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Journal of Virology, November 2006, p. 10436-10456, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01248-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

CD8 T Cells Control Cytomegalovirus Latency by Epitope-Specific Sensing of Transcriptional Reactivation

Institute for Virology, Johannes Gutenberg-University, Mainz, Germany,¹ I. Department of Internal Medicine, Medical Centre Mainz, Mainz, Germany²

Received 14 June 2006/ Accepted 10 August 2006

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Addendum References

 
During murine cytomegalovirus (mCMV) latency in the lungs, most of the viral genomes are transcriptionally silent at the major immediate-early locus, but rare and stochastic episodes of desilencing lead to the expression of IE1 transcripts. This low-frequency but perpetual expression is accompanied by an activation of lung-resident effector-memory CD8 T cells specific for the antigenic peptide 168-YPHFMPTNL-176, which is derivedfrom the IE1 protein. These molecular and immunological findings were combined in the "silencing/desilencing and immune sensing hypothesis" of cytomegalovirus latency and reactivation. This hypothesis proposes that IE1 gene expression proceeds to cell surface presentation of the IE1 peptide by the major histocompatibility complex (MHC) class I molecule L^d and that its recognition by CD8 T cells terminates virus reactivation. Here we provide experimental evidence in support of this hypothesis. We generated mutant virus mCMV-IE1-L176A, in which the antigenic IE1 peptide is functionally deleted by a point mutation of the C-terminal MHC class I anchor residue Leu into Ala. Two revertant viruses, mCMV-IE1-A176L and the wobble nucleotide-marked mCMV-IE1-A176L*, in which Leu is restored by back-mutation of Ala codon GCA into Leu codons CTA and CTT, respectively, were constructed. Pulmonary latency of the mutant virus was found to be associated with an increased prevalence of IE1 transcription and with events of IE3 transactivator splicing. In conclusion, IE1-specific CD8 T cells recognize and terminate virus reactivation in vivo at the first opportunity in the reactivated gene expression program. The perpetual gene expression and antigen presentation might represent the driving molecular force in CMV-associated immunosenescence.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Addendum References

 
After resolution of productive primary infection, in particular by CD8 T cells, cytomegaloviruses (CMVs) establish lifelong latent infections in their respective hosts (for reviews, see references 29, 31, 32, 52, 75, 83-85, and 87). Reactivation of latent human CMV (hCMV) to productive, cytopathogenic infection is still a health risk in immunocompromised patients (9, 57). Hematoablative therapy of leukemias, followed by bone marrow transplantation (BMT) or hematopoietic stem cell transplantation, is associated with a risk of CMV disease resulting from reactivation of latent donor and/or recipient CMV (15, 23). Among the manifestations of CMV disease in humans, interstitial pneumonia is the most dreaded because of its high fatality rate (79). Lungs were also identified as a major organ site of murine CMV (mCMV) disease, latency, and recurrence (4, 43, 70, 78).

Studies in the BALB/c mouse model of CMV infection in the BMT recipient have focused on the lungs for investigating mechanisms of immune control, latency, and reactivation (reviewed in references 25, 75, and 83). In this model, control of productive lung infection and prevention of disseminated viral pneumonia proved to be critically dependent upon the efficient reconstitution of CD8 T cells that infiltrated the lungs, confined infection to inflammatory foci, and eventually resolved productive infection (27, 61, 62). Viral genomes, however, were maintained in the lungs, and disseminated pulmonary CD8 T-cell infiltrates persisted after clearance of productive infection (61). That viral latency is not a static state but involves a permanent immune sensing of reactivation attempts was first suggested by the finding that a high fraction of pulmonary CD8 T cells displayed an activated effector-memory T-cell (T_EM) phenotype characterized by low-to-absent cell surface expression of L-selectin CD62L and by effector function (61). Enrichment of CD8 T cells specific for the IE1 protein-derived major histocompatibility complex (MHC) class I L^d-restricted antigenic peptide 168-YPHFMPTNL-176 (76) in the CD62L^low fraction predicted expression of the ie1 gene and presentation of the IE1 peptide by latently infected lung cells (26).

Studies on viral transcription in latently infected lungs have indeed revealed transcriptional activity at the major immediate-early (MIE) locus from the enhancer-flanking transcription units ie1/3 (m123/M122) and ie2 (m128) giving rise to spliced IE1 and IE2 transcripts but, notably, not to spliced IE3 transcripts (21, 41). Thus, the IE3 protein, the essential transactivator of viral Early (E) genes downstream in the productive cycle (1), was not expressed. Accordingly, the essential gene M55 (gB) was also not expressed, and latency was maintained despite MIE locus activity. MIE locus gene expression, however, is not latency associated in the sense that it is inherent to the latent state. In fact, a great majority of the latent viral genomes are silenced at the MIE locus at any moment. As shown by a statistical analysis of transcriptional events in the lungs (21, 82), MIE locus transcription is a rare event of variegated gene expression, also known as mosaic expression (17). This expression reflects MIE locus desilencing, putatively linked to local opening of the proposed higher-order chromatin-like structure of the latent viral episome (references 5, 47, and 54; for reviews, see references 3, 83, and 84). To give an idea of the "point prevalence," that is, the proportion of viral genomes expressing MIE genes at any moment, a statistical estimate gave 20 events of MIE locus activity per 10⁶ latent viral genomes (82, 83). Activation of the MIE enhancer through the tumor necrosis factor alpha/NF-B/AP-1 signaling pathways (29, 30, 82) led to an 10-fold increase in the frequency of IE1 transcription and to IE3 splicing. However, as it was indicated by the absence of gB transcripts, gene expression did not proceed to complete reactivation and virus recurrence (82).

Combined, these immunological and molecular findings led to the silencing/desilencing and immune sensing hypothesis of CMV latency and reactivation (83). According to this hypothesis, MIE gene expression is sensed and terminated by patrolling CD8 T cells. Although the events are of low frequency at any single time point, the cumulative incidence over a period of months can lead to a substantial activation and expansion of the CD8 T-cell pool. Furthermore, the incidence of MIE gene expression during latency may so far have been underestimated as a consequence of CD8 T-cell function having already terminated MIE gene expression in most cases. As far as we know today, IE2 and IE3 do not contain MHC class I H-2^d-restricted antigenic peptides; therefore, the immunodominant IE1 peptide is the only candidate for CD8 T-cell surveillance of MIE gene reactivation in the BALB/c mouse model. In support of this hypothesis, we provide here experimental evidence to conclude that MIE gene-expressing cells in latently infected lungs indeed present the IE1 peptide for CD8 T-cell sensing of viral reactivation and for intervention at the first opportunity in the viral gene expression program.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Addendum References

 
Construction of plasmids. Recombinant plasmids were constructed according to established procedures, and enzyme reactions were performed as recommended by the manufacturers.

(i) Shuttle plasmid for mutagenesis. pST76KIE1Ala was constructed to introduce a point mutation Ala (codon GCA) in place of Leu codon CTA at the C-terminal MHC class I anchor residue position of the IE1 peptide 168-YPHFMPTNL-176. Briefly, plasmid pUCAMB (21) containing the DNA sequence of the IE1 peptide from nucleotide positions 181,020 to 181,046 (n181,020 to 181,046) of the mCMV genome (GenBank accession no. NC_004065 [complete genome]) (68) served to generate plasmid pIE1_168-176LA. This plasmid was derived from pUCAMB by excision of a 1,005-bp HindIII/BclI fragment containing the DNA sequence corresponding to the IE1 peptide and replacement by a 1,005-bp HindIII/BclI PCR fragment containing the mutated codon GCA, specifying Ala. This fragment was generated with plasmid pUCAMB as template DNA via site-directed mutagenesis by overlap extension using PCR (24) with primers Nona-Ala-f (5'-CTTCATGCCCACTAATGCAGGG-3'[underlining indicates nucleotides implicated in mutagenesis]) (n181,038 to 181,017) and IE1ex4 (5'-ACTGCCTTAGCCAGATTCTCC-3') (n180,697 to 180,717) as well as IE1ex2 (5'-TTTTTAGAGAGATGGAGCCCGC-3') (n181,777 to 181,756) and Nona-Ala-rev (5'-CCCTGCATTAGTGGGCATGAAG-3') (n181,017 to 181,038). In the subsequent fusion reaction, primers IE1ex2 and IE1ex4 were used. PCR was performed with the following cycler conditions: an initial step for 5 min at 95°C for activation of ProofStart Taq DNA polymerase (catalog no. 202205; QIAGEN) followed by 30 cycles for 45 s at 94°C, 60 s at 55°C (or 60°C in the fusion PCR), and 60 s at 72°C. Finally, pIE1_168-176LA was cleaved with BssHII, HpaI, and FspI, and a 6,523-bp BssHII/HpaI fragment was filled up by Klenow DNA polymerase and ligated blunt end into the SmaI-cleaved shuttle plasmid pST76-KSR (8). This plasmid is a derivative of shuttle plasmid pST76K (65) and contains the Bacillus subtilis sacB gene and the Escherichia coli recA gene.

(ii) Shuttle plasmids for revertants. For construction of shuttle plasmid pST76KIE1Leu, pUCAMB25 was cleaved with BssHII, HpaI, and FspI, and a 6,523-bp BssHII/HpaI fragment, encompassing the IE1 peptide-encoding sequence, was filled up by Klenow DNA polymerase and ligated into the SmaI-cleaved vector pST76-KSR. Shuttle plasmid pST76KIE1Leu* was constructed as follows. A 1,471-bp ApaLI fragment carrying a point mutation of nucleotide AT at the wobble position of the Leu codon CTA was generated by mutagenesis PCR with pST76IE1Ala as template DNA and with primers rIE1-revert (5'-ATCTCCTGCTGCTGTTGCTGTTCTTC-3') (n180,313 to 180,338) and Nona-Leu-f* (5'-CTTCATGCCCACTAATCTTGGG-3') (n181,038 to 181,017) as well as Nona-Leu-r* (5'-CCCAAGATTAGTGGGCATGAAG-3') (n181,017 to 181,038) and fIE1-revert (5'-CACAGAGGATTCTGTCTGTGTCAAGG-3') (n181,968 to 181,943). The fusion reaction was performed with primers rIE1-revert and fIE1-revert. pST76KIE1Ala was digested with ApaLI, and the 1,471-bp ApaLI fragment was replaced by the PCR-mutated 1,471-bp ApaLI fragment. Throughout, the fidelity of PCR-based cloning steps and cloning crossings was verified by sequencing (automated DNA sequencing system, model 4000; LI-COR Inc., Lincoln, Nebraska).

(iii) Plasmids for reporter gene assays. Recombinant plasmid pIE1-L176A (for use in firefly luciferase assays; see below) was constructed as follows. Plasmid pST76KIE1Alawas first cleaved with XbaI and Eco47III and then digested with BsmI to eliminate vector sequences. The resulting 1,809-bp fragment, containing the mutated IE1 peptide L176A, was cloned into the XbaI- and Eco47III-digested plasmid pIE100/1 (49). This plasmid contains a genomic fragment of mCMV that encodes the authentic IE1 protein (36, 49). Recombinant plasmid pIE1-A176L* was constructed in an analogous manner, except that pST76KIE1Leu* provided the insert sequence. Plasmids pTLG (2) and pGL3R2 1.5 (12) contain the mouse thymidylate synthase promoter and the mouse ribonucleotide reductase 2 promoter, respectively, each linked to an intronless luciferase reporter gene. Plasmid pRL-TK encoding Renilla luciferase (GenBank accession no. AF025846; catalog no. E2241; Promega) was used to standardize for transfection efficacy.

(iv) Plasmid pDrive-e1 for the synthesis of E1 in vitro transcripts. A cDNA fragment of the e1 gene (11, 13, 68) was amplified by reverse transcription-PCR (RT-PCR) from RNA derived from mouse embryo fibroblasts (MEFs) infected with mCMV-WT.Smith. Oligonucleotides Early1-for1 (5'-GACGACGTTACTTCACCTTCCG-3') and Early1-rev1 (5'-GAACACATTGTCCAAGTCGACC-3') served as forward and reverse primers, respectively. The 1,509-bp amplification product, representing the intronless mCMV e1 sequence from map positions 203 to 2,130 (GenBank accession no. M35146) (11), was cloned into plasmid pDrive by means of UA-based ligation (catalog no. 231122; QIAGEN, Hilden, Germany). For use as a template in the in vitro transcription, plasmid pDrive-e1 was linearized by digestion with SphI. Enzyme reactions were performed as recommended by the manufacturers. Synthetic transcripts were prepared according to the instructions given in the Ambion MEGAscript technical manual no. 1330.

BAC mutagenesis. Mutagenesis of full-length mCMV BAC (bacterial artificial chromosome) plasmid pSM3fr (92) was performed with E. coli strain DH10B (Invitrogen) by using a two-step replacement method (6, 50, 55) with the modifications described previously by Wagner et al. (92) and Borst et al. (8). Shuttle plasmid pST76KIE1Ala was used to generate the BAC plasmid C3XIE1Ala, which contains the mutated codon GCA corresponding to the amino acid point mutation L176A in the IE1 protein and IE1 antigenic peptide sequence. To restore Leu in position 176 of IE1, E. coli DH10B carrying BAC plasmid C3XIE1Ala was used for recombination with shuttle plasmids pST76KIE1Leu and pST76IE1Leu* encompassing the Leu codons CTA and CTT, respectively.

Integrity and sequence analysis of recombinant mCMV BAC plasmids. BAC plasmid DNA was isolated from small-scale cultures (catalog no. BMAX044; Epicenter) and purified (NucleoBond PC 500, catalog no. 740574.25; Macherey-Nagel). The overall integrity of the recombinant BAC plasmids was tested by standard methods of restriction enzyme cleavage, agarose (0.7% [wt/vol]) gel electrophoresis, and ethidium bromide staining. The point mutations in recombinant mCMV BAC plasmids C3XIE1Ala, C3XIE1Leu, and C3XIE1Leu* were verified by sequencing (model 4000; LI-COR) using the Thermo Sequenase fluorescent labeled primer cycle sequencing (7-Deaza-dGPT) kit (catalog no. RPN 2438; Amersham Bioscience). Purified BAC DNA (see above) served as the template and was sequenced in both directions with primers IE1-ex4F1-IR (5'-GAGCGTTCTGTTGTCCTGTAAG-3') (n181,245 to 181,224) and IE1-ex4R1-IR (5'-ACTGCCTTAGCCAGATTCTCCC-3') (n180,697 to 180,718).

Reconstitution of BAC-derived recombinant viruses. Purified DNA of the respective BAC plasmids (see above) was transfected into MEFs by using PolyFect transfection reagent (catalog no. 301107; QIAGEN). To eliminate BAC vector sequences that could attenuate viruses for growth in vivo (92), BAC-derived viruses were subjected to five rounds of passaging in MEF cultures followed by two rounds of plaque purification. Verified BAC vector-free virus clones (see below) were used to prepare high-titered stocks of sucrose gradient-purified viruses (43, 60) mCMV-IE1-L176A (2.3 x 10⁸ PFU/ml), mCMV-IE1-A176L (2.1 x 10⁸ PFU/ml), and mCMV-IE1-A176L* (6.5 x 10⁸ PFU/ml).

Other viruses used in this study include wild-type mCMV-WT.Smith (ATCC VR194/1981, recently reaccessioned as VR-1399), BAC-derived mCMV-WT.BAC MW97.01 (92), and the ie1 gene deletion mutant mCMV-ie1 (19).

Verification of the absence of BAC vector sequences. Total DNA derived from infected cells after the second round of plaque purification was prepared by using a High Pure viral nucleic acid kit (catalog no. 11858874001; Roche). To examine whether a correct excision of the BAC vector sequences from the recombinant mCMV genomes had occurred, PCRs were performed as described by Ghazal et al. (18, 19). Amplification products were analyzed by agarose gel electrophoresis, Southern blotting, and hybridization with the -³²P-end-labeled BAC vector sequence-specific probe 5'-GGATACTCAGCGGCAGTTTGC-3' to detect even traces of amplification products. The mutations were verified again by sequencing as described above for BAC plasmids.

Transient transfections and reporter gene assays. Plasmids (see above) were purified with the QIAGEN plasmid Maxi kit (catalog no. 12163; QIAGEN). A dual-luciferase reporter (DLR) assay (catalog no.1910; Promega Corp.) was employed to standardize for transfection efficacy. NIH 3T3 cells were plated in 60-mm-diameter dishes. On the following day, the cells (ca. 2 x 10⁵ cells per dish) were cotransfected with 20 ng of Renilla luciferase-encoding control plasmid pRL-TK, 2.5 µg of firefly luciferase-encoding reporter plasmid (pTLG or pGL3R2), and 2.5 µg of one of the mCMV IE1 protein-encoding transactivator donor plasmids (pIE100/1, pIE1-L176A, or pIE1-A176L*) or of pUC19 as the negative control. Transfection was performed by using 15 µl PolyFect transfection reagent (catalog no. 301107; QIAGEN). The transactivating effects of the authentic and mutated IE1 proteins on the mouse ribonucleotide reductase 2 promoter activity (45) as well as on the thymidylate synthase promoter activity (20) was tested as follows. The transfected cells were first incubated in Dulbecco's minimal essential medium (DMEM) (5% fetal calf serum) for 5 to 6 h, were then washed twice, and finally were incubated again in DMEM until the next day. For growth arrest, NIH 3T3 cells were then washed three times with low-serum starving medium (DMEM with 0.5% newborn calf serum) followed by an incubation in this starving medium for exactly 48 h. After this starvation period, luciferase activity was measured by the DLR assay as recommended by the manufacturer. The assays were performed with 20-µl samples of cleared cell lysate. Luminescence, expressed as relative luminescence units (RLU), was measured with a single-sample luminometer (Lumat LB 9507; Berthold, Bad Wildbad, Germany). According to the read-inject-read format of the DLR assay, signals from firefly and Renilla luciferase were detected sequentially for each individual sample, with firefly luminescence being measured first. Linear ranges and assay backgrounds for the two luciferases were determined as suggested by the manufacturer. Background RLU were subtracted from assay RLU.

Experimental BMT and establishment of latent mCMV infection. Syngeneic BMT with female BALB/cJ (H-2^d haplotype) mice as bone marrow cell donors and recipients was performed as described previously (60). In brief, hematoablative conditioning of 8- to 9-week-old recipients was achieved by total-body -irradiation with a single dose of 6.5 Gy. BMT was performed 6 h later by intravenous infusion of 5 x 10⁶ femoral and tibial donor bone marrow cells. Shortly after BMT, intraplantar infection of recipients was performed at the left hind footpad with 10⁵ PFU of the recombinant mCMVs indicated. Criteria for the definition of latency were specified previously (for a review, see reference 75) and include absence of infectivity in key organs of CMV tropism (liver, spleen, lungs, and salivary glands) as well as PCR-verified absence of viral DNA from blood (<1 copy per 10⁴ leukocytes). Clearance of viral DNA from the blood is the criterion that takes longest to be fulfilled in the BMT model, usually 8 to 10 months. Animals were bred and housed under specified-pathogen-free conditions in the Central Laboratory Animal Facility (CLAF) of the Johannes Gutenberg University. Animal experiments were approved according to German federal law under permission number 177-07/021-28.

Quantification of productive in vivo infection. Infection of the lungs was assessed by quantification of infectious virus in lung homogenates using a virus plaque assay (PFU assay) on MEFs with the technique of centrifugal enhancement of infectivity as described in greater detail elsewhere (60).

Infection of the liver was assessed by counting of infected cells identified by immunohistological staining of intranuclear IE1 protein pp76/89 in 10 mm² of 2-µm liver tissue sections as described in greater detail previously (60). Growth curves were determined on the basis of three BALB/c mice tested individually per time point after an intraplantar infection. Linear regression lines in a semilogarithmic plot logN(t) = at + logN(0), where N(t) is the number of infected cells at time t after infection, a the slope of the regression line, and logN(0) its ordinate intercept, were calculated by using Mathematica Statistics LinearRegression software, version 5.1 (Wolfram Research, Inc., Champaign, Ill.). The doubling time (DT) of the number of infected cells is then log2/a. Accordingly, the upper and lower 95% confidence limit values of slope a (determined from the ellipsoidal parameter confidence region) give the 95% confidence interval of the DT. The time point of first detection of liver infection as well as its 95% confidence interval is revealed from the points of intersection between the calculated regression lines (slope a and confidence limits of a) and the line logN(t) = 0 (equals one infected cell per detection area).

Quantification of viral genomes in host tissues. For molecular and statistical analysis of latency, the lungs were subdivided into 18 bona fide equally sized pieces as follows: 1 to 3, superior lobe; 4 to 6, middle lobe; 7 to 9, inferior lobe; 10 and 11, postcaval lobe; and 12 to 18, left lung. Each piece represents approximately 3 x 10⁶ to 4 x 10⁶ lung cells containing approximately 18 to 24 µg of DNA. The two pieces of the postcaval lobe were used for determining the load of latent viral DNA in two triplicates for each latently infected mouse. The tissue was minced, and DNA was extracted with a DNeasy tissue kit (catalog no. 69504; QIAGEN) as described previously (82). Latent viral genomes were quantified by real-time PCR using the TaqMan (ABI 7700) system (Applied Biosystems) and the QuantiTect SYBR green PCR kit (catalog. no. 204143; QIAGEN). A 100-ng aliquot of the DNA was added as template DNA to a reaction mixture that included the 2X QuantiTect SYBR green PCR master mix with an initial MgCl₂ concentration of 2.5 mM and 1 µM of each primer. Primers for amplification of a 135-bp fragment of the viral gene M55 (gB) were LCgB-forw and LCgB-rev, and primers for amplification of a 142-bp fragment of the cellular pthrp gene were LCPTHrP-forw and LCPTHrP-rev, as described in greater detail previously (82). PCR was performed with the following cycler conditions: an initial 15 min at 95°C for HotStarTaq DNA polymerase activation followed by 50 cycles of 15 s at 94°C, 30 s at 62°C, and 45 s at 72°C. Data were obtained during the extension period. After amplification, melting curve analysis of the PCR products was performed by raising the temperature to 95°C, cooling down rapidly to 60°C for 30 s, and then slowly raising the temperature again to 95°C, with the fluorescence being recorded continuously. DNA from each of the two lung pieces 10 and 11 was tested in triplicate 100-ng samples. Standard curves for quantification were established by using graded numbers of linearized plasmid pDrive_gB_PTHrP_Tdy (82) as the template.

For quantification of viral replication in the time course of acute infection of footpad and spleen, tissue was processed for DNA extraction essentially as described above for the lungs, except that plantar tissue was homogenized with a QIAGEN MM300 mixer mill at 30 Hz for two periods of 5 min. Real-time quantitative PCR was performed, and log-linear regression lines logN(t) = at + logN(0) as well as the DTs of viral genomes were calculated as described above.

Isolation of poly(A)⁺ RNA from the lungs. Isolation of poly(A)⁺ RNA from lung pieces was performed as described in greater detail previously (82). In essence, lung pieces shock-frozen in liquid nitrogen were homogenized and lysed in a QIAGEN MM300 mixer mill with high-salt lysis/binding buffer (µMACS mRNA isolation kit no. 130-075-201; Miltenyi Biotec, Bergisch Gladbach, Germany). Poly(A)⁺ RNA was isolated as recommended by the manufacturer by binding to paramagnetic oligo(dT)-coated MicroBeads in MACS type µ columns (Miltenyi). Prior to the poly(A)⁺ RNA elution step, digestion of contaminating DNA was conducted with DNase (catalog. no. 27-0514-02; Amersham Biosciences). After the reaction was stopped and after a washing step, bound poly(A)⁺ RNA was eluted. The first drop was discarded, and the second to fourth drops were collected. Collected samples were adjusted to a volume of 75 µl with RNase-free water and were stored at –70°C.

Analysis and quantification of viral transcripts. Quantification of the viral transcripts from genes m123 (ie1), M122 (ie3), M112-113 (e1), and M55 (gB) was performed by real-time one-step RT-PCRs with the primers and probes indicated in the following paragraphs.

(i) IE1 transcripts. For ie1-specific RT-PCR, probe ie1-taq1 directed against the exon 3/4 splicing junction comprised nucleotides 5'-6,338 to 6,328 on exon 4 and 5'-6,205 to 6,192 on exon 3 (GenBank accession no. L06816). Oligonucleotide 5'-6,393 to 6,367 served as forward primer ie1_taq_forw1, and oligonucleotide 5'-6,139 to 6,156 served as reverse primer ie1_taq_rev1, yielding an amplification product of 133 bp.

(ii) IE3 transcripts. For ie3-specific RT-PCR, probe ie3_taq1 directed against the exon 3/5 splicing junction comprised nucleotides 5'-8,079 to 8,061 on exon 5 and 5'-6,205 to 6,198 on exon 3. Oligonucleotide 5'-6,042 to 6,061 served as forward primer HCH17, and oligonucleotide 5'-8,127 to 8,108 served as reverse primer HCH18, yielding an amplification product of 231 bp.

(iii) E1 transcripts. For e1-specific RT-PCR, probe early1-taq-P2 directed against exon 2 comprised nucleotides 5'-1,070 to 1,091 (11) (GenBank accession no. M35146). Oligonucleotide e1_intron1_for1 served as the forward primer spanning the exon 1/2 splicing junction by comprising nucleotides 5'-944 to 955 on exon 1 and 5'-1,049 to 1,058 on exon 2. Oligonucleotide e1_intron2_rev1 served as the reverse primer spanning the exon 2/3 splicing junction by comprising nucleotides 5'-1,552 to 1,562 on exon 3 and 5'-1,217 to 1,225 on exon 2. This resulted in an amplification product of 200 bp.

(iv) gB transcripts. For gB-specific RT-PCR, oligonucleotide 5'-83,175 to 83,200 (68) (GenBank accession no. U68299; complete genome) was used as probe gB-taq-2RT, oligonucleotide 5'-83,137 to 83,156 served as forward primer gB-taq-for2, and oligonucleotide 5'-83,227 to 83,207 served as reverse primer gB_taq_rev2, yielding an amplification product of 91 bp.

Quantifications were performed on an ABI Prism 7700 sequence detection system (Applied Biosystems) by using dual-labeled probes containing fluorescent reporter at the 5' end and a quencher at the 3' end (6-carboxyfluorescein reporter and 6-carboxytetramethylrhodamine quencher system; Operon Biotechnologies Inc., Huntsville, Alabama). The corresponding in vitro transcripts IE1, IE3, and gB were generated as described previously (41) and were used as standards for the quantifications. Standard titrations ranged from 10⁶ to 10¹ in vitro transcripts and were measured in duplicate. The amount of RNA in the standard titrations was adjusted to 4 µg with synthetic poly(A) as the carrier. Quantification of IE1, IE3, and gB transcripts in the lungs was performed for 1/10 aliquots of the yield of poly(A)⁺ RNA from a lung piece (see above).

Reactions for IE1 transcripts were performed in a total volume of 25 µl, containing 5 µl of 5x QIAGEN OneStep RT-PCR buffer, 1 µl of QIAGEN OneStep RT-PCR enzyme mix, 668 µM of each deoxynucleoside triphosphate, 1 µM of each primer, 0.26 µM probe, 1.5 mM additional MgCl₂, and 1.32 µM ROX (5-carboxy-X-rhodamine) as the passive reference. The reaction mixtures for gB, IE3, and E1 transcripts were modified in that primer concentrations were 0.64 µM and, in the case of gB transcripts, the additional MgCl₂ concentration was 3 mM. Reverse transcription was performed at 50°C for 30 min. The cycle protocol for cDNA amplification started with an activation step at 95°C for 15 min followed by 45 cycles of denaturation for 15 s at 94°C and a combined primer annealing/extension step for 1 min at 60°C. The efficiency of the RT-PCRs was >90% throughout.

The sensitivities of the RT-PCRs were determined by limiting dilution analysis (44) performed with graded numbers of synthetic transcripts. Samples were scored as being negative if the cycle threshold value was not <50, that is, if no signal exceeding that of the water control was obtained after 50 amplification cycles. Based on the Poisson distribution function, the maximum likelihood method (16) was used to determine the most probable number (MPN) value and its 95% confidence interval from the fractions of negative samples.

Confocal laser scanning analysis. MEFs were grown for 24 h on acetone-cleaned glass coverslips in 24-well plates at a density of 8 x 10⁴ cells per coverslip. Centrifugal infection with mCMV was performed at a multiplicity of infection (MOI) of 4 (0.2 PFU per cell x enhancement factor of 20). After 4 h of incubation, infected MEFs were washed and fixed in 70% methanol (stored at –20°C) for 90 min at 4°C. Fixed cells were stored in phosphate-buffered saline (PBS). Before use, fixed cells were incubated with 50 µl of blocking buffer (PBS supplemented with 0.3% Triton X-100 and 15% fetal calf serum) for 30 min at room temperature. For the double-immunofluorescence studies, each coverslip was incubated overnight in a humidity chamber with 50 µl of blocking buffer containing one of the following primary antibodies: mouse anti-IE1 monoclonal antibody (CROMA 101) (1:200), mouse anti-E1 monoclonal antibody (CROMA 103) (1:100), or affinity-purified rabbit anti-promyelocytic leukemia (PML) polyclonal antibody H-238 (catalog no. sc-5621, Santa Cruz, Biotech. Inc.) (1:200). After being washed with PBS, each coverslip was incubated for 1 h with the appropriate secondary antibody diluted in blocking buffer. Secondary antibodies used were an Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G (heavy plus light chains) antibody (catalog no. A11001; Molecular Probes) and an Alexa Fluor 546-conjugated goat anti-rabbit antibody (catalog no. A11010; Molecular Probes). The incubations for staining and all subsequent steps were performed in the dark. Coverslips were washed five times in PBS before they were mounted in mounting solution (catalog no. 153-6153; Panbio, Inc.) and stored at 4°C in the dark until the measurements were performed. Throughout, immunofluorescence analyses were followed by staining of the cell nuclei for 5 min at room temperature with 4'-6-diamidino-2-phenylindole (DAPI; Hoechst 333242 dissolved in PBS). Images were acquired using a Zeiss LSM-510 laser scanning microscope and Zeiss software. For the counting of intranuclear PML bodies and exclusion of extranuclear signals, three-dimensional scanning of nuclei was performed with a 100x lens and a Z-step size of 0.3 µm.

Immunological assays. An enzyme-linked immunospot (ELISPOT) assay was used to detect the sensitization, and consequent gamma interferon (IFN-) secretion, of CD8 T cells by viral epitopes. Epitopes were presented after exogenous loading of stimulator cells (P815 mastocytoma cells, H-2^d haplotype) either with the indicated concentrations of synthetic peptides or with naturally processed peptides contained in high-performance liquid chromatography (HPLC) fractions of lysates of infected cells. Alternatively, epitopes were presented after endogenous antigen processing in stimulator cells (BALB/c MEFs, H-2^d haplotype) infected with the indicated recombinant viruses. Second-passage MEFs were infected under conditions of centrifugal enhancement of infectivity with 0.2 PFU per cell, which corresponds to an MOI of 4 (60). To optimize the presentation of the IE1 peptide, viral gene expression was arrested in the IE phase, and MIE gene expression was enhanced by infection in the presence of cycloheximide (50 µg/ml) for 3 h that was then replaced by actinomycin D (5 µg/ml) (60). Custom peptide synthesis in a 1-mg scale and purification to >75% was performed by JERINI Bio Tools GmbH (Berlin, Germany). For a list with the amino acid sequences of the here tested and currently known MHC class I H-2^d-restricted antigenic peptides, see reference 25 or reference 69. HPLC fractionation of lysates from productively infected MEFs was performed as described elsewhere (reference 28 and references therein). The ELISPOT assay was performed as described previously (references 28 and 56 and references therein) with 10⁵ stimulator cells per assay culture and with graded numbers of effector cells seeded in triplicate, except for the analysis of HPLC fractions that was performed with a constant number of 4,000 effector cells. Specifically, effector cells were either cells of an IE1 epitope-specific cytolytic T-lymphocyte (CTL) line, referred to as IE1-CTLL and characterized as shown previously (56), or immunomagnetically purified memory CD8 T cells derived from the spleens or lungs (26, 27, 60). After 18 h of cocultivation, plates were developed and spots were counted. Frequencies of IFN- secreting and spot-forming effector cells and the corresponding 95% confidence intervals were calculated by intercept-free linear regression analysis as described in greater detail previously (56).

Cytolytic activity of cells from CTL lines IE1-CTLL and m164-CTLL (28, 56) was measured in triplicates in a standard 4-h ⁵¹Cr release assay at an effector-to-target cell ratio of 15,000 effector cells to 1,000 ⁵¹Cr-labeled target cells. Target cells were P815 mastocytoma cells pulsed with graded volumes of HPLC-fractionated cell lysate containing naturally processed peptides.

Frequency estimation of transcriptional events in lung tissue. Lungs were subdivided into 18 pieces (see above). Pieces 1 to 9 of the three lobes of the right lung and pieces 12 to 18 of the left lung, altogether 16 lung tissue pieces per mouse, were tested piece by piece with quantitative RT-PCRs for the presence and quantities of IE1, IE3, and gB transcripts. Based on the Poisson distribution function (44), frequencies of transcriptional events, also referred to as foci of transcription, were estimated by the maximum likelihood method (16) from the fractions of lung pieces that were negative for the respective type of transcript. For details of the calculations, see the methodological appendix of reference 21.

Significance analysis. Two independent sets of data with sample sizes n1 and n2, for instance loads of latent virus genomes in two groups of mice infected with different viruses (n^mut, sample size for mutant virus; n^rev, sample size for revertent virus), were compared by using distribution-free Wilcoxon-Mann-Whitney (rank sum) statistics. A calculator is provided on the Web site http://www.socr.ucla.edu/Applets.dir/WilcoxonRankSumTable.html (Ivo Dinov, Statistics Online Computational Resources, UCLA Statistics, Los Angeles, California). Samples are not significantly different if the P value is >0.05 (two-tailed test).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Addendum References

 
Working hypothesis and rationale of the experimental approach. According to the silencing/desilencing and immune sensing hypothesis of CMV latency and reactivation (see the introduction), the presentation of the IE1 peptide by latently infected lung cells that have entered transcriptional reactivation restimulates patrolling IE1 epitope-specific pulmonary memory CD8 T cells, which thereby acquire an activated T_EM state and interrupt the reactivation. It is important to note that all attempts to detect the IE1 protein or the IE1 peptide in latently infected lungs have failed because of the very low frequency of MIE transcription of 10 in the whole organ consisting of 60 million cells. Therefore, a genetic approach was required. The hypothesis postulates that functional deletion of the antigenic IE1 peptide abolishes the immune sensing of MIE gene reactivation. As a consequence, the transcriptional image of latently infected lungs should be altered to more cells expressing MIE genes and, possibly, to a progression of viral gene expression to beyond that of the MIE genes.

We have chosen the reverse genetics approach of constructing a recombinant virus that does not encode the antigenic IE1 peptide. The idea to use a virus with a knockout of the whole exon 4 (m123) of the ie1/3 transcription unit (exons 2, 3, and 4 specifying the IE1 protein and exons 2, 3, and 5 specifying the IE3 protein; see references 36 and 49) was immediately dismissed, because the IE1 protein is known to be involved in the disruption of PML nuclear bodies (19, 90) and in virulence in vivo (19). The aim, therefore, was to selectively eliminate IE1 antigenicity with a minimized influence on IE1 protein structure and function. Soon after the identification of the IE1 peptide sequence 168-YPHFMPTNL-176 (76), systematic replacements of amino acid residues by Ala had identified Pro169 in position 2 of the peptide and Leu176 at its C-terminal position 9 as anchor residues (74) required for efficient binding into hydrophobic pockets of the presenting MHC class I molecule L^d (for a review of MHC binding motifs, see reference 67). Specifically, when measured in a cytolytic assay with the prototype CTL clone IE1 (71), the substitution of Ala for Leu176 led to a reduction of peptide antigenicity by 6 log₁₀ of molar peptide concentration (74). The substitution of other residues for Leu176 showed that Phe can replace Leu without loss of function; that Met, Tyr, and Ile reduce antigenicity by 1 log₁₀ (Met) to 4 log₁₀ (Tyr and Ile); and that Val, Asn, and Trp destroy antigenicity by >6 log₁₀ (80). Notably, removing the branching of the side chain at C by replacing the ßC isopropyl group in Leu by a ßC propyl group in the synthetic Leu isomer Nleu was found to enhance antigenicity by 1 log₁₀ (72). Among the tested substitutions that caused a loss of function (Ala, Val, Asn, and Trp), Ala, in which the hydrophobic side chain is shortened by just one isopropyl group, appeared to be the most conservative modification. The alternative strategy of deleting the core epitope 170-HFMPT-174, which interacts with the T-cell receptor (TCR) (74, 76), a strategy shown previously to destroy IE1 antigenicity and immunogenicity in the respective vaccinia virus recombinant VacV-mCMV-IE1-HFMPT (14), was dismissed, because the deletion of this core epitope or its replacement by a penta-Ala string creates more of a risk of affecting protein function than a point mutation does.

The virus mutant mCMV-IE1-L176A and two revertant viruses were generated by BAC mutagenesis using the two-step replacement method (for a review, see reference 10). As outlined in Fig. 1A, the replacement of Leu by Ala was achieved by mutating codon CTA into GCA. Revertant virus mCMV-IE1-A176L was generated by back-mutation of codon GCA into CTA. Finally, revertant virus mCMV-IE1-A176L* was generated by mutation of codon GCA into CTT, leaving a single nucleotide exchange in the wobble position of the Leu codon as a genetic marker. The three viruses were tested for genomic structural integrity by restriction enzyme cleavage patterns (Fig. 1B), and the fidelity of the mutations was confirmed by sequencing (Fig. 1C).

View larger version (55K): [in this window] [in a new window]   FIG. 1. Construction and verification of recombinant viruses. (A) Maps of the mutagenesis, drawn to scale. The HindIII physical map of the mCMV Smith strain genome is shown at the top with the ie1/3 transcription unit expanded to reveal the location of the authentic antigenic IE1 peptide 168-YPHFMPTNL-176 coding region (WT/Revertant). By means of site-directed mutagenesis, the C-terminal MHC class I anchor residue Leu of the IE1 peptide was point mutated to Ala by mutating the Leu codon CTA into the Ala codon GCA (Mutant). Authentic revertant and wobble revertant (*) were generated by back-mutation of Ala codon GCA into Leu and Leu* codons CTA and CTT. The 6,523-bp BssHII/HpaI fragment comprises the ie1/3 transcription unit with the authentic or mutated IE1 peptide-coding region used in the shuttle plasmids for recombination. (B) Structural analysis of BAC plasmids. Purified DNA of BAC plasmids pSM3fr (lanes 1), C3XIE1Leu (lanes 2), C3XIE1Ala (lanes 3), and C3XIE1Leu* (lanes 4) was subjected to cleavage by EcoRI, HindIII, and XbaI, and fragments were analyzed by agarose gel electrophoresis and staining with ethidium bromide. Lanes M show the size markers. (C) Sequence analysis of mutated BAC plasmids. The fidelity of the sequences is shown between n181,011 and 181,028 for the BAC plasmids C3XIE1Ala (mutant L176A), C3XIE1Leu (revertant A176L), and C3XIE1Leu* (wobble revertant A176L*).

View larger version (9K): [in this window] [in a new window]   FIG. 2. Influence of the Leu codon mutation on viral transcription. Throughout, MEFs were infected in six-well culture dishes at an MOI of 4 (0.2 PFU/cell x 20; centrifugal enhancement of infectivity) for transcriptional analysis. Closed and open symbols indicate infection with revertant virus mCMV-IE1-A176L and with mutant virus mCMV-IE1-L176A, respectively. Ordinate values represent copies per 50 ng of total RNA. Shown are the data from triplicate cultures. The schedules for inhibitor treatment (CH, 50 µg of cycloheximide per ml; ActD, 5 µg of actinomycin D per ml) and virus (V) infection are indicated. Time zero is defined as the end of the 30-min period of centrifugal infection. (A) MIE transcription in the absence of protein synthesis. MEFs were infected in the continuous presence of CH, and spliced IE1 (circles) and IE3 (squares) transcripts were quantified by real-timeRT-PCRs at the indicated time points after infection. (B) Stability of IE1 transcripts. Infection was performed for 3 h in the presence of CH. At the indicated time points after the replacement of CH by ActD, IE1 transcripts were quantified by real time RT-PCR. (C) Viral transcription in absence of metabolic inhibitors. At the indicated time points after infection, IE1 (circles), IE3 (squares), and E1 (diamonds) transcripts were quantified by real-time RT-PCRs.

Functional integrity of the mutated IE1 protein. (i) The mutation L176A does not affect the regulatory function of the IE1 protein in viral gene expression. Functions of the mCMV IE1 protein that are pertinent to viral transcription are the autoactivation of the MIE promoter and the cotransactivation, in cooperation with the IE3 protein, of the e1 promoter (49). We have therefore compared the amounts of IE1, IE3, and E1 transcripts generated after infection of MEFs with mutant virus mCMV-IE1-L176A and revertant virus mCMV-IE1-A176L in the absence of metabolic inhibitor. As shown in Fig. 2C, the mutation L176A had no noticeable influence on the expression kinetics and amounts of these three viral transcripts.

(ii) The mutation L176A does not affect transactivation of cellular promoters. While the IE3 protein is the essential transactivator of mCMV E gene expression (1, 49), IE1 was found to be relevant for the efficacy of virus replication in host tissues (19), a finding that possibly is related to the intrinsic property of IE1 to transactivate promoters of cellular genes of the nucleotide metabolism, such as the ribonucleotide reductase gene (45) and the thymidylate synthase gene (20), in resting cells. Luciferase reporter gene assays with donor plasmids encoding the nonmutated (pIE100/1), the mutated (pIE1-L176A), or the back-mutated (pIE1-A176L*) IE1 protein and with reporter plasmids, in which firefly luciferase gene expression is driven by promoters P^R2 of the ribonucleotide reductase gene (Fig. 3A) and P^TS of the thymidylate synthase gene (Fig. 3B), were employed to test the functional integrity of the mutated IE1 protein. In essence, the transactivating activity of the IE1 protein was not affected by the L176A mutation.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Influence of the L176A mutation on the IE1-dependent transactivation of cellular promoters. Transactivation of reporter genes by the authentic IE1 protein or the mutated IE1 protein was measured by a firefly-Renilla DLR assay. NIH 3T3 cells were cotransfected with reference plasmid pRL-TK encoding Renilla luciferase for standardization of transfection efficacy (not depicted), IE1 protein-encoding donor plasmid, and a reporter plasmid carrying a promoter of interest driving the expression of firefly luciferase. Donor plasmids were pIE100/1, specifying the authentic IE1 protein; pIE1-L176A, specifying the mutated IE1 protein; and pIE1-A176L*, specifying the rescued IE1 protein. Cotransfection with pUC19 in place of the donor plasmid served as a vector control.(A) Transactivation of the ribonucleotide reductase 2 promoter (PR2) by the IE1 protein. Plasmid pGL3R2 1.5 served as the reporter plasmid. (B) Transactivation of the thymidylate synthase promoter (PTS) by the IE1 protein. Plasmid pTLG served as the reporter plasmid. Plasmid maps (not drawn toscale) are illustrated. MIEPE, major immediate-early promoter and enhancer. Ex, exon. Arrows indicate the direction of transcription. Arrowheads mark the location of the mutation in exon 4. Lumines-cence data are expressed as RLU and are normalized for transfection efficacy by forming the quotient of firefly luciferase activity (RLUFL) and Renilla luciferase activity (RLURL). Gray-shaded bars represent the median values of triplicate assay cultures, and the error bars indicate the range.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Dispersion of nuclear domains ND10 by the mutated IE1 protein. MEFs were left uninfected (Ø) or were infected at an MOI of 4 (0.2 PFU/cell x 20; centrifugal enhancement of infection) with the BAC-derived mCMVs indicated. The analysis was performed at 4 h after infection corresponding to an early stage in the E phase. (A) Confocal laser scanning images. (a and e) Green staining for PML protein. Note that red Alexa Fluor 546 fluorescence was electronically converted into green for better contrast. (b and f) Blue counterstaining of DNA in the cell nucleus with DAPI. (c and g) Red staining for intranuclear mCMV proteins E1 and IE1, respectively. Note that green Alexa Fluor 488 fluorescence was electronically converted into red. (d and h) Merge of blue DAPI and green PML staining. Panels a to d and e to h each show an individual, representative cell after double labeling. Note the presence of green-stained intranuclear PML bodies in panels a and d in a cell infected with the ie1 gene deletion mutant and their absence in panels e and h in a cell infected with mutant mCMV-IE1-L176A. The bar marker represents 20 µm. (B) Quantification of intranuclear PML bodies. For statistical significance analysis, intranuclear PML bodies were counted for 30 infected cell nuclei per group. Dots represent the number of PML bodies in individual cell nuclei. The median values are marked by horizontal bars. P values are indicated for comparisons of major interest.

The mutation L176A does not affect virus replication and dissemination in the immunocompromised host. In accordance with the functional integrity of the IE1 protein and the unaltered transactivation of E-phase transcription shown above for the example of E1, virus replication in cell culture was not affected by the mutation. Specifically, the genome-to-infectivity ratios, which for sucrose gradient-purified mono- and multicapsid virions of mCMV is 2-fold the particle-to-infectivity ratio (43), were found to be identical for mutant virus mCMV-IE1-L176A and revertant virus mCMV-IE1-A176L (317 to 683 genomes/PFU and 320 to 555 genomes/PFU, respectively; n^mut = 12, n^rev = 11; P = 0.32; Wilcoxon-Mann-Whitney test; two-tailed).

To test whether this applies also to virus replication in host tissues under conditions of immunoablation by a 7-Gy total-body -irradiation, the virus growth curves were determined for the local site of subcutaneous infection, i.e., the heterogeneous tissue of the footpad, as well as for distant organ sites, exemplified for the spleen and the liver (Fig. 5). The virus growth rates, expressed as doubling times of viral genomes in the case of plantar tissue and spleen and as doubling times of infected cells, mainly hepatocytes, in the liver, revealed no significant differences between the wild-type virus, the mutant virus, and the two revertant viruses. It is interesting to note that with values in the ranges of 22 to 46 h (plantar tissue), 10 to 15 h (spleen), and 13 to 23 h (liver), the doubling times were much longer than one would expect from the virus productivity of an infected cell. These findings suggest that cell-to-cell spread in tissue is a rate-limiting step. All four viruses initiated liver infection between days 3 and 4. Thus, the mutation had no significant influence on the capacity of the virus to disseminate from the local site of intraplantar infection to the liver.

View larger version (28K): [in this window] [in a new window]   FIG. 5. Virus replication and dissemination in vivo. Virus growth curves are shown as log-linear plots of infection load on the ordinate and time after infection on the abscissa. Infection loads in plantar tissue, spleen, and liver were determined at the indicated time points after subcutaneous, intraplantar infection of 7-Gy total-body -irradiated BALB/c recipients with 105 PFU (corresponds to 5 x 107 viral genomes) of the BAC-derived mCMVs indicated. In plantar tissue and in the spleen, the viral loads per 106 tissue cells were quantified by real-time PCR specific for viral gene M55 (gB) normalized to the cellular gene pthrp. Infection of the liver was quantified by counting infected liver cells, which are mostly hepatocytes and some endothelial cells, in representative 10-mm2 areas of tissue sections. Infection of cells was identified by immunohistological staining of intranuclear IE1 protein. Dots represent data from three individual mice per time point, with median values marked. The DTs (95% confidence intervals of DTs are in parentheses) were calculated by log-linear regression analysis. Note that DNA load present on day 1 in plantar tissue (arrows) mostly represents DNA of the virus inoculum. Accordingly, data for day 1 in plantar tissue were excluded from the regression analysis of virus replication. The time point of first detection of infected liver cells is revealed by the intersection between the calculated regression line and ordinate 0 (corresponds to one infected cell per test area), which was between days 3 and 4 for all viruses tested. The horizontal bars indicate the corresponding 95% confidence intervals.

View larger version (16K): [in this window] [in a new window]   FIG. 6. Immunological loss-of-function phenotype of the L176A mutation. Throughout, gray-shaded bars represent the frequencies of CD8 T cells that were successfully sensitized for IFN- secretion in ELISPOT assays. MPN values were determined by intercept-free linear regression analysis of data (spot counts) obtained for graded numbers of effector cells, each assayed in triplicate cultures. Error bars represent the corresponding 95% confidence intervals. (A) Reduction of MHC binding affinity of the IE1 peptide by the mutation. For their use as stimulator cells, P815 (H-2d haplotype) mastocytoma cells were exogenously loaded with synthetic peptides YPHFMPTNL and YPHFMPTNA at the concentrations indicated. Ø, no peptide added. Effector cells were CTLs (200 and 100 cells seeded) of a polyclonal IE1 epitope-specific CTL line (IE1-CTLL) representing a broad spectrum of TCR affinities. (B) Presentation of naturally processed IE1 peptides. Effector cells were CTLs (300, 200, 100, and 50 cells seeded) of the polyclonal IE1-CTLL. Stimulator cells were MEFs that were either left uninfected (n.i.) or were infected under conditions of selective and enhanced MIE gene expression (MOI, 4; cycloheximide replaced after 3 h by actinomycin D) with the viruses indicated. (C) Effect of the mutation on IE1 epitope-specific CD8 T-cell memory. Effector cells were CD8 T cells (104, 5 x 103, and 103 cells seeded) isolated from the pooled spleens of three BALB/c mice at 5 months after intraplantar infection with 105 PFU of wobble revertant virus (top panel) or mutant virus (bottom panel). Target cells were P815 cells exogenously loaded with the indicated synthetic peptides at concentrations of 10–8 M, except for peptide YPHFMPTNA, which was used at 10–6 M. Ø, no peptide added.

For testing immunogenicity (Fig. 6C), BALB/c mice were primed by subcutaneous, intraplantar infection with revertant virus mCMV-IE1-A176L* and with mutant virus mCMV-IE1-L176A. The capacities of the two viruses to prime an IE1 epitope-specific CD8 T-cell response with subsequent generation and maintenance of a memory CD8 T-cell pool were assessed by determining the frequencies of spleen-derived memory CD8 T cells specific for the IE1 epitope. As shown in the top panel of Fig. 6C, 1% of the total CD8 T cells were specific for the IE1 epitope after priming with the revertant virus. This is revealed by their sensitization through stimulator cells presenting exogenously loaded synthetic IE1 peptide 168-YPHFMPTNL-176 or its functional analog, 168-YPHFMPTNF-176. By contrast, as shown in the bottom panel of Fig. 6C, infection with the mutant virus failed to generate IE1 epitope-specific CD8 T cells. That this failure does not reflect a generally low priming efficacy of the mutant virus becomes evident from the successful generation of memory CD8 T cells specific for the coimmunodominant MHC class I D^d-restricted epitope 257-AGPPRYSRI-265 that is derived from the ORFm164 protein(28), meanwhile characterized as glycoprotein gp38/50 expressed in the E phase (T. Däubner and S. A. Oehrlein-Karpi; manuscript in preparation). The observed frequency of m164 epitope-specific memory CD8 T cells of 2%, compared to 1% with the revertant virus, suggests that the m164-specific CD8 T-cell response profits from the absence of IE1-specific priming.

In conclusion, in accordance with the mutagenesis rationale, the point mutation of the C-terminal MHC class I anchor residue of the IE1 peptide in mutant virus mCMV-IE1-L176A has abolished both IE1-specific antigenicity and immunogenicity.

Functional deletion of IE1 antigenicity and immunogenicity has no influence on clearance of productive infection in the lungs during hematopoietic reconstitution. As shown above, a role for the IE1 epitope in the immunological control of mutant virus mCMV-IE1-L176A is precluded for two reasons. First, host cells infected with this virus fail to present the IE1 epitope, and second, this virus fails in priming an IE1-specific CD8 T-cell response. It was clear, however, that this deletion of IE1 antigenicity and immunogenicity would not abolish CD8 T-cell control of virus replication, because security backup is provided by the coimmunodominant m164 peptide as well as by a series of subdominant antigenic peptides known to elicit protective CD8 T cells (for a review, see reference 25). Yet, it remained a reasonable prediction that functional deletion of one out of two immunodominant epitopes will at least reduce the efficacy of the immune control, in particular under conditions of hematoablative treatment and BMT, where the control of productive infection in the BMT recipients by hematopoietic reconstitution of antiviral CD8 T cells is a race against time (25). Admittedly, we were therefore somewhat surprised by the result that peak replication levels as well as the time courses of clearance of productive infection in the lungs of BMT recipients were essentially alike for mutant virus mCMV-IE1-L176A and revertant virus mCMV-IE1-A176L (Fig. 7A). From the virus titer data, this conclusion becomes obvious at a glance, and it is also substantiated by Wilcoxon-Mann-Whitney statistics confirming that virus titers in the lungs were not significantly different between the two groups of BMT recipients at individual time points (except for a marginal significance at 4 weeks) as well as during the whole time course (n^rev = 50; n^mut = 50; P = 0.23; two-tailed). For both viruses, productive infection was found to be resolved at 8 months after BMT and primary infection.

View larger version (28K): [in this window] [in a new window]   FIG. 7. Resolution of productive infection of the lungs and establishment of latency after bone marrow transplantation. (A) Time course of productive infection of the lungs. Infectious virus per lung was monitored by a PFU assay at the indicated time points after BMT and intraplantar infection with 105 PFU of revertant virus mCMV-IE1-A176L (closed circles) or mutant virus mCMV-IE1-L176A (open circles). Symbols represent data for individual BMT recipients with the median values indicated. P values compare mutant virus and revertant virus at each time point of analysis by the distribution-free Wilcoxon-Mann-Whitney rank sum test. p.i., postinfection. (B) Viral DNA load during pulmonary latency. Viral genomes were quantified at 1 year after BMT and infection in the two neighboring lung tissue pieces (10 and 11) of the postcaval lobe for five individual BMT recipients per group. Symbols (closed circles, revertant virus; open circles, mutant virus) represent triplicate real-time PCR data for each lung DNA preparation. Median values are indicated.

Frequencies of epitope-specific CD8 T cells in latently infected lungs. The immunological situations in latently infected lungs were compared for the two viruses at 1 year after BMT and infection (Fig. 8). During latency of the revertant virus mCMV-IE1-A176L, the mCMV-specific fraction of the pulmonary CD8 T-cell pool was dominated by the epitopes IE1 and m164 (Fig. 8A). This was concluded from the frequencies of pulmonary CD8 T cells specific for all currently known H-2^d-restricted antigenic peptides of mCMV tested as synthetic peptides (Fig. 8A, left panel), as well as from the recognition of HPLC-separated naturally processed peptides that might encompass also unidentified antigenic peptides (Fig. 8A, right panel). Indeed, while HPLC fractions 24 and 29 contained the m164 peptide and the IE1 peptide, respectively (Fig. 8A, miniature inserts), unknown minor activities were detected in fractions 25 and 30. During latency of the mutant virus mCMV-IE1-L176A, the mCMV-specific fraction of the pulmonary CD8 T-cell pool consisted almost exclusively of cells specific for the m164 peptide (Fig. 8B). While the frequency of m164-specific CD8 T cells was found to be increased twofold to 3% of all pulmonary CD8 T cells, subdominant epitopes apparently did not profit from the functional deletion of the IE1 peptide.

View larger version (33K): [in this window] [in a new window]   FIG. 8. Frequency and virus epitope specificity of memory CD8 T cells present in latently infected lungs. Pulmonary CD8 T cells were isolated at 1 year after infection of BMT recipients from pools of six lungs and were tested as effector cells in IFN--based ELISPOT assays. (A) BMT and infection with revertant virus mCMV-IE1-A176L. (B) BMT and infection with mutant virus mCMV-IE1-L176A. (Left panels) Stimulator cells were P815 mastocytoma cells exogenously loaded with the indicated synthetic peptides at concentrations of 10–8 M. Ø, no peptide added. Bars represent MPN values determined by intercept-free linear regression analysis of data (spot counts) obtained for graded numbers (8 x 103, 4 x 103, 1 x 103) of effector cells, each assayed in triplicate cultures. Error bars represent the corresponding 95% confidence intervals. (Right panels) Stimulator cells were P815 mastocytoma cells pulsed with 60 µl of 800-µl HPLC fractions containing naturally processed peptides derived from MEFs at 24 h after infection with revertant virus (for testing group A) and mutant virus (for testing group B). Bars represent the median values of triplicate data obtained with a constant number of 4,000 effector cells for testing each HPLC fraction. Error bars indicate the range. Arrowheads mark the HPLC fractions in which the immunodominant peptides m164 and IE1 eluted. Miniature inserts document the identification of the HPLC fractions that contained peptides m164 and IE1. Cytolytic activities of lines m164-CTLL and IE1-CTLL were determined at an effector-to-target cell ratio of 15:1 with 1,000 P815 target cells pulsed with 10 µl of 800-µl HPLC fractions (highest concentration) or with 1:6, 1:36, or 1:216 dilutions thereof. Data represent the mean values of triplicate determinations.

The sensitivity of real-time quantitative RT-PCR specific for the spliced IE1 transcript (36) was calibrated with synthetic polyadenylated transcripts by a limiting dilution assay. As shown in Fig. 9A, eight molecules of synthetic IE1 RNA were detected after 33 to 41 (median value of 35) amplification cycles in all 48 replicates tested, whereas replicates that remained at the level of the water control after 50 amplification cycles occurred at four molecules seeded. There was a clear distinction between negative replicates (n = 6; >50 cycles) and positive replicates (n = 42; 34 to 41 cycles with a median value of 38 cycles). At one molecule seeded per replicate, 20 out of 48 replicates gave an amplification product after 35 to 48 cycles, with a median value of 40 cycles. The fractions of negative replicates exactly followed a Poisson distribution, with an MPN of 1.8 (95% confidence interval, 1.5 to 2.3) molecules required for detection (Fig. 9B). We defined the detection limit of the assay as the next integer number above the upper 95% confidence limit, that is, three mRNA molecules. In the same way, the MPNs of molecules required for detection of IE3 and M55 (gB) mRNA were found to be 1.4 (0.8 to 2.5) and 0.8 (0.5 to 1.3), respectively (data not shown). As transcription assays for latently infected lungs were performed with 1/10 aliquots of the poly(A)⁺ RNA yields of lung pieces, the detection limit for a whole piece was defined as 30 molecules in the case of IE1 as well as IE3 transcripts and as 20 molecules in the case of M55 (gB) transcripts.

View larger version (19K): [in this window] [in a new window]   FIG. 9. Detection limit of the IE1-specific real-time quantitative RT-PCR. (A) Graded numbers of synthetic polyadenylated IE1 transcripts in 48 replicates were amplified by real-time RT-PCR. Dots represent the numbers of cDNA amplification cycles (cycle threshold [Ct] values) required for detection, with the median values marked by horizontal bars. The dotted line indicates the cutoff value defined by the water control. (B) Limiting dilution (Poisson distribution) analysis based on the experimentally determined fraction of negative replicates (see panel A). The log-linear plot shows the Poisson distribution graph (calculated with the maximum-likelihood method) with its 95% confidence region shaded. The MPN (the reciprocal of the Poisson distribution parameter ) is revealed as the abscissa coordinate (dashed arrow) of the point of intersection between 1/e and the regression line; it gives us the number of transcripts that need to be seeded for detection. 95% CI, 95% confidence interval of MPN; P, probability value indicating the goodness of fit.

In a first approach (Fig. 10A), the influence of the L176A mutation on transcriptional activity during latency in the lungs was tested at 1 year after primary infection for mice from the BMT experiment that was characterized in detail as shown above. Although the mutation had not resulted in any phenotype with regard to clearance of productive infection (Fig. 7A) and latent viral genome load in the lungs (Fig. 7B), its impact on MIE gene transcription during latency was striking at a glance. Whereas during latency of the revertant virus only 14 out of 80 lung pieces from five mice tested contained IE1 transcripts, 51 out of 80 lung pieces were positive during latency of the mutant. In addition, splicing to IE3 transcripts was found in 7 out of 80 pieces selectively in lungs that were latently infected with the mutant. In this group, there existed a single piece that contained also M55 (gB) transcripts. As the amounts of IE1 and IE3 transcripts found in this particular piece were clearly far beyond the range of those found in all the other transcriptionally active pieces, this likely represented a solitary case of either persistent or reactivated productive infection.

View larger version (36K): [in this window] [in a new window]   FIG. 10. Contextual analysis of transcription in latently infected lungs. (A) First BMT experiment performed with mutant mCMV-IE1-L176A and the authentic revertant mCMV-IE1-A176L. The analysis was performed at 1 year after BMT and infection. (B) Second BMT experiment performed with mutant mCMV-IE1-L176A and the wobble revertant mCMV-IE1-A176L*. The analysis was performed at 8 months after BMT and infection. Transcripts were quantified for each of the illustrated lung pieces, for seven pieces of each left lung, and for nine pieces of each right lung, comprising superior lobe, middle lobe, and inferior lobe. Color codes are explained by the insert legend. Numbers at the pieces give the numbers of IE1/IE3/gB transcripts determined by the respective real-time quantitative RT-PCRs for 1/10 aliquots of the yield (per piece) of poly(A)+ RNA.