|Screen||MCMV Susceptibility and Resistance Screen|
|Posted On||02/18/2010 12:24 PM|
|Author||Celine Eidenschenk, Duanwu Zhang|
|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. Binary 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). Quantification screen for MCMV susceptibility is carried out by inoculating G3 mice with 1x105 PFU of MCMV, and MCMV titers in the tissues (spleen and liver) are determined by quantitative real-time PCR. High MCMV titers represent susceptible mice.
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|
To make 500 mL:
Cell staining solution
PBND solution (PCR buffer with non-ionic detergents)
To make 1000 mL:
IFNγ, IL-6, IL-12 p70 and TNFα ELISA kits (eBioscience).
Tissue tearor is cleaned with 10% bleach and rinsed with ethanol and sterile PBS.
Generation of MCMV stock
In vitro MCMV titration (Plaque assay)
In vivo MCMV titration
MCMV binary screen
MCMV quantification screen
Analysis of MCMV mutants
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.