The interacting UL31 and UL34 gene products of pseudorabies virus are involved in egress from the host-cell nucleus and represent components of primary enveloped but not mature virions

A 2.6-kbp fragment of the pseudorabies virus (PrV) genome was sequenced and shown to contain the homologues of the highly conserved herpesvirus genes UL31 and UL32. By use of a monospecific antiserum, the UL31 gene product wa s identified as a nuclear protein with an apparent molecular mass of 29 kDa . For functional analysis, UL31 was deleted by mutagenesis in Escherichia c oli of an infectious full-length clone of the PrV genome. The resulting vir us mutants were deficient in plaque formation, and titers were reduced more than 100-fold from those of wild-type PrV. Ultrastructural analyses demons trated that capsid maturation and DNA packaging were not affected. However, neither budding at the inner nuclear membrane nor cytoplasmic or extracell ular virus particles were observed. These replication defects were similar to those of a UL34 deletion mutant (B. G. Klupp, H. Granzow, and T. C. Mett enteiter, J. Virol. 74:10063-10073, 2000) and could be completely repaired in a cell line which constitutively expresses the UL31 protein. Yeast two-h ybrid studies revealed that a UL31 fusion protein specifically interacts wi th plasmids of a PrV genome library expressing the N-terminal part of UL34. Vice versa, UL34 selected UL31-encoding plasmids from the library. Immunof luorescence studies and immune electron microscopy demonstrated that in cel ls infected with wild-type PrV, both proteins accumulate at the nuclear mem brane, whereas in the absence of UL34 the UL31 protein is dispersed through out the nucleus. Like the UL34 protein, the UL31 gene product is a componen t of enveloped virus particles within the perinuclear space and absent from mature virions. Our findings suggest that physical interaction between the se two virus proteins might be a prerequisite for primary envelopment of Pr V at the inner nuclear membrane and that this envelope is removed by fusion with the outer nuclear membrane.