The UL4 gene of pseudorabies virus encodes a minor infected-cell protein that is dispensable for virus replication

Although homologues of the open reading frame (ORF) UL4 of herpes simplex virus 1 (Human herpesvirus 1) have been found in the genomes of all hitherto-analysed alphaherpesviruses, little is known about their function. In a project to analyse systematically, in an isogenic and standardized assay system, the gene products of the alphaherpesvirus pseudorabies virus (PrV; Suid herpesvirus 1), the PrV UL4 gene product was identified using a monospecific rabbit antiserum prepared against a bacterial fusion protein. Western blot and immunofluorescence analyses revealed that the 146 codon UL4 ORF of PrV was translated into a nuclear 15 kDa protein which was detectable from 6 In after infection of rabbit kidney cells, but was not found in purified virus particles. For functional analysis, a UL4-negative virus recombinant (PrV-Delta UL4F) was generated by mutagenesis of an infectious full-length clone of the PrV genome in E coli. PrV-Delta UL4F was replication-competent in rabbit kidney cells, and plaque formation was not affected by the mutation. However, maximum virus titres of PrV-Delta UL4F were decreased about fivefold compared with wild-type PrV, and electron microscopy of infected cells demonstrated an impairment of release of mature virions. This growth defect of PrV-Delta UL4F could be corrected completely by propagation in UI-4-expressing cells. Correlating with the inconspicuous in vitro phenotype, neurovirulence of PrV-Delta UL4F was also not affected significantly. Thus, UL4 encodes a non-structural protein of PrV that enhances virion formation but is not essential for PrV replication in vitro or in vivo