1 Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
2 Instituto Gulbenkian de Ciencia, 2780-156 Oeiras, Portugal
3 Laboratory of Microbiology, Faculty of Medicine, University of Lisbon, 1649-028 Lisbon, Portugal
Correspondence
Stacey Efstathiou
se{at}mole.bio.cam.ac.uk
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ABSTRACT |
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INTRODUCTION |
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From in vitro studies, it is clear that the trans-acting plasmid maintenance proteins encoded by gammaherpesviruses are essential for the stable maintenance of viral genomes during latency. However, the contribution of virus-encoded maintenance functions to virus persistence in vivo is not known. To directly address this question, we have focussed our studies on murine gammaherpesvirus-68 (MHV-68) because the use of this virus enables the study of gammaherpesvirus pathogenesis in an amenable murine host (Simas & Efstathiou, 1998; Nash et al., 2001
; Flano et al., 2002a
). MHV-68 is a natural pathogen of small rodents (Blaskovic et al., 1980
; Blasdell et al., 2003
). Following intranasal infection, MHV-68 replicates in respiratory epithelial cells (Sunil-Chandra et al., 1992
) and establishes latency in lymphoid tissues. Latency is established predominantly in B cells (Marques et al., 2003
) but can also be established in macrophages, dendritic and epithelial cells (Stewart et al., 1998
; Weck et al., 1999
; Flano et al., 2000
). Following infection there is a marked expansion of latently infected B cells in lymphoid germinal centres (Simas et al., 1999
), which allows MHV-68 to gain access to the long-lived memory B cell pool (Flano et al., 2003
). The expansion of latency also drives an infectious mononucleosis-like illness, which peaks 1 month post-infection (p.i.) and is characterized by splenomegaly and lymphocyte activation (Doherty et al., 2001
). Real-time PCR analyses have revealed a restricted pattern of gene expression within B cells during latency (Marques et al., 2003
), which includes transcription of ORF73 in the dividing germinal centre B cell fraction. This observation is consistent with the demonstration that the ORF73 gene products encoded by KSHV and HVS have a latency-associated plasmid maintenance function analagous to that of the EBV-encoded EBNA-1 protein (Ballestas et al., 1999
; Collins et al., 2002
; Grundhoff & Ganem, 2003
). Based on positional homology amongst the gamma-2 herpesviruses, ORF73 genes and C-terminal structural similarity shared between the EBV EBNA-1 and a number of gamma-2 herpesvirus ORF73 gene products (Grundhoff & Ganem, 2003
), it is predicted that ORF73 of MHV-68 is likely to encode a plasmid maintenance function. On this basis, we have constructed and characterized two MHV-68 mutants containing disruptions of the ORF73 gene product in order to determine its role in gammaherpesvirus pathogenesis and to determine whether a gammaherpesvirus defective for episome maintenance can persist in vivo.
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METHODS |
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Recombinant viruses.
ORF73 was manipulated from genomic clone BamG (Efstathiou et al., 1990) (genomic co-ordinates, 101654106903; Virgin et al., 1997
) to make two independent mutations. A frameshift mutation was introduced in the BamG fragment in the vector pACYC184 (New England Biolabs) by digestion with BstEII (genomic co-ordinate, 104379). Terminal 5' overhangs were end-repaired and religated, resulting in the introduction of five novel bases, GTGAC. An independent mutation was made by digestion of BamG in the vector pSP73 (Promega) with BstEII/Bsu36I (New England Biolabs). A 450 bp fragment (genomic co-ordinates, 104379104830) was removed. The BamG fragments mutated in the ORF73 region were excised from their respective vectors using BamHI digestion and gel purification. The gel-purified inserts were then subcloned into the BamHI site of the shuttle vector pST76K_SR (Adler et al., 2000
). BamG fragments were sequenced across the ORF73 region and the integrity of the mutations confirmed. pST76K_SRBamGFS73 and pST76K_SRBamG
73 were then transformed into Escherichia coli strain DH10B containing the MHV-68 BAC (bacterial artificial chromosome) pHA3 (Adler et al., 2000
). Following a multi-step selection procedure, as described previously (Adler et al., 2000
; Messerle et al., 1997
), recombinant BAC clones were selected by restriction enzyme digestion. Revertant viruses were made for both independent mutations using wild-type pST76K_SRBamG. This plasmid was then transformed into E. coli strain DH10B containing the MHV-68 ORF73-mutated BACs. The multi-step recombination procedure was performed as above and recombinant clones screened for wild-type restriction digestion profiles. Mutant and revertant MHV-68 viruses were reconstituted by transfection of BAC DNA into BHK-21 cells. The BAC cassette (flanked by Lox-P sites) was removed by passage through NIH 3T3-Cre cells and limiting dilution to obtain GFP-negative virus. After excision of BAC vector sequences, virus structures were confirmed by restriction enzyme digestion and Southern blot hybridization.
Mice and in vivo infections.
BALB/c mice (Harlan OLAC) were maintained in accordance with UK Home Office guidelines (project licences 80/1378 and 80/1579) at the University of Cambridge, UK, or at the Gulbenkian Institute of Science, Portugal. Mice were infected intranasally with 104 p.f.u. of virus in 20 µl PBS with 1 % FCS after halothane anaesthesia. Intraperitoneal infections used 105 p.f.u. of virus in a volume of 50 µl PBS with 1 % FCS. Infectious virus was assayed on BHK-21 cells following homogenization and freezethaw disruption of tissue. Latent virus from the spleen was assessed by infectious centre assay (Sunil-Chandra et al., 1992). For this assay, single-cell suspensions of splenocytes were co-cultured with BHK-21 cells for 45 days. Monolayers were then fixed with 10 % formal saline and stained with toluidine blue. Plaques were counted using a plate microscope.
In situ hybridization.
Viral tRNAs 14 were detected by in situ hybridization as a marker for latency (Bowden et al., 1997). As described previously (Arthur et al., 1993
), paraffin-embedded spleen sections were de-waxed in xylene, rehydrated through a graded series of ethanol and treated with 100 µg proteinase K ml-1 for 10 min at 37 °C, followed by acetylation with 0·25 % (v/v) acetic anhydride and 0·1 M triethanolamine. Digoxigenin (DIG, Boehringer Mannheim) labelled riboprobes corresponding to MHV-68 viral tRNAs were made by T7 transcription of pEH1.4 (Bowden et al., 1997
). Sections were hybridized with the DIG-labelled riboprobes in 50 % formamide and 1x SSC overnight at 55 °C. The stringent wash was carried out at 58 °C (0·1x SSC, 30 % formamide and 10 mM Tris, pH 7·5). Hybridized probe was detected with alkaline phosphatase-conjugated anti-DIG Fab fragments (Boehringer Mannheim), according to the manufacturer's instructions.
Limiting dilution analysis on different splenocyte populations.
Analysis of virus load in sorted splenocyte populations was carried out as described previously (Marques et al., 2003). Briefly, single-cell suspensions of three to five pooled spleens were prepared per virus, per time-point, and passed through a filter (100 µM). Suspensions were washed in PBS with 2 % FCS and red blood cells were lysed (154 mM ammonium chloride, 14 mM sodium hydrogen carbonate and 1 mM EDTA, pH 7·3). Single-cell suspensions were surface-stained with the following monoclonal antibodies (Pharmingen) and lectins (Vector Laboratories): anti-CD19, anti-CD11b, anti-CD11c, anti-B220 (CD45R) and peanut agglutinin (PNA). A MoFlo cytometer (Cytomation) was used to enrich the following populations: CD19+ for total B cells, CD19+ and PNAhi for germinal centre B cells, B220- and CD11c+ for dendritic cells, and B220-, CD11b+ and CD11c- for macrophages. Sorted populations were analysed using a FACScan flow cytometer and data processed using CellQuest software (Becton Dickinson Immunocytometry Systems). Purities of sorted populations were consistently >95 % and predominantly >98 %.
FACS-purified cell suspensions were serially diluted twofold and eight replicates of each dilution were lysed overnight (0·45 % Tween-20, 0·45 % Nonidet P-40, 2 mM MgCl2, 50 mM KCl, 10 mM Tris, pH 8·3, and 0·5 mg proteinase K ml-1) at 37 °C. After proteinase K inactivation at 95 °C for 5 min, samples were analysed by real-time PCR for the presence of viral DNA. Real-time PCR was performed using a LightCycler from Roche, according to the manufacturer's instructions. Primer/probe sets specific for the MHV-68 K3 gene were used (genome co-ordinates: upper primer, 2483224850; FL-probe, 2497324998; LC-probe, 2500025026; lower primer, 2504925071). The FL-probe is an oligonucleotide labelled at the 3' end with fluorescin (Roche) and the LC-probe is labelled at the 5' end with LC-Red fluorophore and modified at the 3' end by phosphorylation (Roche). Each PCR reaction had a final volume of 10 µl [2 mM MgCl2, 4 ng each primer µl-1, 0·2 µM each internal probe, 1x DNA mixture (Roche) and 1 µl cell lysate]. Amplification used the following programme: melting step of 95 °C for 10 min followed by 45 cycles of 95 °C for 10 s, 55 °C for 10 s and 72 °C for 20 s with a subsequent melting analysis step of 5095 °C at 0·1 °C s-1.
Limiting dilution analysis data were compatible with the single-hit Poisson model (SHPM). This was tested by modelling the data to a generalized linear log-log model fitting the SHPM and checking this model by an appropriate slope test (Bonnefoix et al., 2001). A regression plot of input cell number against log fraction-negative samples was used to estimate the frequency of cells containing viral genomes.
Gardella gels.
Vertical Gardella gels were performed as described (Gardella et al., 1984). NSO cells (25x105) were resuspended in 100 µl sample buffer (15 % Ficoll in TBE, 0·01 mg RNase A ml-1 and 0·01 % bromophenol blue). Cell suspensions were loaded into wells of a 0·8 % TBE agarose gel. A 100 µl volume of lysis buffer (5 % Ficoll, 1 % SDS, 100 µg proteinase K ml-1 and 0·05 % xylene cyanol green) was then overlaid in the sample wells. Samples were electophoresed at 12 V for 35 h and then overnight at 4 °C at 80 V. Gels were stained with ethidium bromide and analysed by Southern blot hybridization.
Southern blot hybridization.
Viral DNA was analysed by Southern blot analysis as described (Bridgeman et al., 2001). In brief, 107 virus-infected cells were resuspended in 500 µl TE (10 mM Tris and 50 mM EDTA, pH 8) and lysed with 0·5 % SDS and 50 µg proteinase K ml-1 at 37 °C overnight. DNA was then purified by phenol/chloroform extraction and ethanol precipitation. For Southern blot analyses, 5 µg viral DNA was digested with an appropriate restriction enzyme and electrophoresed on a 0·8 % agarose Tris/acetate (TAE) gel. DNA was then transferred to a nitrocellulose membrane and probed with a [
-32P]dCTP random prime-labelled fragment; for analysis of ORF73-mutated MHV-68 clones, an ORF73-specific probe was used and Gardella gels were probed with a 1·2 kb Pst repeat fragment.
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RESULTS |
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DISCUSSION |
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In contrast to the relatively normal replication kinetics of ORF73-deficient viruses in BHK cells and lungs of acutely infected mice, these mutants were severely disabled for the establishment of latency in the spleen. Following intranasal infection with either wild-type MHV-68 or 73R, infectious centres could be recovered from the spleen at 7 days p.i., peaked at 14 days p.i. and reached a stable level of approximately 800 infectious centres per spleen thereafter. Following infection with
73, despite the relatively normal seeding of the spleen at 7 days p.i., the characteristic amplification of infectious centres was not observed at 14 days p.i. and by day 21 p.i. infectious centres were not detectable. The critical role of ORF73 in latency was verified using an independently derived frameshift mutant, FS73, administered by the intranasal route and a similar reactivation deficit was observed for both mutants following intraperitoneal injection of virus, a route of infection that efficiently targets virus to the spleen. These data strongly argue that the observed reactivation deficit of the ORF73-deficient mutants is not due to a failure of these viruses to seed the spleen. Given the proposed episome maintenance function of ORF73, we considered it most likely that the failure to detect infectious centres was a failure to efficiently establish latency rather than a deficit in the ability of the mutants to reactivate from latency. To distinguish between an establishment versus a reactivation deficit, the latent virus load was first evaluated by in situ hybridization detection of vtRNA-positive cells (Bowden et al., 1997
). This measure of latency confirmed that the deficit in recovery of infectious centres from the spleen was due to an inability of the ORF73-deficient viruses to colonize germinal centres and thereby efficiently establish latency. This conclusion was supported by determining the genomic DNA load in FACS-purified splenocyte cell populations. Limiting dilution analysis and real-time PCR were used to determine the frequency of genome-positive cells in the following sorted splenocyte populations: total B cells (CD19+), germinal centre B cells (CD19+ and PNAhi), macrophages (B220-, CD11b+ and CD11c-) and dendritic cells (B220-, CD11c+). Wild-type and revertant viruses were able to establish high frequency infection in these cell populations, as described previously (Marques et al., 2003
; Flano et al., 2002b
; Willer & Speck, 2003
). In contrast, no viral genome-positive cells were detected in any of the splenocyte populations derived from ORF73-deficient virus-infected mice. Here, a frequency of less than 1 in 1·5x106 germinal centre B cells, total B cells or macrophages and less than 1 in 7·5x105 dendritic cells (based on the maximum number of cells analysed) was determined at 14 days p.i. Viral genome-positive cells were not detected in total B cells or germinal centre B cells sampled at 21 days p.i., suggesting that the latency deficit was not the result of delay in latent virus amplification. These phenotypes are consistent with the predicted role of ORF73 in plasmid maintenance and imply a critical role for this function in the context of an in vivo infection. Using a variety of independent measures of latency, the most consistent picture to emerge from this study is that ORF73 plays an essential role in host colonization by MHV-68.
At this stage, we cannot formally discount the possibility that the mutations introduced have influenced cis-acting sequences required for virus latency that overlap ORF73 by coincidence. However, we consider it most likely that the phenotypes observed are due to disruption of ORF73, since similar effects are observed with both a deletion and a subtle frameshift mutation. Furthermore, the phenotypes observed are consistent with the predicted role of this gene product in plasmid maintenance. The role of the neighbouring genes, ORF72 and ORF74, have been studied in vivo and neither gene shows a latency phenotype such as the one described in the present study (van Dyk et al., 2000, 2003
; Lee et al., 2003
). It is unlikely therefore that the mutations introduced within ORF73 have influenced neighbouring genes that could have contributed to the phenotype observed. The phenotype of the ORF73-deletion mutant was also confirmed by a subtle frameshift mutation and respective revertant viruses had fully restored wild-type phenotypes. It is, therefore, improbable that secondary mutations might have occurred to influence the phenotype of the ORF73-deficient viruses as described here.
KSHV LANA is a multifunctional protein and has been shown to interact with multiple cellular factors as well as having transcription modulatory activity (Garber et al., 2001; Renne et al., 2001
; Mattsson et al., 2002
; Viejo-Borbolla et al., 2003
). Of particular importance to this study, LANA interacts with histone H1 to mediate tethering of the virus episome to host cell chromosomes (Ballestas et al., 1999
; Cotter & Robertson, 1999
). However, interactions have also been shown between LANA and the tumour suppressor protein p53 (Friborg et al., 1999
) as well as the retinoblastoma protein RB1 (Radkov et al., 2000
). These studies implicate a role for LANA in virus persistence and cell transformation. To date, interactions of ORF73 of MHV-68 with cellular factors have not been dissected, although ORF73 of MHV-68 does share sequence similarity with KSHV LANA at the C terminus, where multiple functions of the protein have been mapped; these functions include DNA binding, dimer formation, transcriptional repression, localization in the nucleus and protein interactions to mediate chromosome binding (Schwam et al., 2000
; Krithivas et al., 2002
; Garber et al., 2002
; Grundhoff & Ganem, 2003
). Further confirmation of the importance of the C terminus is shown in this study; the FS73 mutant encodes 163 amino acids of the N terminus in-frame (approximately the entire N-terminal half of the 314 amino acid protein). This mutant has the same phenotype as the deletion mutant, which only encodes the first 13 amino acids of the N terminus in-frame, suggesting that it is the common deletion of the C terminus that is responsible for the phenotype of the mutant viruses. While the present study suggests that the phenotype observed is consistent with a plasmid maintenance function of ORF73, other functions of the protein that may contribute to the latency phenotype cannot be ruled out. Further analysis of particular functional domains of ORF73 are needed to ascribe particular roles to regions of the protein and this would enable more specific mutations to be made and their contributions to the pathogenesis of MHV-68 assessed. Additionally, for EBV, KSHV and HVS, it has been shown that EBNA-1 and LANA interact with virus origins to support latent replication, and in HVS and KSHV these virus origins map to the terminal repeats (Reisman et al., 1985
; Ballestas & Kaye, 2001
; Collins et al., 2002
). KSHV, HVS and MHV-68 are collinear (Virgin et al., 1997
) and based on such homology it is predicted that the origin of plasmid replication of MHV-68 could also be found in the terminal repeats. Thus, further studies are needed to define the elements of MHV-68 that are both necessary and sufficient for plasmid maintenance.
The data presented in this study are consistent with the prediction that ORF73 of MHV-68 is involved in plasmid maintenance. ORF73 appears to be dispensable for acute phase replication of MHV-68, while, conversely, it is critical for the establishment of latency and hence efficient colonization of the host. As such, it is possible that by abrogating the function of this latency-associated antigen, this latency-deficient virus may provide a novel and safe basis for a gammaherpesvirus vaccine.
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NOTE ADDED IN PROOF |
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ACKNOWLEDGEMENTS |
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Received 22 August 2003;
accepted 22 September 2003.