1 Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
2 Gulbenkian Institute for Science, 2780-156 Oeiras, Lisbon, Portugal
3 Laboratory of Microbiology, Faculty of Medicine, Lisbon University, Portugal
Correspondence
Philip G. Stevenson
pgs27{at}mole.bio.cam.ac.uk
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ABSTRACT |
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Published online ahead of print on 13 October 2004 as DOI 10.1099/vir.0.80480-0.
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INTRODUCTION |
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Several EpsteinBarr virus (EBV) latency gene products have been reported to inhibit apoptosis, including EBNA-1 (Kennedy et al., 2003), EBNA-2 (Lee et al., 2002
), LMP-1 (Henderson et al., 1991
) and LMP-2A (Fukuda & Longnecker, 2004
). Anti-apoptotic activity has also been ascribed to the Kaposi's sarcoma-associated herpesvirus (KSHV) gene products LANA (Friborg et al., 1999
), K15 (Sharp et al., 2002
), v-FLIP (Thome et al., 1997
) and K7 (Feng et al., 2002
; Wang et al., 2002
). In addition to these relatively poorly conserved genes, all gammaherpesviruses encode a homologue of cellular bcl-2 (Hardwick, 1998
; Cuconati & White, 2002
). The EBV bcl-2 homologue is a lytic, rather than a latent, gene product (Pearson et al., 1987
); in latency, EBV inhibits the mitochondrial pathway of apoptosis by inducing cellular bcl-2 expression (Henderson et al., 1991
). The KSHV bcl-2 homologue, encoded by ORF16 (Cheng et al., 1997
; Sarid et al., 1997
), is similarly induced by tetradecanoyl phorbol acetate treatment of latently infected tumour cells (Sarid et al., 1998
; Sun et al., 1999
). However, ORF16 transcripts remain detectable in the absence of induction (Sarid et al., 1997
), suggesting that KSHV may also use its bcl-2 homologue to inhibit apoptosis in latency.
Murine gammaherpesvirus 68 (MHV-68) is a natural murid pathogen that provides an opportunity to identify the in vivo function of a gammaherpesvirus bcl-2 homologue. This function is not necessarily the same in different gammaherpesviruses, but the fact that some kind of bcl-2 homologue has been maintained over tens of millions of years of viral evolution means that broad parallels are likely. M11 transcripts have been detected in lytically infected fibroblasts by RT-PCR (Virgin et al., 1999; Roy et al., 2000
; Marques et al., 2003
) and by microarray analysis (Martinez-Guzman et al., 2003
), but not by Northern blot (Virgin et al., 1999
; Roy et al., 2000
), suggesting that lytic-cycle M11 transcription occurs at only a low level. Interestingly, the M11 microarray signal in MHV-68-infected fibroblasts is resistant to cycloheximide, which, in conjunction with the identification of M11 transcripts in latently infected S11E cells (Martinez-Guzman et al., 2003
), suggests that the M11 promoter is active in latency, as well as in the lytic cycle. In vivo, M11 transcripts have been found in peritoneal cells, but not spleens, of B-cell-deficient mice 6 weeks post-infection (Virgin et al., 1999
); in mediastinal lymph nodes (Rochford et al., 2001
); in the spleens and lungs of persistently infected, immunocompetent mice (Roy et al., 2000
); and in macrophages, dendritic cells, newly formed B cells and marginal-zone B cells at the peak of splenic latency (Marques et al., 2003
). These data are consistent with M11 being transcribed in at least some forms of in vivo MHV-68 latency.
M11 function has been analysed in vitro by overexpressing the isolated gene in fibroblasts. In this setting, M11 inhibits the apoptosis induced by either Fas ligation (Wang et al., 1999), tumour necrosis factor alpha treatment (Wang et al., 1999
; Roy et al., 2000
) or Sindbis virus infection (Bellows et al., 2000
). In vivo, M11-deficient MHV-68 shows normal lytic replication and latency establishment after intraperitoneal infection, but reduced reactivation from latency (Gangappa et al., 2002
). This latter conclusion was based on reduced numbers of infectious centres in the presence of normal viral DNA loads. The present analysis, begun prior to the report of Gangappa et al. (2002)
and using the perhaps more physiological intranasal route of infection, identifies an M11-associated deficit in MHV-68 latency establishment. Stable, long-term latency levels were unaffected, but the peak latent load was reduced. In our hands, there was no evidence of a deficit in ex vivo reactivation from latency. M11 might yet have a role in in vivo reactivation from latency. However, the main impact of a disrupted bcl-2 homologue following intranasal infection of immunocompetent mice was reduced latency amplification in lymphoid germinal centres.
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METHODS |
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Cells and viruses.
Baby hamster kidney cells (BHK-21, ATCC CCL-10), COS-7 cells (ATCC CRL-1651), NIH-3T3-CRE cells (Stevenson et al., 2002) and murine embryonic fibroblasts (MEFs) harvested at 1314 days gestation were all grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 2 mM glutamine, 100 U penicillin ml1, 100 µg streptomycin ml1 and 10 % fetal calf serum (FCS; PAA Laboratories) (complete medium). Medium for MEFs was further supplemented with 50 µM 2-mercaptoethanol. All virus stocks were grown and titrated in BHK-21 cells.
Virus titres.
Lungs were homogenized in complete medium, frozen, thawed and sonicated. Tissue debris was pelleted by brief centrifugation (1000 g, 1 min). Infectious virus in homogenate supernatants was measured by plaque assay of tenfold dilutions of lung homogenates on MEFs, as described previously (de Lima et al., 2004). After 5 days, monolayers were fixed in 10 % formaldehyde and stained with 0·1 % toluidine blue. Plaques were counted with a plate microscope. Latent virus was measured by explant culture of single-cell suspensions of spleens, overlaid onto MEF monolayers and cultured in complete medium with 0·3 % carboxymethylcellulose. The monolayers were fixed and stained after 6 days.
Viral genome quantification.
The viral genome load in individual spleens was measured by real-time PCR. DNA was extracted from each spleen (Wizard genomic DNA purification kit; Promega) and a portion of the MK3 ORF (genomic coordinates 2483225071) was amplified by PCR from 10 ng DNA over 50 cycles (Rotor Gene 3000; Corbett Research). PCR products were quantified by using Sybr green (Invitrogen) and compared with a standard curve of cloned MK3 template, diluted serially in uninfected cellular DNA and amplified in parallel. The MK3 copy number in spleen samples was calculated from the cycle number at which the Sybr green signal crossed a set threshold on the standard curve. Amplified products were distinguished from paired primers by melting-curve analysis. The correct size of the amplified products was confirmed by electrophoresis and staining with ethidium bromide. We determined the frequency of viral genome-positive cells in pooled spleen samples as described previously (Marques et al., 2003). Briefly, cells pooled from five spleens were either used directly or sorted (MoFlo; Cytomation) for germinal-centre B cells (>95 % CD19+PNAhi). Twofold dilutions of cells (eight replicates each) were then lysed overnight with SDS/proteinase K, denatured and used to amplify genomic coordinates 2483225071 (Lightcycler; Roche Diagnostics). Quantification was based on comparison with a standard curve of MK3 plasmid dilutions, amplified in parallel. Specificity of all amplified products was confirmed by melting-curve analysis using internal, fluorochrome-labelled oligonucleotides. The frequency of genome-positive cells was then calculated from a regression plot of input cell number against the fraction of negative samples.
In situ hybridization.
Cells expressing viral tRNAs 14 were detected by in situ hybridization of formalin-fixed, paraffin-embedded spleen-cell sections with a digoxigenin-labelled riboprobe transcribed from pEH1.4 as described previously (Bowden et al., 1997). Hybridized probe was detected with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Boehringer Ingelheim), according to the manufacturer's instructions.
Viral mutagenesis.
We generated two M11 mutations by using the MHV-68 bacterial artificial chromosome (BAC) (Adler et al., 2000). First, we used Rec E/T cloning to interrupt the M11 ORF (genomic coordinates 103418103933). A kanamycin-resistance gene (kanR) flanked by flp recombinase recognition sites was amplified from the plasmid pCP15 by PCR using the primers 5'-TCTCTACATCATCAAACATGAGTCATAAGAAAAGCGGGACTTATTGGGCACACAGGAAACAGCTATGACCATGA-3' and 5'-AACGAGGTGAAAAGTTTGGACAGGTCTCTTTTCCAGTTCTTGAGGGCAGGCCAGGGTTTTCCCAGTCACGACGT-3'. The underlined sequences are homologous to nt 103401103450 and 103650103601 of the MHV-68 genome, respectively (Virgin et al., 1997
). The PCR product was electroporated into Escherichia coli JC8679 containing the MHV-68 BAC. Rec E/T-mediated recombination between the viral genome and the 50 bp sequences at each end of the PCR product inserted the kanR gene into the BAC in place of genomic coordinates 103451103600. DNA was extracted from kanamycin-resistant colonies, screened by restriction-enzyme digestion and electroporated into E. coli DH10B (M11K+). We then removed the kanR-encoding sequence with the flp recombinase expression plasmid pCP20 (Adler et al., 2000
), leaving a single flp recognition site plus short flanking plasmid sequences (167 bp in total), in place of the 150 bp genomic deletion (M11K). This also introduced a stop codon to terminate M11 translation after 11 aa. Secondly, an oligonucleotide encoding stop codons in all reading frames and BamHI and EcoRI restriction sites (underlined) (5'-CTAGCTAGCTAGGATCCGAATTCGGATCCTAGCTAGCTAG-3') was ligated into a BspHI site at genomic coordinate 103751 (M11C). To do this, the BspHI site in pACYC184 (New England Biolabs) was destroyed by digestion with BspHI, blunting with T4 DNA polymerase (New England Biolabs) and religation back to itself. A BamHI-G genomic fragment (Efstathiou et al., 1990
) (genomic coordinates 101653106902) was then cloned into the modified pACYC184. This vector was digested with BspHI and dephosphorylated (Paenibacillus borealis alkaline phosphatase; Roche Diagnostics). The oligonucleotide was heated, reannealed to itself, phosphorylated with polynucleotide kinase (New England Biolabs) and ligated into the BspHI site. Thus, the M11 coding sequence was truncated after 111 aa. The mutant BamHI-G fragment was then subcloned into the SmaI site of the shuttle plasmid pST76K-SR by using EcoRV, which cleaves at genomic coordinate 106316 within BamHI-G and within pACYC184. The oligonucleotide insertion in M11 was then recombined into the MHV-68 BAC by standard methods (Adler et al., 2000
). We also cloned the native BamHI-G fragment into pST76K-SR and reverted each M11 BAC mutant to wild-type in the same way. All BACs were reconstituted to infectious virus by transfecting 5 µg BAC DNA into BHK-21 cells using Fugene-6 (Roche Diagnostics). The loxP-flanked BAC cassette was removed by viral passage through NIH-3T3-CRE cells until cells positive for green fluorescent protein (GFP) were no longer visible.
Plasmids.
Full-length M11 was amplified by PCR from wild-type MHV-68 BAC DNA, including EcoRI and XhoI sites in the respective 5' and 3' primers. The PCR product was cloned into pEGFP-N3 (Becton Dickinson) such that M11 was in-frame with the upstream enhanced GFP (eGFP)-encoding sequence. M11 with stop codons after aa 111 was amplified from M11C BAC DNA by using the same primers and cloned in parallel into pEGFP-N3. All constructs were confirmed as being correct by DNA sequence analysis.
Flow cytometry.
For assays of apoptosis, MEFs were trypsinized, washed in PBS/0·1 % BSA and stained for 1 h on ice with fluorescein isothiocyanate (FITC)-coupled Annexin-V. Cells were washed twice and propidium iodide was added (1 µg ml1) before analysis. For assays of B-cell activation, spleens were disrupted into single-cell suspensions, washed once in PBS/0·1 % BSA and incubated for 30 min on ice with 5 % mouse serum, 5 % rat serum and anti-CD16/32 mAb (Becton Dickinson). Specific staining was done with phycoerythrin-conjugated anti-CD19 and FITC-coupled anti-CD69 (Becton Dickinson). After 1 h incubation on ice, cells were washed twice in PBS/BSA (0·1 %) and analysed on a FACScalibur using CellQuest software (Becton Dickinson). Data were analysed with FCSPress v1.3 (www.fcspress.com).
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RESULTS |
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In addition to lower infectious-centre titres in the spleens of mice infected with the M11 mutant viruses, there was less B-cell activation (Fig. 6a) and lower viral genome loads (Fig. 6b
). B-cell activation correlates with MHV-68 infection both in vitro and in vivo (Stevenson & Doherty, 1999
; Flaño et al., 2000
) and so provides an indication of the extent of host colonization, independently of any requirement for ex vivo viral gene expression or for further manipulation, such as DNA extraction. There was a clear correlation between these three assays (Fig. 6c
), arguing that the reduced infectious-centre titres were not due to a reactivation deficit, but to a reduced amount of latent virus. Flow-cytometric sorting of cells prior to viral genome quantification (Fig. 6d
) indicated that the major deficit was in B-cell colonization.
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DISCUSSION |
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In agreement with Gangappa et al. (2002), we found no evidence for M11 contributing to MHV-68 lytic replication in vitro. This also applied to lytic replication in infected lungs. Although M11 can inhibit the apoptosis of fibroblasts when expressed from a plasmid (Wang et al., 1999
; Roy et al., 2000
; Bellows et al., 2000
), this did not appear to be important in the context of viral infection, either because M11 is not expressed at a sufficient level or because MHV-68 relies mainly on other inhibitors of apoptosis during lytic-cycle replication. No MHV-68 apoptosis inhibitors, apart from M11, have been identified, but the abundant precedents in other herpesviruses would argue that additional inhibitors are inevitable.
In contrast to Gangappa et al. (2002), we found no evidence for ex vivo viral reactivation being dependent on M11. This discrepancy presumably reflects different in vitro assay conditions. It is important to note that explanted murine B cells generally show poor viability, which may be why they reactivate MHV-68 much less efficiently than dendritic cells or macrophages (Marques et al., 2003
). Clearly, any gene that prolongs the survival of latently infected cells could potentially increase the efficiency of ex vivo reactivation. The question is whether the B-cell apoptosis that would otherwise occur is relevant to viral reactivation in vivo. Our data do not rule out an important role for M11 in reactivation in vivo no current assays measure this process directly but, instead, they identify an M11-associated deficit in latency amplification. Such a deficit would presumably also reduce the capacity of MHV-68 to cause chronic disease (Gangappa et al., 2002
).
The lymphoproliferative amplification of MHV-68 latency is proving to be highly complex. Key roles have been identified for the viral ORF73 episome-maintenance protein (Fowler et al., 2003; Moorman et al., 2003
), M2 [a protein of unknown function (Jacoby et al., 2002
; Macrae et al., 2003
; Simas et al., 2004
)] and MK3, a CD8+ T-cell evasion protein (Stevenson et al., 2002
). There may also be a role for the M3 chemokine-binding protein (Bridgeman et al., 2001
), although this remains controversial (van Berkel et al., 2002
). Our data indicate that normal latency amplification also requires M11.
An M11-associated latency deficit was possibly not observed by Gangappa et al. (2002) because they used intraperitoneal infection, which bypasses normal mucosal barriers. Crucially, intraperitoneal virus reaches the spleen when there is little immunity to lytic antigens, allowing lytic viral spread between B cells (Weck et al., 1996
) and less dependence on latency amplification. After intranasal infection, the initial epithelial infection triggers an immune response and infectious virus remains essentially undetectable in lymphoid tissue (Sunil-Chandra et al., 1992
). The natural route of MHV-68 infection is unknown, but the intranasal route does at least reproduce the requirement for mucosal infection to precede lymphoid infection. Notably, M2-deficient MHV-68 shows a defect in latency amplification after intranasal, but not after intraperitoneal, infection (Jacoby et al., 2002
).
An obvious function for M11 would be to promote germinal-centre B-cell survival, as germinal-centre B cells are both a major site of MHV-68 latency (Bowden et al., 1997; Flaño et al., 2002
; Marques et al., 2003
; Willer & Speck, 2003
) and a site of action of pro-apoptotic and anti-apoptotic cellular bcl-2 family members (Marsden & Strasser, 2003
). The marked reduction in the prevalence of viral genome-positive germinal-centre B cells in the absence of M11 (Figs 6d and 7
) was consistent with such a function. However, evidence for M11 transcription in germinal-centre B cells is limited (Marques et al., 2003
). M11 could act instead in latently infected B cells before they acquire a germinal-centre phenotype. Germinal-centre B cells themselves may no longer require M11 if MHV-68, like EBV, induces cellular bcl-2 expression. M11 has an advantage of over cellular bcl-2 in having no pro-apoptotic caspase-cleavage products (Bellows et al., 2000
), but in germinal-centre B cells, there may be a strong, counterbalancing selective pressure to maintain immunological silence by minimizing viral gene expression.
Fas is also a major mediator of germinal-centre B-cell apoptosis (Guzman-Rojas et al., 2002). Despite the predictions of in vitro data (Wang et al., 1999
), M11 did not appear to act by Fas inhibition in vivo (Fig. 8
), which may be consistent with the lack of M11 mRNA in germinal-centre B cells. MHV-68 lacks a v-FLIP, but may encode an as-yet-unidentified inhibitor of Fas. Alternatively, MHV-68 may act in dendritic cells and macrophages (Marques et al., 2003
) to indirectly enhance the survival of MHV-68-infected germinal-centre B cells.
Despite the normal long-term level of splenic latency that is seen in the absence of M11, it seems unlikely that defects in latency amplification have no impact on viral fitness. For example, latency amplification may promote viral seeding to biologically important sites of persistence perhaps submucosal lymphoid tissue or bone marrow that have not yet been identified. Primary infection represents a unique opportunity for herpesviruses to establish a large latent pool for subsequent reactivation and transmission, and one that they evidently exploit to the full. The data presented here indicate that the MHV-68 viral bcl-2 homologue plays an important role in this process.
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ACKNOWLEDGEMENTS |
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Received 29 July 2004;
accepted 5 October 2004.
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