Roles of the different isoforms of the pseudorabies virus protein kinase pUS3 in nuclear egress

Protein kinases homologous to the US3 gene product (pUS3) of herpes simplex virus (HSV) are conserved throughout the alphaherpesviruses but are absent from beta- and gammaherpesviruses. pUS3 homologs are multifunctional and involved in many processes including modification of the cytoskeleton, inhibition of apoptosis and in immune evasion. pUS3 also plays a role in efficient nuclear egress of alphaherpesvirus nucleocapsids. In the absence of pUS3 primary enveloped virions accumulate in the perinuclear space (PNS) in large invaginations of the inner nuclear membrane (INM) pointing to a modulatory function for pUS3 during deenvelopment. The HSV and Pseudorabies virus (PrV) US3 gene is transcribed into two mRNAs encoding different isoforms, which vary in aminoterminal sequences and abundance. To test whether the two isoforms in PrV serve different functions, we constructed mutant viruses expressing exclusively either the larger but minor or the smaller major isoform, a mutant virus with decreased expression of the smaller isoform or a mutant with impaired kinase function. Respective virus mutants were investigated in several cell lines. Our results show that absence of the larger pUS3 isoform has no detectable effect on viral replication in cell culture while full expression of the smaller isoform and intact kinase activity is required for efficient nuclear egress. Absence of pUS3 resulted in only minor titer reduction in most cell lines tested, but disclosed a more severe defect in MDBK cells. However, accumulations of primary virions in the PNS do not account for the observed titer reduction in PrV.