1 Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal
2 Laboratory of Microbiology, Faculty of Medicine, University of Lisbon, Portugal
3 Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
4 GSF-Research Center for Environment and Health, Institute of Molecular Immunology, Clinical Cooperation Group Hematopoietic Cell Transplantation, Munich, Germany
Correspondence
J. Pedro Simas
jpsimas{at}igc.gulbenkian.pt
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ABSTRACT |
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INTRODUCTION |
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Following intranasal inoculation of mice with MHV-68, B lymphocytes constitute the principal target of latent infection (Flano et al., 2002a, 2003
; Marques et al., 2003
; Willer & Speck, 2003
). The establishment of latent infection is characterized by a marked transient expansion of infected germinal centre (GC) B cells in the spleen that peaks at 14 days post-infection (Flano et al., 2002a
; Marques et al., 2003
; Simas et al., 1999
) followed by the development of an infectious mononucleosis-like disease (Tripp et al., 1997
). Later, long-term latent infection is preferentially maintained in GC B cells (Flano et al., 2002a
; Marques et al., 2003
; Willer & Speck, 2003
) and memory B cells (Flano et al., 2002a
; Willer & Speck, 2003
). In a manner similar to EBV (Thorley-Lawson, 2001
), infection of GC B cells by MHV-68 might reflect a strategy to gain access to long-lived memory B cells. Hence, central for an understanding of MHV-68 pathogenesis is the identification of genes expressed during the establishment of latent infection in GC B cells. A recent study has revealed a restricted pattern of virus transcription within GC B cells during the establishment of latency in the spleen, which includes transcription of ORF M2 (Marques et al., 2003
). The M2 gene product encoded by MHV-68 represents a unique protein of unknown function (Virgin et al., 1997
). It has been shown that M2 contains a CD8+ T-cell epitope that is actively recognized, constituting an important target for controlling the establishment of latent infection (Husain et al., 1999
; Usherwood et al., 2000
, 2001
). Although, the function of M2 still remains elusive, two independent studies have investigated the role of this latency-associated gene in replication and pathogenesis (Jacoby et al., 2002
; Macrae et al., 2003
). These studies demonstrated that M2-deficient mutants replicate normally in tissue culture and display normal acute phase replication kinetics in vivo. Nonetheless, consistent with the expression of this gene during the establishment of latency, mutants deficient for M2 are compromised for the transient rise of latency in the spleen, which is normally observed at 14 days post-infection. Despite this failure in expansion of the latently infected cell pool at early times post-infection, long-term latency is relatively unaffected (Jacoby et al., 2002
; Macrae et al., 2003
) and surprisingly a higher number of latently infected splenocytes are detected at late times after infection (Macrae et al., 2003
). These data imply that M2 plays a major role during the establishment phase of latency in the spleen.
In this study, we show that the M2 gene product is required for efficient colonization of splenic follicles but is dispensable for the expansion of latently infected GC B cells at early times post-infection. However, late in infection disruption of M2 resulted in sustained and abnormally high levels of virus persistence in splenic GC B cells but not memory B cells.
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METHODS |
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Recombinant viruses.
An MHV-68 M2 frameshift mutant (M2FS) was generated by a two-step replacement procedure (Adler et al., 2000; Messerle et al., 1997
). For that purpose, an additional guanine was introduced via PCR into the HindIII-E fragment of MHV-68 (Efstathiou et al., 1990
) between nt 4603 and 4604, immediately downstream from the start ATG-codon (Husain et al., 1999
). The PCR fragment was cloned into the HindIII-E fragment using SacII (nt 4313) and SpeI (nt 4631). The resulting mutagenized HindIII-E fragment was then cloned into the shuttle plasmid pST76K-SR and electroporated into E. coli strain DH10B containing the MHV-68 wild-type BAC pHA3 (Adler et al., 2000
). Insertion of the additional guanine generated a new ApaI site, a frameshift and a new stop codon 78 nt downstream from the transcription start site (Fig. 1
). For the purpose of constructing a revertant virus, a 3855 kb FseI-HindIII fragment of MHV-68 (nt 24066261) was cloned into the shuttle plasmid pSP76K-SR (Adler et al., 2000
) and electroporated into E. coli strain DH10B, which contained the M2FS-mutant BAC plasmid. Electroporation of the BAC plasmids into BHK-21 cells resulted in the reconstitution of either M2FS or M2FSR virus expressing green fluorescent protein (GFP). The BAC vector sequence (flanked by loxP sites) was removed by passage through NIH 3T3 Cre cells and limiting dilution to obtain GFP-negative viruses (Adler et al., 2000
, 2001
). The DNA structures of the M2FS and revertant viruses were verified by examination of restriction enzyme digest profiles of E. coli-derived BAC DNA and Southern blot hybridization of reconstituted virus DNA.
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Purification of different splenocyte populations.
Single-cell suspensions of at least four pooled spleens were prepared per time point and passed through a 100 µm filter to remove stromal debris. Cell suspensions were washed in PBS-2 % FCS and red blood cells lysed in 154 mM ammonium chloride, 14 mM sodium hydrogen carbonate, 1 mM EDTA pH 7·3. Single-cell suspensions were preincubated with 2·4 G2 [anti-CD16/CD32 (FcIII/II receptor), rat immunoglobulin G2(
) culture supernatant; Pharmingen] before staining with the following monoclonal antibodies from: Pharmingen anti-CD19 (clone 1D3), -IgG1 (clone A85-1), -IgG2a/2b (clone R2-40) and -IgM (clone R6-60.2); Southern Biotech anti-IgD (clone 11-26) and -CD38 (clone NIMR.5). The lectin peanut agglutinin (PNA) was from Vector Laboratories. Using a MoFlo cytometer (Cytomation) the following enriched cell subpopulations were obtained: CD19+ for total B cells; CD19+ PNAhigh or IgG1/IgG2a/2b+ IgD/IgM CD38low for GC B cells; and IgG1/IgG2a/2b+ IgD/IgM CD38high for memory B cells. Sorted populations were analysed in a FACScan flow cytometer, and the data were processed using CellQuest software (Becton Dickinson Immunocytometry Systems). The purities of sorted populations were usually >95 % and always >90 % except for memory B cells that ranged between 72 and 76 %.
Real-time PCR.
Real-time PCR was performed using a LightCycler from Roche Molecular Biochemicals according to manufacturer's instructions, using sequence-specific fluorescent detection oligonucleotide hybridization probes coupled to suitable fluorophores as described (Marques et al., 2003).
Limiting dilution analysis.
FACS purified single-cell suspensions were serially diluted twofold and eight replicates of each cell dilution were lysed overnight (0·45 % Tween-20, 0·45 % NP-40, 2 mM MgCl2, 50 mM KCl, 10 mM Tris pH 8·3 and 0·5 mg ml1 Proteinase K) at 37 °C. Proteinase K was then inactivated (5 min at 95 °C) and the samples were analysed by real-time PCR, with primer/probe sets specific for K3, in a final volume of 10 µl per PCR reaction [2 mM MgCl2, 4 ng ml1 each primer, 0·2 µM each internal probe and 1x DNA mix (Roche) and 1 µl cell lysate]. This protocol detected S11 cells (approximately 40 MHV-68 genomes per cell) at a frequency of 1 : 1x105 in an uninfected control population. Our data were compatible with the single-hit Poisson model (SHPM) as tested by modelling the limiting dilution data according to a generalized linear log-log model fitting the SHPM (log(log(µi))=log (f)+log (xi)) and checking this model by an appropriate slope test as described by Bonnefoix et al. (2001). A regression plot of input cell number against log-fraction-negative samples was used to estimate the frequency of cells with virus genomes. Estimation of the cell subset frequency of MHV-68 infection, f, consisted of computation by maximal-likelihood estimation. The standard error of f was calculated as the square root of the negative reciprocal of the second derivative of log(L),
RT-PCR analysis of virus transcription.
Quantification of virus transcription was performed as described before (Marques et al., 2003). RNA was isolated from splenocytes purified by sorting from pools of five spleens and NIH 3T3 cells using the RNeasy mini kit with the RNase-free DNase set protocol (Qiagen) according to the manufacturer's instructions. For cDNA synthesis, 2 µg RNA was incubated at 70 °C for 10 min with 500 µg pd(T)1218, 5' PO4 sodium salt in a total volume of 23 µl. Samples were then reverse transcribed in a total reaction volume of 40 µl containing 0·5 mM each dNTP, 1x first strand buffer (Gibco-BRL), 1 U RNase OUT ribonuclease inhibitor (Gibco-BRL), and 400 U superscript II RT (Gibco-BRL). Reactions were performed for 50 min at 37 °C followed by a step of 5 min at 90 °C. Viral cDNAs were quantified using real-time PCR, as described above. Primer and probe genome coordinates used were according to Marques et al. (2003)
. Tenfold serial dilutions of plasmid template spiked into total splenic cDNA equivalent to a minimum of 10 000 copies of Hprt were used to establish, for each gene, a linear relationship between the input template copy number and the cycle number, at which an arbitrary fluorescence threshold was crossed. This gave the relative quantity of each viral transcript in each sample. The signal in RT-negative controls, indicative of residual viral DNA, was subtracted from the total to give a cDNA-specific signal. In this study, only RT-PCR reactions yielding 10 or more copies of viral transcript per RT-PCR reaction were considered.
In situ hybridization.
Digoxigenin (Boehringer Mannheim)-labelled riboprobes corresponding to MHV-68 vtRNAs (tRNA-like transcripts) 14 were generated by T7 transcription of pEH1.4 (Simas et al., 1999). In situ hybridization was performed as previously described (Bowden et al., 1997
). Briefly, 5 µm paraffin embedded sections were dewaxed in xylene, rehydrated through graded ethanol solutions, treated with 100 mg ml1 proteinase K for 10 min at 37 °C and acetylated with 0·25 % (v/v) acetic anhydride-0·1 M triethanolamine. Sections were hybridized with labelled riboprobes in 50 % formamide, 1x SSC overnight at 55 °C. The stringent wash (0·1xSSC, 30 % formamide, 10 mM Tris pH 7·5) was carried out at 58 °C. Hybridized probe was detected with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Boehringer Mannheim) according to the manufacturer's instructions.
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RESULTS |
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M2 is dispensable for lytic replication
Two independent studies have investigated the role played by M2 in replication (Jacoby et al., 2002; Macrae et al., 2003
). These studies demonstrated that recombinant viruses deficient in M2 replicate normally in tissue culture and display normal acute phase replication kinetics in lung tissue upon infection of mice. Consistent with these studies, no significant difference could be detected in the growth kinetics of the M2FS and M2FSR mutant viruses in comparison to wild-type MHV-68, derived from pHA3 (Adler et al., 2001
) (data not shown). Moreover, in agreement with the in vitro replication kinetics of the M2FS mutant, no significant difference was observed in the ability of this mutant or the M2FSR versus wild-type MHV-68 to productively replicate in lung tissue upon intranasal inoculation of mice (data not shown).
M2 is required for efficient establishment of latency but dispensable for long-term latency
To analyse the role played by M2 during the establishment and maintenance of latency, mice were infected with M2FS mutant or wild-type MHV-68 and the latent load in spleen was examined by quantification of reactivation-competent virus by infectious centre assay. The results obtained showed that early during the establishment of latency, 14 and 21 days post-infection, the numbers of infectious centres were reduced by about 10-fold for animals infected with M2FS in comparison with MHV-68 (Fig. 2). In contrast, at later time points, 43 and 71 days post-infection, the mean number of infectious centres was consistently higher for M2FS versus wild-type MHV-68 (Fig. 2a
). In a separate experiment the phenotype of the M2FSR virus was examined in the spleen at 14 days post-infection and no difference was detected in the number of infectious centres in comparison to wild-type MHV-68, indicating a reverted phenotype (Fig. 2b
).
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Reduced levels of M2FS mutant in memory B cells
We next sought to assess if the observed increased frequency of M2FS persistence in GC B cells resulted in a concomitant increase in the levels of infection established in memory B cells. To this end, the frequency of virus genome-positive cells was determined for GC B cells and memory B cells (Table 3). Enriched populations of GC B cells (IgG1/IgG2a/2b+ IgD/IgM CD38low) and memory B cells (IgG1/IgG2a/2b+ IgD/IgM CD38high) were obtained (Fig. 3
) as previously described by others (Flano et al., 2002a
). During the establishment of latency at 14 days post-infection, the ratio of the reciprocal frequencies of viral DNA-positive GC B cells versus memory B cells was 0·5 and 0·3 for wild-type MHV-68 and M2FS, respectively. Later in infection, however, the ratios were 0·9 and 0·08 for wild-type virus and M2FS mutant, respectively. That is, late in infection in comparison with MHV-68, a 20-fold increase in GC B cells harbouring M2FS resulted in only a twofold increase in frequency of infection in memory B cells. These data showed that the sustained high levels of M2FS persistence in GC B cells were not accompanied by a simultaneous increase in the levels of virus persistence in memory B cells indicating that M2 is important for efficient exiting of infected GC B cells into the memory compartment.
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DISCUSSION |
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The role played by M2 following the establishment of latency in the spleen was evaluated next. Remarkably, at late times post-infection disruption of M2 resulted in an increase in the frequency of virus-positive total B cells and GC B cells. In agreement, and consistent with a non-reactivation deficit phenotype, the levels of M2FS infectious centres, the percentage of infected follicles and the mean number of infected cells per follicle late during infection were higher than those obtained for M2FSR and MHV-68. The finding that disruption of M2 leads to increased levels of infectious centres at late times post-infection has been previously reported (Macrae et al., 2003). Notably, the sustained high levels of M2FS persistent infection of GC B cells did not result in a concomitant increase in the frequency of infection in memory B cells. This finding suggests that M2 might facilitate entry of infected cells into the memory B-cell pool.
In the absence of M2, the high levels of infection of GC B cells could in part reflect an overall cessation of GC reaction or be a consequence of an increased number of follicles infected. However, in situ hybridization analysis revealed that the mean number of infected follicles and the mean number of infected cells within each follicle remained stable throughout infection. These data were further evidence that absence of M2 does not impair expansion of latently infected GC B cells.
Analysis of M2FS transcription during persistent infection of GC B cells revealed a selective pattern of virus transcription coherent with an overall state of latent infection. Amongst those genes transcribed during M2FS persistent infection, ORF73 showed the highest number of detectable transcripts. This result is consistent with previous data showing that during the establishment of MHV-68 latency in GC B cells, transcripts corresponding to ORF73 were also the most abundant (Marques et al., 2003). Recently it has been shown that in the absence of ORF73, MHV-68 is unable to establish latent infection in the spleen (Fowler et al., 2003
; Moorman et al., 2003
). Thus, ORF73 transcription during persistent M2FS infection of GC B cells is further evidence that, like for ORF73 of KSHV and herpesvirus saimiri, ORF73 of MHV-68 is involved in genome episome maintenance in proliferating cells, namely latently infected GC B cells.
During long-term persistent infections, herpesviruses typically show low levels of transcription activity. In the case of EBV it has been proposed that infected cells enter the long-lived memory pool by shutting down transcription of all the viral latent-proteins, probably a pre-requisite for evasion from immune system elimination (Thorley-Lawson, 2001). Therefore, it is somehow surprising that in the absence of M2, MHV-68 could persist at high levels in GC B cells, although in a restricted fashion, in nevertheless a transcriptionally active state. It is possible that immune evasion activity afforded by MHV-68 is sufficient to avoid immune clearance; namely that mediated by the viral-encoded M3 chemokine-binding protein and MK3 protein that has been implicated in CD8+ CTL evasion (Bridgeman et al., 2001
; Stevenson et al., 2002
). Another factor that might be involved in facilitating M2FS persistence could be attributed to the absence of M2, itself an important target for CD8+ CTL control of infection (Usherwood et al., 2000
, 2001
).
It is not immediately apparent why disruption of M2 results in the observed phenotype. It is possible that M2 is required for infected B cells to enter splenic follicles. Once inside follicles, however, M2 is dispensable for the expansion of latently infected cells. The combined observation of sustained high levels of persistent M2FS infection in GCs and the lower frequency of infection of memory B cells relative to GC B cells may be indicative that M2 is thereafter required for the transition of latently infected cells into the long-lived memory B-cell compartment and eventual cessation of virus driven expansion of infected GC B cells. Future experiments directly addressing the molecular function of M2 and analysing further the kinetics and tropism of infection of M2-deficient recombinant viruses will elucidate further the role played by this gene product in infection. In particular, they will reveal if M2, in a manner functionally similar to the EBV LMP1 and LMP2a, can modulate B-cell function by providing surrogate signals necessary for antigen-independent activation of naive B cells.
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ACKNOWLEDGEMENTS |
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Received 26 March 2004;
accepted 22 June 2004.