Interdependent Regulation of Cytomegalovirus Proteins in Complex

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes significant morbidity and mortality in the immune compromised. Using the mutine cytomegalovirus (MCMV) model, we identified two genes, M140 and M141, which are determinants of macrophage tropism. Monocytes/macrophages are a critical cell type for cytomegalovirus (CMV) pathogenesis as sites of viral latency and by supporting viral replication and disseminating virus throughout the body. We previously found that the localization of the M140/M141 gene products (pM140/pM141) is different when the proteins are co-expressed as compared to their individual expression. When the proteins are individually expressed pM141 localizes diffusely throughout the cytoplasm and, to a lesser extent, the nucleus while pM140 localizes almost exclusively in the nucleus. Co-expression alters the localization of either protein, as they co-localize to a distinct perinuclear region. The proteins form a stable complex and in the absence of pM140, pM141 is significantly destabilized. These results imply a complex regulation and interdependence of the proteins. The purpose of this study was to identify protein domains within pM140 and pM141 that are required for the interdependent regulation of the proteins and to determine the mechanism of pM141 protein degradation. We identified an interaction domain within pM140 at the extreme N terminus of the protein that is required foroptimal complexing with in vitro expressed pM141. We found that pM141 is degraded in a proteasome-dependent, but ubiquitin-independent pathway. The M141 protein is stabilized by pM140 in a concentration-dependent manner and we identified the region of pM140 required to stabilize pM141. However, direct complexing between pM140 and pM141 is not sufficient to stabilize the protein. Thus, our results support a model in which pM140 protects pM141 from proteasomal degradation by recruiting an additional viral or cellular protein to the pM140 and pM141 complex and this as yet unidentified protein is strictly ...