An Essential Role of the Enhancer for Murine Cytomegalovirus In Vivo Growth and Pathogenesis

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Abstract
The transcription of cytomegalovirus (CMV) immediate-early (IE) genes is regulated by a large and complex enhancer containing an array of binding sites for a variety of cellular transcription factors. Previously, using bacterial artificial chromosome recombinants of the virus genome, it was reported that the enhancer region of murine CMV (MCMV) is dispensable but performs a key function for viral multiplication (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72: 8502-8509, 1998). In the present study, we defined, through the reconstitution of infectious enhancerless MCMVs, the growth requirement for the enhancer in tissue culture and explored its significance for steering a productive infection in vivo. A comparison of cis and trans complementation systems for infection of enhancerless virus in permissive fibroblasts revealed a multiplicity-dependent growth phenotype that is severely compromised in the rate of infectious-virus multiplication. The in vivo impact of viruses that have an amputated enhancer was investigated in an extremely sensitive model of MCMV infection, the SCID mouse. Histological examination of spleens, livers, lungs, and salivary glands from animals infected with enhancer-deficient MCMV demonstrated an absence of tissue damage associated with CMV infection. The lack of pathogenic lesions correlated with a defect in replication competence. Enhancerless viruses were not detectable in major target organs harvested from SCID mice. The pathogenesis and growth defect reverted upon restoration of the enhancer. Markedly, while SCID mice infected with 5 PFU of parental MCMV died within 50 days postinfection, all mice infected with enhancerless virus survived for the duration of the experiment (1 year) after infection with 5 × 10 5 PFU. Together, these results clarify the importance of the enhancer for MCMV growth in cell culture and underscore the in vivo significance of this region for MCMV virulence and pathogenesis.

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