Murine gammaherpesvirus 68 glycoprotein 150 does not contribute to latency amplification in vivo
© Ruiss et al.; licensee BioMed Central Ltd. 2012
Received: 18 January 2012
Accepted: 3 June 2012
Published: 9 June 2012
Murine gammaherpesvirus 68 (MHV-68) is used as a model to study the function of gammaherpesvirus glycoproteins. gp150 of MHV-68, encoded by open reading frame M7, is a positional homolog of gp350/220 of EBV and of gp35/37 of KSHV. Since it had been proposed that gp350/220 of EBV might be a suitable vaccine antigen to protect from EBV-associated diseases, gp150 has been applied as a model vaccine in the MHV-68 system. When analyzing the function of gp150, previous studies yielded conflicting results on the role of gp150 in latency amplification, and disparities between the mutant viruses which had been analyzed were blamed for the observed differences.
To further develop MHV-68 as model to study the function of gammaherpesvirus glycoproteins in vivo, it is important to know whether gp150 contributes to latency amplification or not. Thus, we re-evaluated this question by testing a number of gp150 mutants side by side. Our results suggest that gp150 is dispensable for latency amplification. Furthermore, we investigated the effect of vaccination with gp150 using gp150-containing exosomes. Vaccination with gp150 induced a strong humoral and cellular immune response, yet it did not affect a subsequent MHV-68 challenge infection.
In this study, we found no evidence for a role of gp150 in latency amplification. The previously observed contradictory results on the role of gp150 in latency amplification were not related to differences between the mutant viruses which had been used.
Infection of cells by herpesviruses is orchestrated by several viral glycoproteins which function either separately or within multiprotein-complexes to mediate virus entry . Murine gammaherpesvirus 68 (MHV-68) has been extensively used as a model to study the function of gammaherpesvirus glycoproteins [2–5]. Glycoprotein 150 (gp150) of MHV-68 is encoded by open reading frame M7  and has been shown to be virion-associated . It is a positional homolog of gp350/220 of EBV and of gp35/37 of KSHV , and it has been shown to be important for virion release [8, 9] and antibody evasion . When studying the function of gp150, both de Lima et al.  and Stewart et al.  found that mutation of gp150 did not considerably affect lytic replication in vivo and in vitro, except from a moderate deficit in release of extracellular virus. However, Stewart et al. observed a reduction in splenomegaly and in the number of latently infected cells during latency amplification. In contrast, de Lima et al. reported normal latency amplification by gp150-deficient MHV-68. Clearly, to further develop MHV-68 as model for studying the function of gammaherpesvirus glycoproteins in vivo, it is important to know whether gp150 reliably contributes to latency amplification. Here, we present data supporting that gp150 is dispensable for latency amplification but it remains possible that it might play a role under different, so far ill-defined circumstances.
Results and discussion
Previously, conflicting results have been published on the role of gp150 in latency amplification [8, 9], and several hypotheses were raised to explain these discrepancies: i) Stewart et al. introduced a large deletion in the MHV-68 genome to disrupt gp150 which might have inadvertently disrupted additional viral functions, e.g. unknown latency transcripts . On the other hand, the mutations made by de Lima et al. might allow the expression of a truncated but still functional gp150 protein . ii) Stewart et al. investigated only a single mutant while de Lima et al. constructed two independent mutants. The latter can be considered to be a superior strategy to study a gene function . Although Stewart et al. carefully analyzed their mutant by various standard procedures, and even analyzed the viral transcriptome by DNA oligonucleotide microarray, it is formally possible that subtle defects escaped their analysis. iii) Stewart et al. also constructed a BAC-based revertant but BAC-based revertants do not control for problems after virus reconstitution .
gp150 and latency amplification
To address the above mentioned points, we re-evaluated whether gp150 plays a role during latency amplification. For this purpose, we analyzed the following viruses: i) the original mutant (vgp150Δ) which was constructed in our own laboratory and which has been described in Stewart et al. ; ii) a second gp150 mutant (vgp150Δ_2) which we independently reconstituted from the same BAC clone from which the original mutant vgp150Δ had been derived - to test for aberrations which might have occurred during reconstitution; iii) a M7-FRT mutant constructed exactly as described by de Lima et al. , to test whether the different size and position of the deletion affect the phenotype. All mutant viruses were compared to the revertant virus vgp150R, which had been constructed in our own laboratory and shown to behave exactly like parental (wild-type) virus .
gp150 and vaccination
Taken together, our data suggest that gp150 is dispensable for latency amplification. Vaccination with gp150-containing exosomes did not affect a subsequent MHV-68 challenge infection, even though it induced a strong humoral and cellular immune response.
Cell lines and virus stocks
BHK-21 cells (ATCC CCL-10) were grown in Glasgow-MEM (PAN Biotech, Aidenbach, Germany) supplemented with 5% fetal calf serum (FCS), 5% tryptose phosphate broth, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. NIH3T3 cells (ATCC CRL-1658) and HEK293 cells (ATCC CRL-1573) were grown in DMEM (Invitrogen, Darmstadt, Germany) supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Working stocks of viruses were prepared as previously described . Briefly, stocks were grown by infecting BHK-21 cells. After showing complete cytopathic effect (CPE), BHK-21 cells were harvested and the supernatant was used as working stock after two times freezing/thawing the cells and removing cell debris by centrifugation. Virus titers were determined by plaque assays. Briefly, 10-fold dilutions were incubated on BHK-21 cells for 90 min at 37 °C. After removing the inoculum, cells were incubated for 5 days at 37 °C with fresh medium containing methylcellulose. Cells were stained with 0.1% crystal violet solution to determine the number of plaques.
In vivo experiments
Six to eight weeks old C57BL/6 or BALB/c mice were purchased from Charles River Laboratories (Sulzfeld, Germany) and housed in individually ventilated cages (IVC) during the MHV-68 infection period. Mice were infected intranasally (i.n.) with 5 × 104 plaque forming units (PFU) of MHV-68. Prior to i.n. infection, mice were anesthetized with ketamine and xylazine. To determine lytic virus titers, the lungs were harvested and homogenized using the FASTPREP®-24 instrument (MP Biomedicals, Heidelberg, Germany). After two times freezing and thawing the homogenates, plaque assays were performed with 10-fold dilutions as described above. To determine the frequency of cells carrying virus reactivating from latency, spleens were harvested, single splenocyte suspensions were prepared and analyzed in an ex vivo limiting dilution reactivation assay as described . Briefly, serial threefold dilutions of infected mouse splenocytes were plated on monolayers of 1 × 104 low-passage NIH3T3 cells per well in 96-well tissue culture plates. 24 wells were plated per dilution (starting with 1,5 × 105 cells/well). NIH3T3 cells were screened microscopically for a viral cytopathic effect (cpe) for up to 3 weeks. To differentiate between latently infected cells and infectious virus in the samples, serial threefold dilutions of spleen cells were plated before or after mechanical disruption of viable cells (by two freeze-thaw cycles). No infectious virus was detected in samples of mechanically disrupted cells (data not shown). Frequencies of reactivating cells were calculated on the basis of the Poisson distribution by determining the cell number at which 63.2% of the wells scored positive for CPE. All animal experiments were in compliance with protocols approved by the local Animal Care and Use Committee (District Government of Upper Bavaria; permit number 124/08).
Measurement of latent viral load by quantitative real time PCR
Viral load in the spleens of infected mice was determined by quantitative real-time PCR using the ABI 7300 Real Time PCR System (Applied Biosystems, Foster City, CA) as described . Briefly, DNA was extracted from spleen cells using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) and quantified by UV spectrophotometry. Amplification of 100 ng of DNA per reaction was performed with Taqman universal PCR master mix and universal cycling conditions (Applied Biosystems, Foster City, CA). Using primers and probes as described , a 70 bp region of the MHV-68 glycoprotein B (gB) gene was amplified and viral DNA copy numbers were quantified. A standard curve was created using known amounts of a plasmid containing the Hin dIII-N fragment of MHV-68 encompassing the gB gene. The murine ribosomal protein L8 (rpl8) was amplified in parallel and used to normalize for input DNA between samples. The primer and probe sequences for L8 were as follows: Forward: 5’-CATCCCTTTGGAGGTGGTA-3’; Reverse: 5’-CATCTCTTCGGATGGTGGA-3’ and Probe: 5’-ACCACCAGCACATTGGCAAACC-3’. A standard curve for rpl8 was generated by serial 10 fold dilution of a plasmid containing rpl8 (RZPD clone IRAVp968B01123D6, RZPD, Berlin, Germany). The data are presented as viral genome copy numbers relative to the copy number of L8. The quantification limit was set at 50 copies per sample, according to published recommendations .
Production and characterization of gp150-carrying exosomes
To generate gp150-carrying exosomes, 293 cells were transfected with an expression plasmid coding for gp150, and three days later, exosomes were prepared from the supernatant. Control exosomes were prepared in the same way from untransfected 293 cells. Briefly, exosomes were subjected to sequential centrifugation steps (300 g for 10 min, 5000 g for 10 min and 100.000 g for 120 min). The pelleted exosomes were washed and resuspended in PBS containing proteinase inhibitors (Complete Mini Proteinase Inhibitor Cocktail, Roche, Penzberg, Germany). The protein content was analyzed in a BCA protein microassay (Pierce, Rockford, USA). Exosomes were analyzed for overall gp150 content by Western Blot and for gp150 surface content by FACS analysis. For Western Blotting, 30 μg of exosomes were lysed in sample buffer, separated by SDS-PAGE and transferred onto a Hybond-ECL membrane (GE Healthcare, Munich, Germany). gp150 was detected by incubating the membrane with polyclonal rabbit anti-MHV-68 antibody , followed by peroxidase-conjugated donkey anti-rabbit antibody (Jackson Laboratories, Newmarket, UK). Blots were developed using ECL reagent (GE Healthcare, Munich, Germany) according to the instructions of the manufacturer. For FACS analysis, flow cytometry Protein G antibody binding beads (Polysciences Europe GmbH, Eppelheim, Germany) were coated with polyclonal rabbit anti-MHV-68 antibody for 30 min at room temperature. After washing with PBS and blocking with RPMI/10% FCS, beads were incubated with exosomes for two hours at room temperature. After washing, bound exosomes were detected by incubation with PE-conjugated anti CD63 monoclonal antibody (Immunotools, Friesoythe, Germany). CD63, a member of the tetraspanin family, is a marker protein for exosomes .
Vaccination with exosomes and challenge infection
Mice were vaccinated twice (day 0 and 14) with gp150-containing exosomes (293/gp150 exosomes) or as a control, with exosomes prepared from untransfected 293 cells (293 exosomes). Vaccination was performed i.p. with a mixture of 10 μg of exosomes and 10 nmol CpG-ODN 1668 (InvivoGen, San Diego, USA) in a total volume of 200 μl PBS per mouse. Fourteen days after the second vaccination (day 28), mice were challenged with 5 × 104 PFU of MHV-68.
ELISPOT assay for determination of gp150-specific T cells
To determine gp150-specific T-cells, splenocytes from vaccinated mice were plated onto precoated ELISPOT plates in a densitiy of 106 cells/well and restimulated in vitro with 10 μg of exosomes (+/− gp150). After 24 h, activated T-cells were quantified using a mouse IFN-γ-ELISPOT Kit (eBioscience, Frankfurt, Germany) as recommended by the manufacturer, and spots were counted by inspection under a dissecting microscope.
ELISA for determination of gp150-specific antibodies
To determine gp150-specific antibodies in sera of vaccinated mice, Nunc Maxisorp plates (Nunc, Wiesbaden, Germany) were coated overnight with 3 μg/ml of MHV-68 lysate. The lysate was prepared by disruption of MHV-68 via dilution in PBS with 0.05% Triton X-100 as described . After washing with PBS/Tween, plates were blocked with RPMI containing 10% FCS for 1 hour and washed again. Then, fifty-fold serum dilutions were incubated for two hours. After washing, bound antibody was detected with HRP-conjugated rat anti-mouse antibody (Promega, Mannheim, Germany) using TMB (Becton Dickinson, Heidelberg, Germany) as substrate and, after stopping with 1 M phosphoric acid, reading the absorbancy at 450 nm.
If not otherwise indicated, data were analyzed by two-tailed Student’s t-test.
This work was supported by grants from the BMBF (NGFNplus, FKZ PIM-01GS0802-3) and the Wilhelm Sander-Stiftung (grant 2009.046.1) to H.A., from the Japan Herpesvirus Infections Forum to S.O., and from the Deutsche Krebshilfe (grant 107793) to R.Z.. We are grateful to the members of our animal facilities for expert technical assistance. We also thank the members of our group for helpful discussions and B. Adler for critically reading of the manuscript.
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