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...

Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and pseudorabies virus (PrV), was investigated by electron microscopy of infected cell lines of different origins. In all virus-cell systems analyzed, similar observations were made concerning the different stages of virion morphogenesis. After intranuclear assembly, nucleocapsids bud at the inner leaflet of the nuclear membrane, resulting in enveloped particles in the perinuclear space that contain a sharply bordered rim of tegument and a smooth envelope surface. Egress from the perinuclear cisterna primarily occurs by fusion of the primary envelope with the outer leaflet of the nuclear membrane, which has been visualized for HSV-1 and EHV-1 for the first time. The resulting intracytoplasmic naked nucleocapsids are enveloped at membranes of the trans-Golgi network (TGN), as shown by immunogold labeling with a TGN-specific antiserum. Virions containing their final envelope differ in morphology from particles within the perinuclear cisterna by visible surface projections and a diffuse tegument. Particularly striking was the addition of a large amount of tegument material to ILTV capsids in the cytoplasm. Extracellular virions were morphologically identical to virions within Golgi-derived vesicles, but distinct from virions in the perinuclear space. Studies with gB-and gH-deleted PrV mutants indicated that these two glycoproteins, which are essential for virus entry and direct cell-to-cell spread, are dispensable for egress. Taken together, our studies indicate that the deenvelopment-reenvelopment process of herpesvirus maturation also occurs in EHV-1, HSV-1, and ILTV and that membrane fusion processes occurring during egress are substantially different from those during entry and direct viral cell-to-cell spread.Herpesviruses are large DNA-containing enveloped viruses that replicate in the nuclei of infected cells. Based on biological parameters and sequence data, the family Herpesviridae is divided into the subfamilies Alpha-, Beta-, and Gammaherpesvirinae (58). Despite their biological diversity, many steps of herpesvirus morphogenesis seem to be conserved. Attached herpesvirus virions penetrate the cell membrane by direct fusion between the viral envelope and the plasma membrane, and the deenveloped nucleocapsids reach the nucleopores by movement along cellular microtubuli, where the genomic DNA is released into the nucleus (19,50,51,52). Progeny nucleocapsids assemble in the nucleus and exit this compartment by budding at the inner nuclear membrane into the perinuclear space (35,47). The subsequent events of envelopment and egress remain controversial. Whereas for herpes simplex virus type 1 (HSV-1), a model that entails vesicular transport of enveloped virions through the secretory pathway with concomitant in situ modification of virion glycoproteins was initially proposed, for varicella-zoster virus (VZV), pseudorabies virus (PrV), and human cytomegalovir...

Although the nuclear envelope is a dynamic structure that disassembles and reforms during mitosis, the formation of membranous vesicles derived from the nuclear envelope has not yet been described in noninfected cells. However, during herpesvirus maturation, intranuclear capsids initiate transit to the cytosol for final maturation by budding at the inner nuclear membrane. Two conserved herpesvirus proteins are required for this primary envelopment, designated in the alphaherpesviruses as pUL31 and pUL34. Here, we show that simultaneous expression of pUL31 and pUL34 of the alphaherpesvirus pseudorabies virus in stably transfected rabbit kidney cells resulted in the formation of vesicles in the perinuclear space that resemble primary envelopes without a nucleocapsid. They contain pUL31 and pUL34 as shown by immunolabeling and are derived from the nuclear envelope. Thus, coexpression of only two conserved herpesvirus proteins without any other viral factor is sufficient to induce the formation of vesicles from the nuclear membrane. This argues for the contribution of cellular factors in this process either recruited from their natural cytoplasmic location or not yet identified as components of the nuclear compartment.nuclear envelope ͉ primary envelopment ͉ pseudorabies virus ͉ herpesvirus egress H erpesvirus particles are complex macromolecular assemblies consisting of Ͼ30 virally encoded proteins, which make up four morphologically distinct structures, core, capsid, tegument, and envelope. Herpesvirus morphogenesis proceeds in two different cellular compartments. While capsid assembly and DNA packaging take place in the nuclei of infected cells, acquisition of the majority of tegument and final envelopment occur in the cytoplasm. To gain access to the cytoplasm the nucleocapsid has to traverse the nuclear lamina and the inner and outer nuclear membranes, because the diameter of the nuclear pores is too small to allow exit of the Ϸ110-nm particle. Although alternative mechanisms for nuclear egress have been proposed, most data support a model that entails primary envelopment by budding of capsids at the inner nuclear membrane resulting in the formation of primary virions in the perinuclear space whose envelope then fuses with the outer nuclear membrane to release the capsid into the cytoplasm (reviewed in ref. 1).The molecular mechanism of this budding, scission, and fusion reaction is unknown. Glycoproteins gB and gH, which are essential for fusion of the viral envelope with the plasma membrane during entry (2) as well as for cell-cell spread, are not required for virion formation indicating that a different molecular mechanism is responsible for fusion during nuclear egress (3). Cellular fusion processes involving the nuclear membranes occur during mitosis. However, the fusion machinery involved is unknown. Soluble N-ethylmaleimide sensitive factor attachment protein receptors ( Two conserved herpesvirus proteins, in the alphaherpesviruses designated as pUL31 and pUL34, are required for nuclear egress of herpesv...

The UL36 open reading frame encoding the tegument protein ICP1/2 represents the largest open reading frame in the genome of herpes simplex virus type 1 (HSV-1). Polypeptides homologous to the HSV-1 UL36 protein are present in all subfamilies of Herpesviridae. We sequenced the UL36 gene of the alphaherpesvirus pseudorabies virus (PrV) and prepared a monospecific polyclonal rabbit antiserum against a bacterial glutathione S-transferase (GST)-UL36 fusion protein for identification of the protein. The antiserum detected a >300-kDa protein in PrV-infected cells and in purified virions. Interestingly, in coprecipitation analyses using radiolabeled infected-cell extracts, the anti-UL36 serum reproducibly coprecipitated the UL37 tegument protein, and antiserum directed against the UL37 protein coprecipitated the UL36 protein. Infectious herpesvirus particles contain more than 30 virusencoded proteins which are assembled into the four morphologically differentiable components of the herpesvirus virion: the inner nucleoprotein core containing the double-stranded DNA genome, the icosahedral capsid shell, the tegument located between the capsid and envelope, and the lipid envelope containing viral (glyco)proteins (reviewed in reference 31). The requirements and molecular interactions which result in formation of herpesvirus capsids are well characterized (reviewed in references 16 and 36). However, much less is known about the molecular details of tegumentation or envelopment. It is now generally accepted that herpesvirus particles mature via an envelopment-deenvelopment-reenvelopment pathway (reviewed in reference 28). Intranuclear capsids bud through the inner nuclear membrane, thereby acquiring a primary envelope and, presumably, also a primary tegument (17). This budding process requires the presence of the products of the UL31 and UL34 genes of herpes simplex virus type 1 (HSV-1) (6, 30, 32) and pseudorabies virus (PrV) (14,20). Primary enveloped virions differ in morphology (15, 17) and biochemical composition (20, 35) from mature virus particles. This can be explained by loss of the primary envelope and at least part of the primary tegument by fusion of the primary envelope with the outer leaflet of the nuclear membrane and by translocation of capsids into the cytoplasm. Final tegumentation then occurs in the cytoplasm, and capsids acquire a final envelope by budding into trans-Golgi vesicles (reviewed in references 13 and 28).Although the overall pathway of herpesvirus virion maturation appears clear, the molecular details are largely unknown. For example, in HSV-1, more than 15 proteins have been hypothesized or demonstrated to be components of the tegument, and more than 11 virally encoded proteins reside in the virion envelope (reviewed in references 28 and 36). How these complex structures are assembled into a functional virus particle is currently under intense study. Whereas herpesvirus capsids exhibit icosahedral symmetry, it has long been assumed on the basis of electron microscopic evidence that the tegumen...

The pseudorabies virus (PrV) homolog of the tegument protein encoded by the UL48 gene of herpes simplex virus type 1 (HSV-1) was identified by using a monospecific rabbit antiserum against a bacterial fusion protein.UL48-related polypeptides of 53, 55, and 57 kDa were detected in Western blots of infected cells and purified virions. Immunofluorescence studies demonstrated that the PrV UL48 protein is predominantly localized in the cytoplasm but is also found in the nuclei of infected cells. Moreover, it is a constituent of extracellular virus particles but is absent from primary enveloped perinuclear virions. In noncomplementing cells, a UL48-negative PrV mutant (PrV-⌬UL48) exhibited delayed growth and significantly reduced plaque sizes and virus titers, deficiencies which were corrected in UL48-expressing cells. RNA analyses indicated that, like its HSV-1 homolog, the PrV UL48 protein is involved in regulation of immediate-early gene expression. However, the most salient effect of the UL48 gene deletion was a severe defect in virion morphogenesis. Late after infection, electron microscopy of cells infected with PrV-⌬UL48 revealed retention of newly formed nucleocapsids in the cytoplasm, whereas enveloped intracytoplasmic or extracellular complete virions were only rarely observed. In contrast, capsidless particles were produced and released in great amounts. Remarkably, the intracytoplasmic capsids were labeled with antibodies against the UL36 and UL37 tegument proteins, whereas the capsidless particles were labeled with antisera directed against the UL46, UL47, and UL49 tegument proteins. These findings suggested that the UL48 protein is involved in linking capsid and future envelope-associated tegument proteins during virion formation. Thus, like its HSV-1 homolog, the UL48 protein of PrV functions in at least two different steps of the viral life cycle. The drastic inhibition of virion formation in the absence of the PrV UL48 protein indicates that it plays an important role in virion morphogenesis prior to secondary envelopment of intracytoplasmic nucleocapsids. However, the UL48 gene of PrV is not absolutely essential, and concomitant deletion of the adjacent tegument protein gene UL49 also did not abolish virus replication in cell culture.Pseudorabies virus (PrV, suid herpesvirus 1), the causative agent of Aujeszky's disease of pigs (41), has been classified as a member of the Varicellovirus genus within the Alphaherpesvirinae subfamily of Herpesviridae (49). Although the DNA sequence of the ca. 150-kbp genome of PrV has not yet been elucidated completely, gene content and arrangement appear to be widely similar to those of other alphaherpesviruses including herpes simplex virus type 1 (HSV-1) (38), and therefore the gene nomenclature established for HSV-1 has been adopted (41).PrV virions exhibit the typical morphology of herpesvirus particles (26). The nucleoprotein core containing the DNA genome is enclosed in an icosahedral capsid shell. The capsid is surrounded by a protein layer named the tegument a...