This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The human cytomegalovirus particle is structurally complex and consists of at least 30 different proteins. The viral genome is contained within an icosahedral nucleocapsid that is surrounded by an amorphous layer called the tegument. The tegument is enclosed within a lipid membrane containing virus-encoded glycoproteins. The assembly of the virion is complicated because viral replication and encapsidation occur in the nucleus of the infected cell but acquisition of some tegument proteins and a mature envelope takes place in the cytoplasm. While a mechanism by which the virion escapes the nucleus through the nuclear membrane has recently been proposed, the pathways that regulate transport of the subviral particle to the site of final envelopment have not been definitively identified. Because the cytoplasmic assembly compartment overlaps the microtubule-organizing center (MTOC) of the infected cell, we hypothesize that the particles track to this site on microtubules. The transport of cargo towards the MTOC is accomplished by minus-end directed microtubule motors of which cytoplasmic dynein is the most extensively studied. In uninfected cells, dynein activity mediates many processes including import of material from the cell periphery, retrograde vesicle traffic, positioning of cellular organelles, nuclear envelope breakdown, and cell division. It therefore seems likely that the virus has evolved mechanisms to exploit dynein and other components of the microtubule network for efficient assembly of viral progeny and release from the infected cell. Along the same lines, it is well established that HCMV infection leads to dysregulation of key cell cycle proteins and cell cycle arrest. The perturbation of cellular signaling pathways and cell cycle progression provides an environment conducive to viral gene expression and replication; however, our recent studies using inhibitors of the cyclin-dependent kinases (cdks) suggest that these proteins also play a role in virus assembly. Because the activity of microtubule motors is regulated by phosphorylation, we hypothesize that motor proteins are substrates for cdk activity in infected cells. To elucidate the roles of microtubule motors, specifically dynein, in the assembly of HCMV particles we propose to accomplish the following specific aims: 1) Characterization of dynein and dynactin expression and localization in HCMV-infected cells. 2) Determination of the effects of dynein inhibition on nuclear egress and envelopment. 3) Elucidation of the regulation of dynein function by cdks.
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