The study of viral genetics via the construction of recombinant murine cytomegaloviruses
Craggs, Megan Mari
Cytomegaloviruses are highly host specific, and murine cytomegalovirus (MCMV) has been widely used as a model for studying human cytomegalovirus (HCMV) infections to overcome the difficulty of experimentation with HCMV in vivo. The ability to manipulate the viral genome and introduce specific mutations into viral genes should facilitate the investigation of mechanisms governing cytomegalovirus host specificity. Our laboratory has utilized a modified MCMV genome lacking a SwaI site to construct recombinant MCMVs through homologous recombination. However, it was not known whether this modification of the MCMV genome by removing the SwaI site would affect the biological properties of MCMV, or whether the SwaI mutation could be reversed should the need arose. To this end, two recombinant bacterial artificial chromosomes, pMCMV_ETwt and pMCMV_ETSwa-*, were constructed and characterized by restriction endonuclease analysis, demonstrating that only the expected genome modifications were present. Recombinant viruses were then reconstituted in tissue culture and their biological properties were compared to the Smith strain of MCMV as well as the parental MCMV_EGFP virus. Viral DNA isolated from infected cells showed the expected restriction patterns for MCMV-ETwt and MCMV-ETSwa-. The expression of MCMV proteins M112-113, ppM44 and gB, representative of those expressed in the early, delayed early, and late phases of infection, respectively, did not differ significantly between the recombinant viruses or the Smith strain of MCMV. In addition, virus growth in permissive Balb/3T3 cells at both low and high multiplicities of infection, and semi-permissive COS-1 cells at a high multiplicity of infection showed growth kinetics or patterns of infection that were similar to the Smith strain of MCMV. Results from these preliminary experiments suggested that the modification of the MCMV genome by removal of the SwaI site did not appear to affect the biological properties of MCMV in vitro. Furthermore, analysis of MCMV_ETwt demonstrated that we could reconstitute the SwaI site in the MCMV genome if necessary, resulting in a virus that should be identical to the Smith strain of MCMV. Therefore, these results suggest that the SwaI- MCMV genome will enable easier construction of recombinant MCMVs with desired alterations in any region of the viral genome.
DegreeMaster of Science (M.Sc.)
CommitteeWarrington, Rob C.; Wang, Hong; Tikoo, Suresh K.; Roesler, William J.; Misra, Vikram; Khandelwal, Ramji L.; Xiang, James