Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
Correspondence
Philip Stevenson
pgs27{at}mole.bio.cam.ac.uk
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ABSTRACT |
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INTRODUCTION |
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The expression of a key viral transactivator, for example BZLF-1 of EpsteinBarr virus (EBV), is generally sufficient to initiate the lytic cycle of a gammaherpesvirus. The promoters of gammaherpesvirus transactivators are therefore silenced in latently infected cells until turned on by as yet poorly defined extracellular signals (Binne et al., 2002). The key lytic cycle transactivator in gamma-2-herpesviruses is encoded by ORF50 (Sun et al., 1998
). ORF50 transcription is both necessary and sufficient to drive the entire lytic cycle of either the Kaposi's sarcoma-associated herpesvirus (KSHV) (Gradoville et al., 2000
; Lukac et al., 1999
) or the related murine gammaherpesvirus-68 (MHV-68) (Wu et al., 2000
, 2001
). Silencing of the KSHV ORF50 promoter contributes to the maintenance of latency in vitro (Chen et al., 2001
). Here we have used MHV-68 to study the role of ORF50 transcriptional control in host colonization.
MHV-68 is a natural pathogen of small rodents (Blaskovic et al., 1980), including Apodemus sylvaticus (field mice) (Blasdell et al., 2003
). After intranasal infection of inbred strains of laboratory mice (Mus musculus/domesticus), MHV-68 replicates lytically in respiratory epithelial cells and then establishes life-long latency in lymphoid tissue. As with EBV and KSHV, B cells provide the principal latent reservoir of MHV-68 (Sunil-Chandra et al., 1992a
). MHV-68 can also be latent in macrophages (Weck et al., 1999
), epithelial cells (Stewart et al., 1998
) and dendritic cells (Flano et al., 2000
), and still persists in B cell-deficient mice. However, this persistence seems to reflect chronic lytic infection more than the establishment of a stable latent viral reservoir (Gangappa et al., 2002
). Latency in B cells is therefore crucial to normal host colonization. Another feature MHV-68 shares with the human gammaherpesviruses is its capacity to drive B cell proliferation. There is a massive expansion of latently infected B cell numbers in lymphoid germinal centres after primary infection (Simas & Efstathiou, 1998
), which probably allows MHV-68 to disseminate to sites such as the bone marrow (Sunil-Chandra et al., 1992b
) and to establish a latent reservoir for life-long persistence. The latency expansion also drives an infectious mononucleosis-like illness, characterized by lymphadenopathy, splenomegaly and widespread lymphocyte activation (Doherty et al., 2001
).
MHV-68 infection of conventional mice is an established model of gammaherpesvirus pathogenesis. Thus by defining the functions that contribute to MHV-68 lymphocyte proliferation and persistence in vivo, we can identify potential therapeutic targets in the clinically important gammaherpesvirus infections of humans and the economically important gammaherpesvirus infections of domesticated ungulates. We have previously used the deletion of immune evasion genes to generate MHV-68 mutants with a limited capacity for latency amplification (Bridgeman et al., 2001; Stevenson et al., 2002
). However, the equivalent immune evasion functions in other gammaherpesviruses are not always obvious, perhaps limiting the general applicability of this approach to vaccine design. Consequently, we have now used a more general strategy of attenuation, and deregulated the transcription of the MHV-68 ORF50 (Liu et al., 2000
) by inserting a truncated murine cytomegalovirus (MCMV) IE1 promoter (Dorsch-Hasler et al., 1985
) in its 5'-untranslated region. Our aim has been to force entry into the viral lytic cycle and to determine the effect this has on host colonization. In the accompanying paper (Boname et al., 2004
) we have explored the vaccine potential of the deregulated virus.
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METHODS |
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Cell lines.
Baby Hamster Kidney cells (BHK-21) (ATCC CCL-10), L929 cells (ATCC CCL-1), NIH-3T3 cells (ATCC CRL-1658), the cre-expressing derivative NIH-3T3-CRE (Stevenson et al., 2002) and murine embryonic fibroblasts (MEFs) were all grown in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 2 mM glutamine, 100 U penicillin ml-1, 100 µg streptomycin ml-1 and 10 % fetal calf serum (PAA Laboratories) (complete medium).
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 on MEF monolayers as described previously (Stevenson et al., 2002). After 6 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 splenocyte suspensions on MEF monolayers, which were fixed and stained after 7 days.
Viral mutagenesis.
A portion of the MCMV IE1 promoter/enhancer (genomic co-ordinates -380 to +36 relative to the IE1 transcription start site) (Dorsch-Hasler et al., 1985) was amplified from MCMV genomic DNA using Pfu polymerase (Promega). We chose the MCMV IE1 promoter because it is highly active in a range of murine cells (Addison et al., 1997
) and because it has evolved to function in the context of a viral genome. We chose a truncated form because the upstream enhancer region does not seem to improve transcription (Addison et al., 1997
) some upstream elements in the equivalent human cytomegalovirus IE1 enhancer may even mediate silencing (Liu et al., 1994
). We also wanted to minimize any disruption of the MHV-68 genome. Primers were designed to include BsgI restriction sites at each end. The PCR product was then cloned into the BsgI site of the BamN genomic clone in pUC19 (Efstathiou et al., 1990
) (genomic co-ordinates 64 76568 813) (Virgin et al., 1997
). The mutant BamN fragment was subcloned into the shuttle vector pST76K-SR and inserted into the MHV-68 BAC by transient RecA-mediated recombination (Adler et al., 2000
). A revertant virus, with the unmutated BamN fragment recombined into the BAC in place of the mutant form, was then made in a similar way. A control mutant virus was made by inserting part of an MHV-68 intron upstream of ORF73 (genomic co-ordinates 105 095104 879; H. M. Coleman & P.G. Stevenson, unpublished data) into the same site.
To make an ORF48-deficient virus, we cloned a ClaI genomic fragment (genomic co-ordinates 62 12669 177) into pUC19 and removed part of ORF48 (65 58166 582) by digestion with PpuMI (65991) and AflII (66463), in-filling with Klenow fragment DNA polymerase and religation. The mutant ClaI fragment was excised, in-filled and subcloned into the SmaI site of pST76K-SR, followed by RecA-mediated recombination into the MHV-68 BAC as above. Infectious virus was reconstituted from each BAC by transfecting 5 µg BAC DNA into NIH-3T3 cells with Fugene-6 (Roche Diagnostics). The loxP-flanked BAC/GFP cassette was then removed by viral passage through NIH 3T3-CRE cells until GFP+ cells were no longer visible. The identity of each recombinant virus was confirmed by DNA sequence analysis of a PCR product spanning the mutation site. Viral stocks were grown and titrated in BHK-21 cells.
Flow cytometry.
Spleens were disrupted into single-cell suspensions, washed in PBS/0·1 % BSA/0·01 % azide and incubated for 15 min on ice with 5 % mouse serum, 5 % rat serum and anti-CD16/32 mAb. Specific staining was with phycoerythrin (PE)-conjugated anti-CD8 and fluorescein isothiocyanate (FITC)-coupled anti-TcR V4, anti-CD69-FITC and anti-CD19-PE, or anti-CD62L-PE (all from BD-Pharmingen), anti-CD4-FITC (Serotec), and anti-CD8-tricolor (Caltag Laboratories). After 1 h on ice, unbound antibody was removed by washing twice in PBS/BSA (0·1 %)/azide (0·01 %) and cells were analysed on a FACS Calibur using Cellquest software (BectonDickinson). Data were graphed with FCSPress v1.3 (www.fcspress.com). For flow cytometric sorting, red cells were removed by flash lysis in water and spleen cell suspensions were stained with anti-CD19-PE and anti-I-Ab-FITC (Serotec). After washing, CD19+I-Ab+ and CD19-I-Ab+ populations were separated using a FACStar plus (BectonDickinson).
Analysis of viral RNA.
RNA was extracted from MHV-68-infected cells with RNAzol-B (Tel-Test Inc.). Samples were then processed for Northern blotting or cDNA synthesis. For Northern blotting, total RNA was electrophoresed (5 µg per lane) on a 1 % formaldehyde agarose gel and blotted overnight onto positively charged nylon membranes (Roche Diagnostics). Probes for -actin, ORF48, ORF49, ORF50 and M7 were generated by PCR of either cellular cDNA or MHV-68 BAC DNA, gel-purified and random-prime-labelled (Qbiogene) with [32P]dCTP (Amersham Biosciences). Blots were washed (0·2x SSC, 0·1 % SDS, 65 °C) and exposed to X-ray film. For cDNA synthesis, any contaminating DNA was first removed with RNase-free DNase (Promega). For the detection of ORF48 and ORF49 transcripts, cDNA was synthesized with AMV reverse transcriptase (Promega) using an oligo-dT primer and amplified with Taq polymerase (Amersham Biosciences), according to the manufacturers' instructions. The primers used corresponded to genomic co-ordinates 66 58966 565 and 65 58165 604 for ORF48, and 67 66067 639 and 66 73566 753 for ORF49. Reactions were run for 30 cycles with an annealing temperature of 55 °C. To identify the ORF50 transcription start site we used 5' RACE (5'/3' RACE kit; Roche Diagnostics), according to the manufacturer's instructions. RNA was reverse-transcribed with a primer corresponding to genomic co-ordinates 68 05068 030, a poly-dA tail was added to the 5' end with terminal transferase and the product was amplified with a tail-specific primer and primers corresponding to ORF50 exon 2 (genomic co-ordinates 68 02668 005 or 67 99767 976).
Analysis of viral DNA.
For Southern blot analysis of viral genomes, DNA was purified from infected BHK-21 cells by Proteinase K digestion, phenol/chloroform extraction and salt/ethanol precipitation. DNA (5 µg) was restriction digested, electrophoresed, transferred onto Hybond nylon filters and hybridized to a probe prepared by random-primed [32P]dCTP labelling of the BamN genomic fragment. Filters were washed (0·2x SSC, 0·1 % SDS, 65 °C) and exposed to X-ray film. For PCR detection of viral genomes, DNA was purified from tissue fragments using the Wizard DNA genomic purification kit (Promega). We used either Taq polymerase to amplify part of the viral M7 gene (genomic co-ordinates 69 52669 913) over 30 cycles, or AmpliTaq Gold (Roche Diagnostics) to amplify part of ORF57 (genomic co-ordinates 75 84176 202) over 50 cycles. The latter was sufficient for single-copy template detection. All PCR products were visualized by agarose gel electrophoresis and ethidium bromide staining.
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 previously described (Bridgeman et al., 2001). Bound probe was detected with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Boehringer Ingelheim) according to the manufacturer's instructions.
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RESULTS |
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The M50 virus recovered from the spleen at days 5 and 7 of infection (Fig. 4A) was apparently latent, since no pre-formed infectious virus was detected in freeze-thawed spleen cells. However, extrapolating a single-cell definition of latency to a whole organ virus recoverable from live but not from killed cells is problematic with low levels of virus. For example, a rapid uptake of new virions by uninfected spleen cells a short half-life of infectious particles rather than latency could also have accounted for the absence of pre-formed infectious virus. In situ hybridization of mediastinal lymph nodes for viral tRNA expression 7 days after infection showed no evidence of the M50 infection compared to WT and M50R controls (Fig. 5
). Thus, by this marker of latency (Bowden et al., 1997
), M50 lymphoid colonization was below detectable limits even at its peak.
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In addition to achieving lower infectious centre titres, the M50 virus caused little of the splenomegaly (not shown) or virus-driven T cell and B cell activation and CD8+V4+ T cell expansion (Fig. 6
) that normally characterize the MHV-68-associated infectious mononucleosis illness. Since it is the amplification of latent virus that drives this host immune activation (Doherty et al., 2001
), the relative lack of immune activation was further evidence that the M50 virus was unable to maintain normal latency.
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DISCUSSION |
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Even though MHV-68 fairly readily enters the lytic cycle in in vitro cell lines, it was evident that ORF50 availability is still a limiting factor. Clearly a similar modification of the EBV BZLF-1 or KSHV ORF50 promoters may be one means of generating an in vitro lytic replication system for these viruses as well. Up-regulating ORF50 production presumably increased the probability of initiating lytic infection rather than speeding up the lytic cycle itself, which is likely to be limited by substrate supply for virion assembly. The fact that up-regulating ORF50 transcription reduced rather than increased lytic spread in vivo argued that here exogenous host factors are the dominant influence on virus replication, even before the onset of adaptive immunity (Fig. 4a, days 35). The interferon system is one likely candidate (Dutia et al., 1999
).
The insertion of self-contained expression cassettes into the left end of the MHV-68 genome can cause latency-associated virus attenuation (Jacoby et al., 2002; Adler et al., 2001
), perhaps as a consequence of driving the high-level expression of reporter genes (
-galactosidase or green fluorescent protein) in latently infected cells. However, two viruses with left-end lacZ expression cassettes have established normal latency (Simas et al., 1998
) and a green fluorescent protein expression cassette inserted into the MHV-68 K3 ORF caused no more attenuation than a small non-coding insertion in the same site (Stevenson et al., 2002
). Thus another possible explanation for the attenuating effect of certain insertions is that they disrupt specific viral functions at the left end of the genome. Even this attenuation was much less severe than seen with the M50 virus a latency reduction of one rather than three or four orders of magnitude. Thus it seemed unlikely that the MCMV IE1 promoter insertion caused attenuation by a non-specific effect of increased transcription.
The incapacity of the M50 virus to amplify latently infected B cell numbers was presumably due to the MCMV IE1 promoter maintaining ORF50 transcription and hence continually driving lytic virus replication. This would be consistent with the increased ORF50 transcription and lytic replication observed with the M50 virus in vitro. We were not able to define directly the in vivo behaviour of the M50 MCMV IE1 promoter fragment because so few infected lymphoid cells were ever found. However, the selective reduction of viral DNA in B cells and fact that the M50 virus inevitably lysed NS0 cells supported the idea of destabilized B cell latency.
The presence of low levels of M50 viral DNA in spleens (Fig. 7b) raised the possibility that the M50 virus was perhaps still able to establish some kind of latency, perhaps in myeloid cells (Fig. 7c
). However, if so it is difficult to see why it did not reactivate in vitro after day 10 of infection. WT MHV-68 reactivates relatively efficiently from macrophages and dendritic cells (Marques et al., 2003
), and transcription from the native ORF50 promoter should not have been compromised in the M50 virus: the MCMV IE1 promoter element would simply have extended the mRNA 5' untranslated region. An alternative explanation for the viral DNA detected at low levels by PCR is that this was non-functional genomic debris, left over after the immune clearance of infected cells. Regardless of whether the M50 virus persisted at all in a viable form, the key point is that it failed to drive in vivo B cell proliferation, the pathological process usually associated with gammaherpesvirus-associated tumours. Thus we could define with MHV-68 the role of a fundamental gammaherpesvirus function lytic cycle repression in pathogenesis, and so demonstrate a possible general means of gammaherpesvirus attenuation. In the accompanying paper (Boname et al., 2004
) we report the capacity of infection with the M50 virus to protect against a subsequent WT MHV-68 challenge.
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ACKNOWLEDGEMENTS |
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Received 22 August 2003;
accepted 10 October 2003.