Point mutations in BHV-1 Us3 gene abolish its ability to induce cytoskeletal changes in various cell types.

The Us3 gene is conserved among alphaherpesviruses and codes for a protein kinase, a multifunctional protein involved in many phases of virus infection, like nuclear egress, modulation of apoptosis and modification of the cellular cytoskeleton. Bovine herpesvirus (BHV-1), a member of the Alphaherpesvirinae, contains an open reading frame homologous to Us3 of other herpesviruses, which has been identified as a serine/threonine kinase (Takashima, Y., Tamura, H., Xuan, X., Otsuka, H., 1999. Identification of the Us3 gene product of BHV-1 as a protein kinase and characterization of BHV-1 mutants of the Us3 gene. Virus Res. 59, 23-34). To study the activity of BHV-1 Us3, we have cloned its sequence under control of the human cytomegalovirus (HCMV) promoter/enhancer and introduced it into a recombinant baculovirus (Bac Us3). Confocal microscopy analysis showed profound cytoskeletal modifications in various BHV-1-permissive and non-permissive cells transduced with BacUs3. We observed that Us3 expression changed cellular shape and induced formation of long microtubule-containing cell projections, a phenomenon which had also been observed in cells expressing pseudorabies virus Us3. The intracellular localization of Us3 was mostly nuclear but when the protein accumulated it could be detected in the cytoplasm, cell membranes and projections. Mutated forms of BHV-1 Us3 with point mutations near or within the kinase catalytic domain did not affect cell morphology indicating that kinase activity of BHV-1 Us3 is required for its cytoskeleton remodelling function.

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