Virion assembly and egress are among the least understood areas of virology. This is particularly true for human cytomegalovirus (HCMV), whose nucleocapsids must exit from the nucleus, then proceed through complex steps in the cytoplasm acquiring the tegument layer and envelope before they exit from the cell. The lack of understanding of HCMV assembly and egress presents a significant barrier to understanding HCMV pathogenesis and developing anti-viral treatments that might target these processes. The significance of this proposal is that it brings together data showing that HCMV utilizes both viral and cellular proteins to remodel the nucleus and the cytoplasm, forming a highly interconnected and interdependent "assembly-egress continuum" between the nucleus, the cytoplasmic viral assembly compartment (AC), and the cytoskeleton. Our previous studies of the assembly-egress continuum (5) have shown that the nuclear periphery is dramatically altered in HCMV infected cells, especially near the AC. We have demonstrated that the endoplasmic reticulum chaperone glucose regulated protein 78 (GRP78) functions as a component of the AC (6). Depletion of GRP78 results in the loss of AC integrity, virus-induced nuclear lamin phosphorylation, lamina rearrangement and the nuclear morphology characteristic of HCMV infection. Our data also confirmed the role of dynein in assembly compartment formation, and showed that the nuclear morphology characteristic of an HCMV infection is, in part, due to virus-mediated alterations in dynein function (5).
The specific aims will build on these observations and further define the assembly-egress continuum.
In Aim 1 we will define HCMV- mediated reorganization of the shape and size of the nucleus and AC formation. We will focus on dynein's function in these processes, as well as the potential role of fatty acid synthesis with respect to the production of nuclear membranes to maintain the enlarged nuclei in infected cells.
In Aim 2 we will define the role of GRP78, SUN domain and KASH domain proteins in the assembly-egress continuum. We previously established the importance of GRP78 in assembly compartment formation/integrity and have established a link between assembly compartment-localized GRP78, lamin phosphorylation and lamina reorganization characteristic of infected cells (5, 6). We propose mutational analysis of GRP78 to further understand the role(s) of its functional domains (DNAJ, protein binding, ATPase and the KDEL ER retention signal)(1, 64) in AC formation/localization, lamina phosphorylation/rearrangement and nuclear morphology. SUN and KASH domain proteins span the nuclear envelope and connect with the cytoskeleton. Our data (5) show that SUN domain protein depletion during HCMV infection facilitates alteration in nuclear membrane structure. The role of SUN and KASH domain proteins in the assembly-egress continuum will be further examined.
In order to better understand the molecular pathogenesis of human cytomegalovirus (HCMV) it is essential to determine the means by which the virus alters key cellular features such as nuclear structure and cytoplasmic organization. Such understanding will elucidate new aspects of HCMV virology and pathogenesis, and will highlight new targets for therapeutic intervention in HCMV disease.
|Alwine, James C (2012) The human cytomegalovirus assembly compartment: a masterpiece of viral manipulation of cellular processes that facilitates assembly and egress. PLoS Pathog 8:e1002878|
|Clippinger, Amy J; Alwine, James C (2012) Dynein mediates the localization and activation of mTOR in normal and human cytomegalovirus-infected cells. Genes Dev 26:2015-26|