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

Abstract

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 was identified as a nuclear protein with an apparent molecular mass of 29 kDa. For functional analysis, UL31 was deleted by mutagenesis in Escherichia coli of an infectious full-length clone of the PrV genome. The resulting virus mutants were deficient in plaque formation, and titers were reduced more than 100-fold from those of wild-type PrV. Ultrastructural analyses demonstrated that capsid maturation and DNA packaging were not affected. However, neither budding at the inner nuclear membrane nor cytoplasmic or extracellular virus particles were observed. These replication defects were similar to those of a UL34 deletion mutant (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063-10073, 2000) and could be completely repaired in a cell line which constitutively expresses the UL31 protein. Yeast two-hybrid studies revealed that a UL31 fusion protein specifically interacts with plasmids of a PrV genome library expressing the N-terminal part of UL34. Vice versa, UL34 selected UL31-encoding plasmids from the library. Immunofluorescence studies and immune electron microscopy demonstrated that in cells infected with wild-type PrV, both proteins accumulate at the nuclear membrane, whereas in the absence of UL34 the UL31 protein is dispersed throughout the nucleus. Like the UL34 protein, the UL31 gene product is a component of enveloped virus particles within the perinuclear space and absent from mature virions. Our findings suggest that physical interaction between these two virus proteins might be a prerequisite for primary envelopment of PrV at the inner nuclear membrane and that this envelope is removed by fusion with the outer nuclear membrane.Pseudorabies virus (PrV, suid herpesvirus 1) is the causative agent of Aujeszky's disease in pigs (36). It is classified as a member of the Varicellovirus genus within the Alphaherpesvirinae subfamily of Herpesviridae (43). Herpesvirus particles are composed of an icosahedral capsid which encloses the doublestranded DNA genome and which in turn is surrounded by a layer of numerous viral gene products named tegument and a lipid membrane of cellular origin containing mostly glycosylated virus-encoded proteins (36, 43). After DNA replication and encapsidation in the host-cell nucleus, virus egress starts with budding of nucleocapsids at the inner nuclear membrane, leading to enveloped particles in the perinuclear space (44). It has been proposed that these perinuclear virions are released by vesicular transport through the endoplasmic reticulum and Golgi apparatus (26, 51), which would permit processing of viral glycoproteins, but not substitution of envelope or tegument proteins. According to another hypothesis, the primary envelope of herpesviruses is lost by fusion with the outer nuclear membrane, followed by release of naked nucleocapsids i...

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Transport and egress of herpes simplex virus in neurons

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