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Background
Screen	MCMV Susceptibility and Resistance Screen
Posted On	02/18/2010 12:24 PM
Author	Celine Eidenschenk
Science Writer	Nora G. Smart
Mouse cytomegalovirus (MCMV) is a β-herpes virus that is contained by the host through the action of natural killer (NK) cells of the innate immune system before the onset of the adaptive immune response. Mice of the C57BL/6 or C57BL/10 backgrounds do not become sick in response to MCMV inoculated at a dose of 1x10⁵ PFU. The virally encoded m157 protein is detected by NK cell–activating receptor Ly49H, and as a consequence, infected cells are lysed (1;2). In BALB/c mice, which lack the Ly49H-encoding gene, MCMV is frequently lethal within one week following inoculation of 1 x 10⁵ PFU. Although C57BL/6 mice are completely resistant to challenge with 1x10⁵ PFU of MCMV, higher doses cause sickness and death in these animals. A few mice die when MCMV is inoculated at a dose of 2 x 10⁵ PFU, and nearly all mice appear sick; ~90% of mice die when inoculated with 4 x 10⁵ PFU (Figure 1). The exceptionally sharp dose-lethality characteristics of the virus permit sensitive screens for enhanced susceptibility or resistance. Screens for MCMV susceptibility and resistance are carried out by inoculating G3 germline mutants on a C57BL/6 background with 1x10⁵ and 1x10⁶ PFU of MCMV, and either severe illness and death, or lack of illness and survival, are taken as endpoints, respectively. Wild type C57BL/6 and BALB/c mice are used as controls (3). Mice susceptible to MCMV may have mutations in genes required to sense virally encoded nucleic acids and proteins, in genes encoding cytokine mediators along with their receptors and transducers, and in genes encoding components required for proper NK cell granule exocytosis (4;5). In addition, mutations may affect proteins that support homeostasis during the innate immune response (6), or genes required for the ontogeny of NK cells or other cells that support the innate immune response. Many of the mutations causing MCMV susceptibility or resistance can be distinguished by testing mutants for standard cytokine responses and NK cell function (see NK Cytotoxicity Protocol). Resistance to MCMV may be caused by mutations affecting host proteins that are needed by the invading virus for infection and replication. MCMV has evolved evasion mechanisms to avoid detection by the host by targeting virus-recognition mechanisms, and antigen processing and presentation (5). MCMV resistance mutants may also have mutations that allow the host to overcome evasion.
Reagents and Solutions
MCMV medium Dulbecco’s modified eagle medium (Mediatech Inc., Herndon, VA) 3% (v/v) heat-inactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) 200 IU/mL penicillin (Gibco) 200 mg/mL streptomycin (Gibco) 3T3 medium Dulbecco’s modified eagle medium 10% (v/v) heat-inactivated fetal bovine serum 200 IU/mL penicillin 200 mg/mL streptomycin Agar medium Dulbecco’s modified eagle medium 3% (v/v) heat-inactivated fetal bovine serum 2% (v/v) gentamycin (Gibco) 200 IU/mL penicillin 200 mg/mL streptomycin To make 500mL: 4g carboxy methyl cellulose (Sigma) Autoclave 5mL L-glutamine (Gibco) 5mL gentamycin 5mL 100x penicillin/streptomycin stock 15mL serum 470mL DMEM Shake everyday for one week until the medium is homogenized. 10% formalin Dilute 37% formaldehyde stock (Sigma) to 10% with water. Cell staining solution 1% crystal violet (w/v) in 20% ethanol (store up to 1 yr at RT) Before use, dilute 1 part with 9 parts milli-Q water. IL12p70, IFNγ and IL-6 ELISA kits (eBioscence) tissue tearor cleaned with bleach and rinsed with ethanol and sterile PBS
Method
Generation of MCMV stock Save an aliquot from old stock to make new stock. Inject 3 week old BALB/c females with 10³ PFU of MCMV stock (depending on age, mice are injected with 1-5000 PFU of MCMV stock). After two weeks, harvest salivary gland and homogenize in 1mL of MCMV medium using the tissue tearor, spin at 200 g for 3 min to pellet cellular debris, remove supernatant. Spin supernatant at 1500 g for 5 min, eliminate upper lipidic layer by aspiration, and freeze the rest at -80^oC. In vitro MCMV titration Day 1: Plate 3T3 cells in a 24 well plate at 40,000 cells/well in 1mL of 3T3 medium. Make sure to have 6 wells per sample (non-diluted, 1/10, and 1/100 in duplicate). Day 2: In a 96 well plate, make 1:10 and 1:100 dilutions of organ supernatants from step 3 (ie use 25μL sample + 225μL medium). Infect cells with MCMV by first removing cell media, and then adding 200μl of the undiluted and diluted samples. Incubate 2 hours at 37^oC. Add 1mL of agar medium to each well, and incubate for 5 days at 37^oC. Note: For liver samples, take off the samples before adding the agar medium (otherwise, enzymes from the liver will kill the cells). Agar medium can be added directly to cells incubated with all other samples (supernatants of cell culture, spleen, salivary glands). Day 6: Fix the cells by adding 200μl 10% formalin to each well, and incubating 6 hours at room temperature (RT). Remove fix and add 200μl cell staining solution per well. Incubate 2 minutes, RT. Wash the plate well 3-4x. Let dry and count the number of plaques. In vivo MCMV titration Inject 6-week old mice of each strain (Balb/c and C57BL/6) with 1x10⁵, 2x10⁵, 5x10⁵, and 1x10⁶ PFU of MCMV (in vitro titer) using 4 mice per dose. Check mice every day for appearance and death. Discard cages by day 7. Adjust in vitro titer if needed. In vivo MCMV screening Use in vivo titer to inoculate ENU-mutagenized G3 C57BL/6 mice at 1x10⁵ PFU and 1x10⁶ PFU. As described above, wild type C57BL/6 mice are completely resistant to the low dose of MCMV, but die at day 5 post-infection with a dose of 1x10⁶ PFU. Check mice every day for sickness or death (both of which are scorable outcomes of infection). Discard cages by day 7. Breed and fix mutants, and confirm the MCMV phenotype. Please see the Genetic Mapping protocol for breeding schemes and mapping strategies. Analysis of MCMV mutants Test mutant tissues 5 days post-MCMV infection for viral titers (see steps 3-10) . Test mutants for standard cytokine responses using ELISA kits 36 hours after inoculation with 1x10⁵PFU of MCMV. Test mutants for NK cell function. For an example of an in vivo NK cell cytotoxicity protocol, see the In Vivo NK Cell and CD8+ Cell Cytotoxicity Screen. In vitro methods are listed in the In Vitro NK Cell Cytotoxicity Protocols.
Alleles Identified
3d aki aoba bullet gray caruso concrete CpG1 CpG2 CpG3 CpG5 daniel domino elektra Endeka Feckless Gemini goodnight grey wolf Havelock Helens honey rider jinx Joker Lps2 mayday miklos moneypenny pam bouvier Paris plenty pococurante poison salt and pepper slumber solitaire sooty souris sphinx Stamper-immunologic teflon toffee tumormouse Viper warmflash
References
1. Lee, S. H., Girard, S., Macina, D., Busa, M., Zafer, A., Belouchi, A., Gros, P., and Vidal, S. M. (2001) Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily, Nat. Genet. 28, 42-45. 2. Brown, M. G., Dokun, A. O., Heusel, J. W., Smith, H. R., Beckman, D. L., Blattenberger, E. A., Dubbelde, C. E., Stone, L. R., Scalzo, A. A., and Yokoyama, W. M. (2001) Vital involvement of a natural killer cell activation receptor in resistance to viral infection, Science 292, 934-937. 3. Crozat, K., Georgel, P., Rutschmann, S., Mann, N., Du, X., Hoebe, K., and Beutler, B. (2006) Analysis of the MCMV resistome by ENU mutagenesis, Mamm. Genome 17, 398-406. 4. Crozat, K., Hoebe, K., Ugolini, S., Hong, N. A., Janssen, E., Rutschmann, S., Mudd, S., Sovath, S., Vivier, E., and Beutler, B. (2007) Jinx, an MCMV susceptibility phenotype caused by disruption of Unc13d:a mouse model of type 3 familial hemophagocytic lymphohistiocytosis, J. Exp. Med. 204, 853-863. 5. Beutler, B., Jiang, Z., Georgel, P., Crozat, K., Croker, B., Rutschmann, S., Du, X., and Hoebe, K. (2006) Genetic analysis of host resistance: Toll-Like receptor signaling and immunity at large, Annu. Rev. Immunol. 24, 353-389. 6. Croker, B., Crozat, K., Berger, M., Xia, Y., Sovath, S., Schaffer, L., Eleftherianos, I., Imler, J. L., and Beutler, B. (2007) ATP-sensitive potassium channels mediate survival during infection in mammals and insects, Nat. Genet. 39, 1453-1460.