822B Mary Ellen Jones, CB #7290
We have recently been addressing to aspects of herpes simplex virus type 1 (HSV-1) replication: the mechanism of induction of NFkB and the activation of the DNA double-strand break response (DBS). We have evidence that failure to induce NFkB leads to decreased viral yields, and therefore that activation is critical for efficient virus replication. In the case of the DNA damage response, HSV-1 infection leads to destruction of Mre11, a component of the DNA damage sensor MRN. This result suggests that HSV-1 has evolved a mechanism for inactivation DDR because it otherwise targets for inactivation a critical step in virus replication.
The activation of NFkB following infection is bi-phasic: an immediate activation dependent on attachment and entry of HSV-1 into cells, and a delayed activation dependent on the expression of early and/or late viral proteins. We have identified the viral immediate-early protein ICP27 as sufficient, and specifically mapped a small acidic domain near the N-terminus as necessary, for delayed activation of NFkB. Our current studies are focused on how this viral protein activates signal transduction pathways leading to NFkB activation.
We have continued to characterize the host DNA double-strand break response following HSV infection. We previously demonstrated that HSV induced the DBS response, including activation of (i) ATM, (ii) the heterotrimeric MRN damage sensor, and (iii) CHK2. We demonstrated that Mre11, a critical component MRN, was considerably reduced, coincident with the onset of viral DNA replication, and that this loss occurred through a proteosome-independent mechanism. ATM kinase is required for activation of MRN; treatment of infected cells with a highly specific ATM inhibitor prevented phosphorylation of Nbs1, another component of the MRN, and also prevented loss of MRE11. Under these conditions virus yield was also reduced ~99%, suggesting that activation of MRN was a critical cellular function required for virus replication.
We previously observed that HSV-1 infected cells accumulate in the G1 phase of the cell cycle, and that cdk2, an important effector kinase in G1-S phase progression is inhibited. Current studies are focusing on the mechanism of cyclin kinase inhibition, and the identity of viral proteins and cellular pathways required for cell cycle regulation. The phosphatase cdc25A is required for G1-S progression through its ability to remove inhibitory phosphates from cdk2. CHK2, activated as part of the DBS response is also part of the cell cycle checkpoint pathway through its ability to phosphorylate and inactivate cdc25A, thus blocking G1 to S phase progression. We are investigating the possibility that cdc25A is inactivated in a CHK2-dependent manner leading to the inhibition of cdk2 activity.
Gregory, D.A., and S.L. Bachenheimer (2008) Characterization of Mre11 loss following HSV-1 infection. /Virology/ 373:124-136.
Hargett, T., S. Rice, and S.L. Bachenheimer (2006) Herpes simplex virus type 1 ICP27-dependent activation of NF-kB. J. /Virology/ 80: 10565-10578.
Hargett, D., T. McLean, and S. L. Bachenheimer. (2005) Herpes simplex virus ICP27 activation of stress kinases JNK and p38. /J. Virology/ 79: 8348-8360.
Gregory, D., D. Hargett, D. Holmes, E. Money, and S.L. Bachenheimer. (2004) Efficient replication by herpes simplex virus type 1 involves activation of the IKK-IkB-p65 pathway. /J. Virology/ 78: 13582-13590.
Karaca, G., D. Hargett, T.I. McLean, J.S. Aguilar, P. Ghazal, E.K. Wagner, and S.L. Bachenheimer. (2004) Inhibition of the stress-activated kinase, p38 does not affect the virus transcriptional program of herpes simplex virus type 1. /Virology/ 329:143-156.