Cellular Substrates of the Herpes Simplex Virus Ser/Thr Kinase Us3: Identification, Characterization and Implications for Virus Biology

It is common for viruses to encode multifunctional proteins to extract maximum functionality from limited coding regions. One great example is the HSV Ser/Thr kinase Us3. The 66kDA protein is required for nuclear egress, prevention of apoptosis and as we show here, for proper viral protein synthesis. Although Us3 displays no sequence homology with any cellular kinases, Us3 mimics Akt activity. Direct inhibitory phosphorylations of Akt substrates TSC2 and PRAS40 by Us3 are required for constitutive activation of mTORC1, a major protein synthesis regulator. Importantly, mTORC1 inhibition reduces viral protein synthesis and replication. Further upstream, knockdown of TSC2 improves replication of Us3-deficient virus, suggesting the attenuation of Us3 deficient virus is at least in part due to its inability to counteract TSC2.;We also show Us3 is responsible for the down-regulation of MAPK signaling. Unlike p38 and JNK, which require ICP27 for activation in infection, Erk activity is suppressed in HSV1 infection by an unknown mechanism. Here, we establish that Us3 is necessary and sufficient to suppress Erk in absence of other viral factors. Although we do not know the substrates involved, regulation of Erk signaling defines yet another role for the viral kinase.;While prior studies have identified single Us3 substrates, we combined substrate label and covalent capture techniques to identify over 220 cellular candidate substrates, including known targets TSC2 and Lamin A/C. These candidate substrates represented an impressive array of biological functions ranging from apoptosis to chromatin remodeling. The challenge now is to tease apart those phosphorylation events that are meaningful to virus biology from those that are simply biological noise associated with the catalytic activity of Us3.;Lastly, given the importance of Us3 in replication and the uniqueness of its sequence among viral and cellular Ser/Thr kinases, Us3 could make an ideal antiviral target. Others have suggested the use of mTORC1 inhibitors in antiviral strategies, however direct inhibition of mTORC1 is associated with immunosuppressive side effects. Agents targeting Us3, the factor responsible for mTORC1 activation in HSV-infected cells, have the potential to achieve the same efficacy without the safety concerns associated with targeting a crucial host factor.