCAAGTCGATATCGAAAAAGAAGAGC-3′. Lowercase letters in 83Stop F indicate mutated base pairs. Real-time PCR primers were as follows: IFN-β F, 5′-CAGCAATTTTCAGTGTCAGAAGCT-3′; IFN-β R, 5′-TCATCCTGTCCTTGAGGCAGTAT-3′; GAPDH F, 5′-CTGGGCTACACTGAGCACCAG-3′; GAPDH R, 5′-CCAGCGTCAAAGGTGGAG-3′; RANTES F, 5′-TGCTGCTTTGCCTACATTGC-3′; RANTES R, 5′-TTGCCACTGGTGTAGAAATACTCCTT-3′; MCP2 F, 5′-AGCAGAGAGGTTGAGAACAACCCA-3′; MCP2 R, 5′-AGCGCTGCAGAAACCTTCATCTTG-3′; MIP1A F, 5′-TGTCCTGTCTCTCCTCA-3′; MIP1A R, 5′-CATTGGTGCTGAGAGCG-3′; MIG F, 5′-TTGAATCAGCCTACAGGCCTCACA-3′; MIG R, 5′-TGCACTGGAGAGAAGAAAGGCACT-3′; IL-8 F, 5′-AGAAACCACCGGAAGGAACCATCT-3′; and IL-8 R, 5′-AGAGCTGCAGAAATCAGGAAGGCT-3′.

Shuttle vectors for allelic exchange.To construct the pGS284ΔUL83 shuttle vector, UL83 flanking regions corresponding to nucleotides 118881 to 119388 and 121000 to 121500 of the HCMV AD169 strain genome were amplified by PCR using primers 83RF F and 83RF R for the right flanking region and 83LF F and 83LF R for the left flanking region of UL83. These PCR products were then cloned into the pGEMT-Easy vector (Clontech) to yield pUL83Flanks. pUCK4 (52) was digested with BamHI to excise the kanamycin resistance cassette, which was then cloned into BamHI-digested pUL83Flanks to yield pΔUL83Kan. pΔUL83Kan was digested with BglII (which drops out the UL83 flanks and kanamycin cassette) and was cloned into BglII-digested pGS284 (48) to yield pGS284ΔUL83. pGS284ΔUL83 contains a kanamycin cassette in place of the UL83 coding region from nucleotide 121000 to nucleotide 119388. To construct the pGS284-UL83Stop shuttle vector, the entire UL83 open reading frame (ORF) with both flanks was first amplified with 83RF R and 83LF F primers and then cloned into the pGEMT-Easy cloning vector to create pGEMTE-UL83R. The stop codon mutations were created by using PCR product generated with the 83Stop F and 83Stop R primers, thereby mutating the UL83 sequence to create stop codons in all three open reading frames starting at nucleotide 119388. This PCR product was TA cloned into pGEMT-Easy to create pGEMTE-UL83StopRV. pGEMTE-UL83Stop was created by replacing the EcoRV fragment of pGEMTE-UL83R with the EcoRV fragment from pGEMTE-UL83StopRV. Additionally, this mutation created a novel SpeI site to facilitate screening. pGEMTE-UL83Stop was then digested with BbsI to remove the region containing the stop codons and cloned into pGS284ΔUL83, which was digested with BbsI to create pGS284-UL83Stop. All constructs used in these studies were sequence verified.

BAC mutagenesis.All viral mutants were generated using previously reported allelic exchange protocols (48, 56). Briefly, the pADCREGFP (12), pADCREGFPΔUL83, and pADCREGFP-UL83Stop bacterial artificial chromosomes (BACs) were generated by standard allelic exchange procedures described previously. The shuttle vector pGS284ΔUL83 was used for recombination with thepADCREGFP BAC to generate the ΔUL83 virus. The shuttle vector pGS284-UL83Stop was used for recombination with the pADCREΔUL83 BAC to generate the UL83Stop virus. Following allelic exchange, all mutant BACs were screened by restriction enzyme digestion, Southern blot analysis, and direct sequencing to confirm proper recombination and incorporation of the desired mutations. Recombinant viruses were generated as described previously (48, 56). Briefly, BAC DNA was transfected (∼10 μg) into 5 × 10⁶ human foreskin fibroblasts via electroporation (950 μF, 260 V). Cells were seeded into dishes and infectious virus harvested when 100% cytopathic effect was observed. Wild-type and UL83 recombinant viruses generated from BAC DNA were propagated as described previously (9). Infectious titers for all viruses were determined at the same time by plaque assay as described previously (9).

Recombinant adenoviruses.pAdIE2 was constructed by cloning the IE2 cDNA from pCGNIE2 (60) into pADTrack (22) via KpnI to create pADTrack-IE2.Adenovirus was generated according to the AdEasy protocol as described previously (22). Adpp65 and AdGFP have previously been described (25). Adenoviruses were propagated and titered on 293 cells. Adenovirus transduction has been previously described (35) and was enhanced by adding 1 μl/ml Lipofectamine (Invitrogen) to the virus inoculum. Expression of viral proteins following transduction was confirmed by Western blotting.

Western blot analysis.Western blotting was conducted as previously described (7). Briefly, cells were harvested by trypsinization, collected by centrifugation, and lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, 1% NP-40, 0.25% Na-deoxycholate) containing a protease inhibitor cocktail (Roche) added at the time of lysis. Cellular debris was removed by centrifugation, and the supernatant fluids were reserved. The protein concentration was determined by use of Bradford assay (6). Equal amounts (40 μg) of protein were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 8.5% to 10% gels. Proteins were transferred to nitrocellulose membrane (Optitran; Schleicher & Schuell) and probed with primary and secondary antibodies. Immunoreactive proteins were detected by use of an ECL system (Amersham).

Antibodies.The following antibodies were obtained from commercial sources: α-pp65 (1205-S; Rumbaugh-Goodwin Institute); α-pp28 (1207; Rumbaugh-Goodwin Institute), α-tubulin (TU-02; Santa Cruz); and α-IE1/2 (MAb810; Chemicon). pp71 antibodies were a generous gift from T. Shenk and have been previously described (24).

Northern blot analysis.RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's protocol and quantitated on a Nanodrop spectrophotometer. Northern blot analysis was performed as previously described (23). Briefly, total RNA (6 to 10 μg) was separated by electrophoresis on a 1% formaldehyde gel and transferred to Nytran SuperCharge membranes using a TurboBlotter device (Schleicher & Schuell) according to the manufacturer's instructions. Membranes were cross-linked using a Stratalinker instrument and probed with ³²P-labeled probes generated by random priming in ULTRAhyb (Ambion) hybridization buffer at 48°C. Membranes were then washed twice in low-stringency wash buffer (0.1× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% SDS) and twice in high-stringency wash buffer (2× SSC, 0.1% SDS) at 45°C and exposed to film for autoradiography.

Real-time PCR.RNA was DNase treated (DNA free; Ambion), and 2 μg was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's protocol. cDNA was then used as the template for real-time PCR. All reactions were performed in duplicate using SYBR green dye (ABgene) and standard conditions on a Bio-Rad iCycler instrument.

HCMV gene expression blocks cytokine and chemokine induction.Previous reports have demonstrated that infection with transcriptionally inactive UV-irradiated HCMV or infection with wild-type HCMV in the presence of cycloheximide results in a dramatic induction of cytokine and chemokine expression compared with wild-type infection (11, 23, 45, 59). These results suggest that a newly synthesized HCMV gene product can inhibit the induction of these genes during infection. To confirm the previous results, we assayed for the expression of key cytokines and chemokines by Northern blotting. HFF cells were infected for 8 h at a multiplicity of 5 PFU per cell with either purified wild-type HCMV or purified UV-inactivated HCMV. As shown in Fig. 1, infection with UV-inactivated HCMV results in a robust induction of IFN-β, RANTES, MIG, and MCP-2 expression compared with wild-type infection. Blots were also probed for the immediate-early 1 (IE1) transcript to confirm that our UV irradiation protocol efficiently blocks viral gene expression.

• Open in new tab
• Download powerpoint

FIG. 1.

HCMV gene expression is required to block chemokine expression. Telomerase 12 HFF cells were mock infected (M), infected with WT HCMV, or infected with UV-HCMV at a multiplicity of 5 PFU/cell. RNA was isolated 8 h postinfection and analyzed for IFN-β, RANTES, MIG, MCP-2, IE1, and GAPDH transcript accumulation by Northern blotting.

Delayed IE86 expression correlates with increased cytokine induction.We have reported that the HCMV IE2 gene product IE86 can efficiently block the induction of IFN-β following viral infection (50). In addition, two previous reports using a viral deletion mutant and gene array analysis demonstrated that HCMV UL83 gene product pp65 could also attenuate the expression of IFN-β and of a number of chemokines during HCMV infection (1, 10). Using mutant viruses, we attempted to elucidate the individual contributions of both IE86 and pp65 with regard to the inhibition of IFN-β and chemokine expression during HCMV infection. To accomplish this, we used two viral mutants. The first was an IE2 mutant virus termed IE2ΔSX, which has amino acids 136 to 290 deleted from exon 5 of IE2 and is fused at its carboxy terminus to green fluorescent protein (GFP). IE2ΔSX is viable but expresses IE86 at dramatically reduced levels and with delayed kinetics compared to IE86 expression during a wild-type or revertant virus infection (43). The second mutant was a UL83 deletion mutant termed ΔUL83. The ΔUL83 mutant has the UL83 ORF corresponding to nucleotides 119388 to 121000 of the AD169 genome replaced with a kanamycin cassette. The ΔUL83 virus was constructed to be identical to the previously described RVAd65 UL83 deletion mutant (44). To determine the relative roles that IE86 and pp65 play in the attenuation of cytokine and chemokine expression, cells were infected with either purified UV-irradiated HCMV, IE2ΔSX virus, IE2-Rev virus (a revertant virus of IE2ΔSX), or ΔUL83 virus. RNA was isolated at various times after infection and assayed for IFN-β and RANTES expression by Northern blot analysis. As shown in Fig. 2A, infection with both UV-irradiated HCMV and the IE2ΔSX virus resulted in a robust induction of IFN-β and RANTES expression. However, infection with the IE2 revertant virus did not induce the expression of either gene. Interestingly, the level of induction observed following infection with the IE2ΔSX virus closely paralleled that observed following infection with UV-inactivated virus. As previously described (1, 10), infection with the ΔUL83 virus also resulted in an increase in both IFN-β and RANTES expression (Fig. 2B). However, this increase in expression is delayed and significantly reduced compared to the response following infection with either the IE2ΔSX virus or UV-inactivated HCMV. This suggests that there is a greater cytokine response following infection with a virus that does not express IE86, at early times during infection, than after infection with a virus that does not express pp65.

• Open in new tab
• Download powerpoint

FIG. 2.

Cytokine response following infection with HCMV IE2ΔSX and ΔUL83 mutant viruses. Telomerase 12 HFF cells were mock infected (lanes M), infected with IE2ΔSX or IE2ΔSX-REV virus (A), or infected with ΔUL83 or WT virus (B) at a multiplicity of 5 PFU/cell. RNA was isolated 4, 8, and 24 h postinfection and was assayed for IFN-β, RANTES, and GAPDH expression by Northern blot analysis. IFN-β and RANTES expression was quantified by phosphorimaging analysis and standardized to GAPDH expression. Numbers below the blots represent the increase (n-fold) over mock-infected cells. Results for UV-HCMV are also shown.

The expression of pp65 could be affected during infection with the IE2ΔSX virus, or deletion of pp65 could somehow alter the expression of IE86 during infection. To address these possibilities, we examined the protein levels of IE86 and pp65 following infection with the IE2ΔSX, ΔUL83, or wild-type virus. As previously reported, infection with the IE2ΔSX virus resulted in a dramatic reduction and delay in IE86 expression compared to infection with wild-type HCMV (43). The truncated form of IE86 was not detected until 24 h after infection, whereas IE86 was abundantly expressed by 4 h following wild-type infection. There was also a slight decrease in the expression of pp65 at 4 h postinfection with the IE2ΔSX virus compared to the wild-type virus (Fig. 3A). These results are consistent with previous reports demonstrating the IE2ΔSX virus expresses lower levels of pp65 (43). However, pp65 was clearly present within the infected cell by 4 h postinfection with the IE2ΔSX virus. The levels of two other tegument proteins, pp71 and pp28, were approximately the same following infection with either the IE2ΔSX or wild-type virus (Fig. 3A). As expected, the ΔUL83 virus did not express pp65. Surprisingly, there was a delay in the expression of IE86 following infection with the ΔUL83 virus relative to that following infection with the wild-type virus (Fig. 3B). IE86 was abundantly expressed by 4 h postinfection following wild-type infection, whereas IE86 was not expressed until 8 h postinfection with the ΔUL83 virus. We obtained similar results when a second UL83 deletion mutant virus termed RVAd65 (44) was used (data not shown). Interestingly, there was also a significant delay in the expression of pp71 following infection with the ΔUL83 virus (Fig. 3B). Taken together, these results identify a connection between delayed IE86 expression and increased accumulation of IFN-β and RANTES transcripts.

• Open in new tab
• Download powerpoint

FIG. 3.

Expression of IE and tegument proteins following infection with IE2ΔSX or ΔUL83 mutant viruses. Telomerase 12 HFF cells were mock infected (lanes M), infected with IE2ΔSX or IE2ΔSX-REV virus (A), or infected with ΔUL83 or WT virus (B) at a multiplicity of 5 PFU/cell. Cell lysates were harvested 4, 8, and 24 h postinfection and assayed for IE86, pp65, pp71, pp28, and tubulin expression by Western blot analysis.

IE86 blocks virus-induced IFN-β and RANTES transcript expression.Since there was a decrease in the expression of pp65 following infection with the IE2ΔSX virus, we could not rule out the possibility that decreased expression of pp65 was also contributing to the cytokine induction observed during infection with the IE2ΔSX virus. Therefore, we wanted to determine whether expression of IE86 or pp65 alone was capable of blocking virus-induced cytokine expression. To accomplish this, we used replication-deficient adenoviruses that express IE86, pp65, or GFP. Cells were transduced with adenoviruses that express either IE86, pp65, or GFP at a multiplicity of infection of 3 PFU/cell. Twenty-four h after transduction, the cells were infected either with UV-HCMV (Fig. 4A and B) or with Sendai virus (Fig. 4C and D). RNA was isolated 8 h postinfection and reverse transcribed, and real-time PCR was performed to quantitate the abundance of IFN-β and RANTES transcripts. As shown in Fig. 4A to D, infection with either UV-irradiated HCMV or Sendai virus alone resulted in a dramatic accumulation of IFN-β and RANTES message. However, expression of IE86 prior to infection with UV-HCMV or Sendai virus greatly reduced or abolished the induction of IFN-β or RANTES (Fig. 4A-D). Prior expression of either pp65 or GFP had no effect on the induction of IFN-β or RANTES following either type of infection. A Western blot is also shown (Fig. 4E) to confirm the expression of IE86, pp65, and GFP at the time of infection. These results confirm the role of IE86 as an IFN-β antagonist and also demonstrate its ability to block the induction of RANTES following virus infection.

• Open in new tab
• Download powerpoint

FIG. 4.

Expression of IE86 blocks the induction of IFN-β and RANTES during virus infection. Telomerase 12 HFF cells were transduced with replication-defective adenoviruses expressing IE86, pp65, or GFP. Twenty-four h later, the transduced cells were infected with UV-HCMV (A and B) or with 100 HA units/ml of Sendai virus (C and D). RNA was isolated 8 h postinfection and analyzed for IFN-β (A and C) and RANTES (B and D) expression by real-time PCR. Transcript levels were standardized to GAPDH levels and represent the average of two independent experiments. Data represent the increase (n-fold) over mock-infected cells. (E) Telomerase 12 cell lysates were harvested 24 h after transduction with the given adenovirus and assayed for IE86, pp65, and GFP expression by Western blot analysis.

Generation of a UL83 stop codon mutant.The previous results demonstrate that ectopic expression of pp65 does not block the induction of IFN-β or RANTES during virus infection. They further suggest the increased cytokine response observed in Fig. 2B following infection with the ΔUL83 virus is not the result of abolishing pp65 expression but is likely due to the delayed expression of IE86. The delay in pp71 protein expression following infection with the ΔUL83 virus (Fig. 3D) also suggests that deletion of the UL83 ORF from the viral genome in some way alters expression of the downstream UL82 ORF, which codes for pp71. Interestingly, pp71 is a critical regulator of immediate-early gene expression (9). In order to determine whether loss of pp65 expression or deletion of the UL83 gene sequence from the genome is responsible for the cytokine induction observed following infection with the ΔUL83 virus, we constructed a second UL83 viral mutant. This mutant, termed UL83Stop, contains point mutations that introduce stop codons in all three reading frames 35 bp downstream from the UL83 start codon. Therefore, like the ΔUL83 virus, the UL83Stop virus will not be capable of expressing pp65. However, unlike the ΔUL83 virus, the UL83Stop virus does not delete any genomic sequences and still contains the entire UL83 ORF (Fig. 5A) and therefore should not interfere with the expression of pp71. The UL83Stop virus was constructed by standard allelic exchange protocols described previously (48, 56). To confirm proper recombination and insertion of the stop codons within the UL83 ORF, restriction enzyme analysis of wild-type, ΔUL83, and UL83Stop BAC DNA was performed. As indicated in Fig. 5B, wild-type and UL83Stop DNA digested with XhoI generates the predicted 9.5-kb fragment that is absent from ΔUL83-digested DNA due to a novel XhoI site in the kanamycin cassette. The stop codon mutations within UL83Stop also create a novel SpeI restriction enzyme site in the UL83 ORF (Fig. 5A). To confirm deletion of UL83 from ΔUL83 and distinguish UL83Stop DNA from wild-type DNA, PCR was used to amplify across the recombination junction to produce a specific fragment using purified wild-type, ΔUL83, or UL83Stop BAC DNA as the template. As shown in Fig. 5C, there is a 1.5-kb PCR product generated by using wild-type and UL83Stop DNA as the template. No amplification product was observed using ΔUL83 BAC DNA. Digestion of the 1.5-kb UL83Stop PCR fragment with SpeI produced a 1.0-kb fragment and a 0.5-kb fragment, indicating the stop codon mutations were properly located within the viral genome. The fragment amplified from WT DNA was not digested with SpeI. Finally, to confirm that the UL83Stop is unable to express pp65, cells were infected with either wild-type, ΔUL83, or UL83Stop virus, and cell lysates were prepared 72 h postinfection and subjected to Western blot analysis. As expected, neither the ΔUL83 virus nor the UL83Stop virus expresses pp65, whereas pp65 is abundantly expressed following infection with the wild-type virus (Fig. 5D).

• Open in new tab
• Download powerpoint

FIG. 5.

Construction of a UL83 stop codon mutant. (A) Schematic representation of the UL83 genomic region depicting the UL83 and UL82 ORFs, the 4.0-kb UL82 and UL83 coterminal transcript, the XhoI (X) and SpeI (S) restriction sites, and the organization of the ΔUL83 and UL83Stop mutant viruses. The asterisk represents the stop codon mutation within the UL83Stop virus. (B) Wild-type, ΔUL83, or UL83Stop BAC DNA was digested with XhoI, and the fragments were separated by agarose gel electrophoresis. The asterisk indicates the diagnostic fragment which is absent in the ΔUL83 digest and present in the WT and UL83Stop digests. (C) WT, ΔUL83, or UL83Stop BAC DNA was used as the template to amplify a specific genomic region spanning the 5′ end of the UL83 ORF and the adjoining flanking sequence. The PCR product was then digested with SpeI to confirm that the stop codon was properly inserted within the UL83 ORF. (D) HFF cells were either mock infected or infected with WT, ΔUL83, or UL83Stop virus. Cell lysates were prepared 72 h postinfection and assayed for pp65 expression by Western blot analysis. Tubulin expression was included as an internal loading control.

We then examined the expression of IE86 and pp71 following infection with either wild-type or UL83Stop virus. Fibroblasts were infected with either virus, and cell lysates were prepared at various times postinfection. As shown in Fig. 6, the expression of IE86 and pp71 following UL83Stop virus infection occurred with kinetics the same as those observed following wild-type infection; the expression levels were also similar to those observed following wild-type infection. These results suggest that the delay in IE86 and pp71 expression observed with the ΔUL83 virus (Fig. 3B) is the result of deleting the UL83 ORF from the genome and was not due to abolishing pp65 expression.

• Open in new tab
• Download powerpoint

FIG. 6.

UL83Stop mutant exhibits wild-type immediate-early gene expression. Telomerase 12 HFF cells were either mock infected or infected with wild-type HCMV or the UL83Stop virus at a multiplicity of 5 PFU/cell. Cell lysates were prepared at the indicated times postinfection and assayed for IE86, pp65, pp71, pp28, and tubulin expression by Western blot analysis.

pp65 is not responsible for blocking IFN-β and RANTES expression.We next compared the cytokine responses following infection with the UL83Stop and ΔUL83 mutants. Fibroblasts were infected with wild-type, UV-HCMV, ΔUL83, or UL83Stop virus. Cells were harvested and RNA was extracted 8 h postinfection and assayed for IFN-β expression by Northern blot analysis. As shown in Fig. 7A, infection with UV-HCMV resulted in a strong induction of IFN-β transcript. Infection with ΔUL83 also resulted in IFN-β transcript accumulation, although it was not nearly as robust as that observed for UV-HCMV. Interestingly, infection with either wild-type or UL83Stop virus completely blocked the induction of IFN-β (Fig. 7A). Similar results were obtained for RANTES expression (data not shown). Northern blotting was used to determine if prior infection with the UL83Stop virus could block Sendai virus-induced cytokine expression. Cells were infected with either wild-type, ΔUL83, or UL83Stop virus for 8 h and then infected with Sendai virus. RNA was isolated 8 h after Sendai virus infection and assayed for IFN-β and RANTES expression. As shown in Fig. 7B, infection with Sendai virus alone results in a dramatic induction of both IFN-β and RANTES expression. However, when cells were infected with either the wild-type virus or the UL83Stop virus prior to Sendai virus infection, the induction of both IFN-β and RANTES was almost completely blocked, demonstrating that an HCMV gene other than pp65, which is expressed within the first 8 h of infection, was responsible for this inhibition. Prior infection with the ΔUL83 virus also resulted in an attenuated cytokine response. However, this inhibition was not as dramatic as that observed for the wild-type virus or the UL83Stop virus. Since the UL83Stop and ΔUL83 viruses do not express pp65, the difference in their abilities to inhibit the cytokine response following viral infection is likely due to the different mutation strategies adopted to prevent pp65 expression.

• Open in new tab
• Download powerpoint

FIG. 7.

The UL83Stop mutant blocks the induction of IFN-β and RANTES as effectively as wild-type virus. (A) Telomerase 12 HFF cells were mock infected or infected with wild-type, ΔUL83, or UL83Stop virus at a multiplicity of 5 PFU/cell. RNA was isolated 8 h postinfection and assayed for IFN-β and GAPDH by Northern blot analysis. (B) Telomerase 12 HFF cells were either mock infected or infected with wild-type, ΔUL83, or UL83Stop virus at a multiplicity of 5 PFU/cell. Eight h postinfection, the cells were superinfected with Sendai virus (100 HA U/ml). RNA was isolated 8 h after Sendai virus infection and assayed for IFN-β, RANTES, and GAPDH by Northern blot analysis. The expression of IFN-β and RANTES was quantified by phosphorimaging analysis and standardized to GAPDH expression. Numbers below the blots represent the increase (n-fold) over mock-infected cells. Results for UV-HCMV are also shown.

HCMV IE86 blocks induction of multiple proinflammatory chemokines.We also examined the ability of IE86 to block the induction of a number of other chemokines that were previously reported to be blocked by pp65 following infection with the ΔUL83 virus. Cells were transduced with adenoviruses that express either IE86, pp65, or GFP and then infected with UV-HCMV for 8 h. RNA was isolated and assayed by real-time PCR for expression of IFN-β, RANTES, MCP-2, MIG, MIP-1α, and IL-8. As shown in Table 1, infection with UV-HCMV resulted in a robust induction of all cytokines and chemokines tested compared to wild-type infection. All chemokines were induced to levels similar to that observed for UV-HCMV in cells expressing either pp65 or GFP, demonstrating that these gene products are not capable of blocking chemokine induction during HCMV infection. However, cells expressing IE86 effectively blocked chemokine induction following UV-HCMV infection. ISG56, which is induced by both WT HCMV and UV-HCMV (11, 47), was included to show that the effect of IE86 is specific and that not all antiviral genes are regulated by IE86. These results demonstrate that IE86 not only functions as an IFN-β and RANTES antagonist but also functions to inhibit the expression of numerous chemokines during HCMV infection.