Cells infected with wild-type herpes simplex virus type 1 (HSV-1) show disruption of the organization of the nuclear lamina that underlies the nuclear envelope. This disruption is reflected in changes in the localization and phosphorylation of lamin proteins. Here, we show that HSV-1 infection causes relocalization of the LEM domain protein emerin. In cells infected with wild-type virus, emerin becomes more mobile in the nuclear membrane, and in cells infected with viruses that fail to express UL34 protein (pUL34) and US3 protein (pUS3), emerin no longer colocalizes with lamins, suggesting that infection causes a loss of connection between emerin and the lamina. Infection causes hyperphosphorylation of emerin in a manner dependent upon both pUL34 and pUS3. Some emerin hyperphosphorylation can be inhibited by the protein kinase C␦ (PKC␦) inhibitor rottlerin. Emerin and pUL34 interact physically, as shown by pull-down and coimmunoprecipitation assays. Emerin expression is not, however, necessary for infection, since virus growth is not impaired in cells derived from emerin-null transgenic mice. The results suggest a model in which pUS3 and PKC␦ that has been recruited by pUL34 hyperphosphorylate emerin, leading to disruption of its connections with lamin proteins and contributing to the disruption of the nuclear lamina. Changes in emerin localization, nuclear shape, and lamin organization characteristic of cells infected with wild-type HSV-1 also occur in cells infected with recombinant virus that does not make viral capsids, suggesting that these changes occur independently of capsid envelopment.During primary envelopment, herpes simplex virus type 1 (HSV-1) nucleocapsids translocate from the nucleus to the cytoplasm by budding into the inner nuclear membrane and then fusing with the outer nuclear membrane. The capsid does not, however, have unimpeded access to the inner nuclear membrane. Lining the inside of the inner nuclear membrane is the nuclear lamina, which is composed of a meshwork of proteins with spaces too small for the capsid to move through without some disruption (2,19,65). The lamina meshwork is made up of intermediate filament family proteins called lamins that are linked to the inner nuclear membrane and to intranuclear proteins by association with lamin-associated proteins (LAPs) (reviewed in reference 65). Connection of the network of lamin proteins to the inner nuclear membrane is mediated by integral membrane LAPs, including emerin, lamin B receptor, LAP2-␤, and MAN-1 (26).Emerin is a member of a family of nuclear envelope proteins that share a common sequence called the LEM domain that mediates association with BAF (barrier to autointegration factor) and is important for the assembly of LEM domain proteins into the re-forming nuclear envelope following mitosis (21,37,39). Emerin also contains a lamin-binding domain that helps retain it in the interphase nuclear envelope (6,14,25,37). Emerin is ubiquitously expressed but is not essential for the viability of cells in culture (36). Failure to ...

Hsp90 functions to facilitate the folding of newly synthesized and denatured proteins. Hsp90 function is modulated through its interactions with cochaperones and the binding and hydrolysis of ATP. Recently, novobiocin has been shown to bind to a second nucleotide binding site located within the C-terminal domain of Hsp90. In this report, we have examined the effect of novobiocin on Hsp90 function in reticulocyte lysate. Novobiocin specifically inhibited the maturation of the heme-regulated eIF2alpha kinase (HRI) in a concentration-dependent manner. Novobiocin induced the dissociation of Hsp90 and Cdc37 from immature HRI, while the Hsp90 cochaperones p23, FKBP52, and protein phosphatase 5 remained associated with immature HRI. Proteolytic fingerprinting of Hsp90 indicated that novobiocin had a distinct effect on the conformation of Hsp90, and molybdate lowered the concentration of novobiocin required to alter Hsp90's conformation by 10-fold. The recombinant C-terminal domain of Hsp90 adopted a proteolytic resistant conformation in the presence of novobiocin, indicating that alteration of Hsp90/cochaperone interactions was not the cause of the novobiocin-induced protease resistance within Hsp90's C-terminal domain. The concentration dependence of this novobiocin-induced conformation change correlated with the dissociation of Hsp90 and Cdc37 from immature HRI and novobiocin-induced inhibition of Hsp90/Cdc37-dependent activation of HRI's autokinase activity. The data suggest that binding of novobiocin to the C-terminal nucleotide binding site of Hsp90 induces a change in Hsp90's conformation leading to the dissociation of bound kinase. The unique structure and properties of novobocin-bound Hsp90 suggest that it may represent the "client-release" conformation of the Hsp90 machine.

The nuclear lamina is thought to be a steric barrier to the herpesvirus capsid. Disruption of the lamina accompanied by phosphorylation of lamina proteins is a conserved feature of herpesvirus infection. In HSV-1-infected cells, protein kinase C (PKC) alpha and delta isoforms are recruited to the nuclear membrane and PKC delta has been implicated in phosphorylation of emerin and lamin B. We tested two critical hypotheses about the mechanism and significance of lamina disruption. First, we show that chemical inhibition of all PKC isoforms reduced viral growth five-fold and inhibited capsid egress from the nucleus. However, specific inhibition of either conventional PKCs or PKC delta does not inhibit viral growth. Second, we show hyperphosphorylation of emerin by viral and cellular kinases is required for its disassociation from the lamina. These data support hypothesis that phosphorylation of lamina components mediates lamina disruption during HSV nuclear egress.

First, I would like thank Rich for his guidance along this path. My character is exponentially larger after these four years, although so are the number of wrinkles on my brow. Second, my thesis committee has been extremely instrumental in the development of my scientific and professional identity. Thank you for sending my comp's abstract back! My wonderful and caring father deserves all the credit for my work ethic and love of biology. Thank you for helping me with my homework, driving me to Wal-mart for science project supplies, and always believing in me even when I did not. Thank you, Aunt Barbara for being my protector and teacher. Your true unconditional love and confidence in me has made a world of difference in my life. To all my teachers, thank you for helping me build confidence, grow under your love (or hate), and for taking the extra step to inspire a geeky kid from the sticks. Here's to you Mrs, Carmichael, Dr. Nelson, Dr. Hartson, Mrs. Blanton, Mrs. King, and Mrs. Howard. To Sarah, my best friend, thank you for always being there for me and for always being you. I would not have made it with out you. To the love of my life, Dan, thank you for coffee, inspiration and helping me see reality. Let us always share science and humor together.