Identification, subviral localization, and functional characterization of the pseudorabies virus UL17 protein

Homologs of the UL17 gene of the alphaherpesvirus herpes simplex virus 1 (HSV-1) are conserved in all three subfamilies of herpesviruses. However, only the HSV-1 protein has so far been characterized in any detail. To analyze UL17 of pseudorabies virus (PrV) the complete 597-amino-acid protein was expressed in Escherichia coli and used for rabbit immunization. The antiserum recognized a 64-kDa protein in PrV-infected cell lysates and purified virions, identifying PrV UL17 as a structural virion component. In indirect immunofluorescence analyses of PrV-infected cells the protein was predominantly found in the nucleus. In electron microscopic studies after immunogold labeling of negatively stained purified virion preparations, UL17-specific label was detected on single, mostly damaged capsids, whereas complete virions and the majority of capsids were free of label. In ultrathin sections of infected cells, label was primarily found dispersed around scaffold-containing B-capsids, whereas on DNA-filled C-capsids it was located in the center. Empty intranuclear A-capsids were free of label, as were extracellular capsid-less L-particles. Functional characterization of PrV-DeltaUL17F, a deletion mutant lacking codons 23 to 444, demonstrated that cleavage of viral DNA into unit-length genomes was inhibited in the absence of UL17. In electron microscopic analyses of PrV-DeltaUL17F-infected RK13 cells, DNA-containing capsids were not detected, while numerous capsidless L-particles were observed. In summary, our data indicate that the PrV UL17 protein is an internal nucleocapsid protein necessary for DNA cleavage and packaging but suggest that the protein is not a prominent part of the tegument.     

The UL7 gene of pseudorabies virus encodes a nonessential structural protein which is involved in virion formation and egress

Homologues of the UL7 gene of herpes simplex virus type 1 are conserved in alpha-, beta-, and gammaherpesviruses. However, little is known about their functions. Using a monospecific rabbit antiserum raised against a bacterial fusion protein, we identified the UL7 gene product of the neurotropic alphaherpesvirus pseudorabies virus (PrV). In Western blot analyses of infected cells and purified PrV particles the serum specifically detected a 29-kDa protein, which matches the calculated mass of the 266-amino-acid translation product of PrV UL7. For functional analysis, UL7 was deleted by mutagenesis of an infectious full-length clone of the PrV genome in Escherichia coli. The obtained recombinant PrV-DeltaUL7F was replication competent in rabbit kidney cells, but maximum virus titers were decreased nearly 10-fold and plaque diameters were reduced by ca. 60% compared to wild-type PrV. Electron microscopy of infected cells revealed that in the absence of UL7, formation and nuclear egress of nucleocapsids were not affected, whereas secondary envelopment of cytoplasmic nucleocapsids appeared to be delayed and release of mature virions was less efficient. The observed replication defects were corrected by repair of the viral UL7 gene or by propagation of PrV-DeltaUL7F in UL7-expressing cells. PrV-DeltaUL7F was moderately attenuated in mice. Compared to wild-type virus, mean survival times were prolonged from 2 to 3 days after intranasal infection. However, neuroinvasion and transneuronal spread of PrV were not abolished in the absence of UL7. Thus, UL7 encodes a virion protein of PrV, which plays a role during virion maturation and egress both in vitro and in vivo.     

Egress of alphaherpesviruses: comparative ultrastructural study

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.     

Essential function of the pseudorabies virus UL36 gene product is independent of its interaction with the UL37 protein

The large tegument protein encoded by the UL36 gene of pseudorabies virus (PrV) physically interacts with the product of the adjacent UL37 gene (B. G. Klupp, W. Fuchs, H. Granzow, R. Nixdorf, and T. C. Mettenleiter, J. Virol. 76:3065-3071, 2002). To analyze UL36 function, two PrV recombinants were generated by mutagenesis of an infectious PrV full-length clone in Escherichia coli: PrV-DeltaUL36F exhibited a deletion of virtually the complete UL36 coding region, whereas PrV-UL36BSF contained two in-frame deletions of 238 codons spanning the predicted UL37 binding domain. Coimmunoprecipitation experiments confirmed that the mutated gene product of PrV-UL36BSF did not interact with the UL37 protein. Like the previously described PrV-DeltaUL37 (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 75:8927-8936, 2001) but in contrast to PrV-DeltaUL36F, PrV-UL36BSF was able to replicate in rabbit kidney (RK13) cells, although maximum virus titers were reduced ca. 50-fold and plaque diameters were reduced by ca. 45% compared to wild-type PrV. PrV-DeltaUL36F was able to productively replicate after repair of the deleted gene or in a trans-complementing cell line. Electron microscopy of infected RK13 cells revealed that PrV-UL36BSF and phenotypically complemented PrV-DeltaUL36F were capable of nucleocapsid formation and egress from the nucleus by primary envelopment and deenvelopment at the nuclear membrane. However, reenvelopment of nucleocapsids in the cytoplasm was blocked. Only virus-like particles without capsids were released efficiently from cells. Interestingly, cytoplasmic nucleocapsids of PrV-UL36BSF but not of PrV-DeltaUL36F were found in large ordered structures similar to those which had previously been observed with PrV-DeltaUL37. In summary, our results demonstrate that the interaction between the UL36 and UL37 proteins is important but not strictly essential for the formation of secondary enveloped, infectious PrV particles. Furthermore, UL36 possesses an essential function during virus replication which is independent of its ability to bind the UL37 protein.     

Effects of Simultaneous Deletion of pUL11 and Glycoprotein M on Virion Maturation of Herpes Simplex Virus Type 1

The conserved membrane-associated tegument protein pUL11 and envelope glycoprotein M (gM) are involved in secondary envelopment of herpesvirus nucleocapsids in the cytoplasm. Although deletion of either gene had only moderate effects on replication of the related alphaherpesviruses herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV) in cell culture, simultaneous deletion of both genes resulted in a severe impairment in virion morphogenesis of PrV coinciding with the formation of huge inclusions in the cytoplasm containing nucleocapsids embedded in tegument (M. Kopp, H. Granzow, W. Fuchs, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 78:3024-3034, 2004). To test whether a similar phenotype occurs in HSV-1, a gM and pUL11 double deletion mutant was generated based on a newly established bacterial artificial chromosome clone of HSV-1 strain KOS. Since gM-negative HSV-1 has not been thoroughly investigated ultrastructurally and different phenotypes have been ascribed to pUL11-negative HSV-1, single gene deletion mutants were also constructed and analyzed. On monkey kidney (Vero) cells, deletion of either pUL11 or gM resulted in ca.-fivefold-reduced titers and 40- to 50%-reduced plaque diameters compared to those of wild-type HSV-1 KOS, while on rabbit kidney (RK13) cells the defects were more pronounced, resulting in ca.-50-fold titer and 70% plaque size reduction for either mutant. Electron microscopy revealed that in the absence of either pUL11 or gM virion formation in the cytoplasm was inhibited, whereas nuclear stages were not visibly affected, which is in line with the phenotypes of corresponding PrV mutants. Simultaneous deletion of pUL11 and gM led to additive growth defects and, in RK13 cells, to the formation of large intracytoplasmic inclusions of capsids and tegument material, comparable to those in PrV-ΔUL11/gM-infected RK13 cells. The defects of HSV-1ΔUL11 and HSV-1ΔUL11/gM could be partially corrected in trans by pUL11 of PrV. Thus, our data indicate that PrV and HSV-1 pUL11 and gM exhibit similar functions in cytoplasmic steps of virion assembly.     

Pseudorabies virus UL36 tegument protein physically interacts with the UL37 protein

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. This physical interaction could be verified using yeast two-hybrid analysis which demonstrated that the UL37 protein interacts with a defined region within the amino-terminal part of the UL36 protein. By use of immunogold labeling, capsids which accumulate in the cytoplasm in the absence of the UL37 protein (B. G. Klupp, H. Granzow, E. Mundt, and T. C. Mettenleiter, J. Virol. 75:8927-8936, 2001) as well as wild-type intracytoplasmic and extracellular virions were decorated by the anti-UL36 antiserum, whereas perinuclear primary enveloped virions were not. We postulate that the physical interaction of the UL36 protein, which presumably constitutes the innermost layer of the tegument (Z. Zhou, D. Chen, J. Jakana, F. J. Rixon, and W. Chiu, J. Virol. 73:3210-3218, 1999), with the UL37 protein is an important early step in tegumentation during virion morphogenesis in the cytoplasm.     

Identification and characterization of the pseudorabies virus tegument proteins UL46 and UL47: role for UL47 in virion morphogenesis in the cytoplasm

Proteins encoded by the UL46 and UL47 genes of herpes simplex virus type 1 (HSV-1) constitute major components of the viral tegument. However, their functions have so far not been elucidated in detail. By use of monospecific antisera directed against bacterially expressed glutathione-S-transferase fusion proteins, the homologous UL46 and UL47 proteins of the alphaherpesvirus pseudorabies virus (PrV) were identified in virus-infected cells and in virions. The PrV UL46 gene product of 693 amino acids (aa) exhibits an apparent molecular mass of 95 kDa, whereas the UL47 product of 750 aa was identified as a 97-kDa protein. Both are present in purified virions, correlating with their role as tegument proteins. Immunofluorescence analysis by confocal laser scan microscopy showed that late in infection the UL46 product is detectable in the cytoplasm, whereas the UL47 product was observed to be diffuse in the cytoplasm and speckled in the nucleus. Virus mutants lacking either the UL46 or the UL47 gene or both were isolated on noncomplementing cells, demonstrating that these genes either singly or in combination are not required for productive viral replication. However, plaque sizes were decreased. Interestingly, in one-step growth analysis, UL47 deletion mutants exhibited an approximately 10-fold decrease in final titers, whereas the UL46 deletion mutant was not affected. This finding correlated with ultrastructural observations which showed unimpaired virion morphogenesis in the absence of the UL46 protein, whereas in the absence of the UL47 protein intracytoplasmic aggregates of partially tegumented capsids were observed. In summary, we identified the PrV UL46 and UL47 proteins and show that the UL47 protein plays an important role in virion assembly in the cytoplasm.     

The capsid-associated UL25 protein of the alphaherpesvirus pseudorabies virus is nonessential for cleavage and encapsidation of genomic DNA but is required for nuclear egress of capsids

Homologs of the UL25 gene product of herpes simplex virus (HSV) have been identified in all three subfamilies of the Herpesviridae. However, their exact function during viral replication is not yet known. Whereas earlier studies indicated that the UL25 protein of HSV-1 is not required for cleavage of newly replicated viral DNA but is necessary for stable encapsidation (A. R. McNab, P. Desai, S. Person, L. Roof, D. R. Thompson, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998), viral DNA packaging has recently been demonstrated to occur in the absence of UL25, although at significantly decreased levels compared to wild-type HSV-1 (N. Stow, J. Virol. 75:10755-10765 2001). To clarify the functional role of UL25 we analyzed the homologous protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL25 was found to be essential for viral replication, as a mutant virus lacking the UL25 protein required UL25-expressing cells for productive propagation. In the absence of the UL25 protein, newly replicated PrV DNA was cleaved and DNA-containing C-type capsids were detected in infected cell nuclei. However, although capsids were frequently found in close association with the inner nuclear membrane, nuclear egress was not observed. Consequently, no capsids were found in the cytoplasm, resulting in an inhibition of virion morphogenesis. In contrast, the formation of capsidless enveloped tegument structures (L particles) in the cytoplasm was readily observed. Thus, our data demonstrate that the PrV UL25 protein is not essential for cleavage and encapsidation of viral genomes, although both processes occur more efficiently in the presence of the protein. However, the presence of the PrV UL25 protein is a prerequisite for nuclear egress. By immunoelectron microscopy, we detected UL25-specific label on DNA-containing C capsids but not on other intranuclear immature or defective capsid forms. Thus, the PrV UL25 protein may represent the hitherto missing trigger that allows primary envelopment preferably of DNA-filled C capsids.     

Functional analysis of the pseudorabies virus UL51 protein

Homologs of the UL51 protein of herpes simplex virus have been identified in all herpesvirus subfamilies, but until now, no function has been assigned to any of them. To investigate function of the UL51 gene product of the alphaherpesvirus pseudorabies virus (PrV), we isolated and analyzed a mutant lacking the major part of the open reading frame, PrV-DeltaUL51F, and a rescuant. One-step growth analysis of PrV-DeltaUL51F revealed only slightly reduced titers, but plaque size was notably diminished and reached only approximately 30% the plaque size of wild-type PrV. Ultrastructurally, intracytoplasmic capsids were found in large numbers either without envelope or in different stages of envelopment, indicating that secondary envelopment in the cytoplasm was less efficient. However, neuroinvasion in the mouse trigeminal pathway after intranasal infection was only slightly delayed. A PrV UL11 mutant also showed a defect in secondary envelopment (M. Kopp, H. Granzow, W. Fuchs, B. G. Klupp, E. Mundt, A. Karger, and T. C. Mettenleiter, J. Virol. 77:5339-5351, 2003). Since both proteins are part of the viral tegument and are predicted to be membrane associated, they may serve similar, possibly redundant functions during viral morphogenesis. Therefore, we also isolated a mutant simultaneously lacking UL51 and UL11. This mutant exhibited further reduced plaque size compared to the single-deletion mutants, but viral titers were comparable to those for the UL11 mutant. In electron microscopic analyses, the observed defect in secondary envelopment was similar to that found in the UL11 single-deletion mutant. In conclusion, both conserved tegument proteins, either singly or in combination, are involved in virion morphogenesis in the cytoplasm but are not essential for viral replication in vitro and in vivo.     

Identification and characterization of a novel structural glycoprotein in pseudorabies virus, gL.

Herpesvirus envelope glycoproteins play important roles in the interaction between virions and target cells. In the alphaherpesvirus pseudorabies virus (PrV), seven glycoproteins that all constitute homologs of glycoproteins found in herpes simplex virus type 1 (HSV-1) have been characterized, including a homolog of HSV-1 glycoprotein H (gH). Since HSV-1 gH is found associated with another essential glycoprotein, gL, we analyzed whether PrV also encodes a gL homolog. DNA sequence analysis of a corresponding part of the UL region adjacent to the internal inverted repeat in PrV strains Kaplan and Becker revealed the presence of two open reading frames (ORF). Deduced proteins exhibited homology to uracil-DNA glycosylase encoded by HSV-1 ORF UL2 (54% identity) and gL encoded by HSV-1 ORF UL1 (24% identity), respectively. To identify the PrV UL1 protein, rabbit antisera were prepared against two synthetic oligopeptides that were predicted by computer analysis to encompass antigenic epitopes. Sera against both peptides reacted in Western blots of purified virions with a 20-kDa protein. The specificity of the reaction was demonstrated by peptide competition. Since the PrV UL1 sequence did not reveal the presence of a consensus N-linked glycosylation site, concanavalin A affinity chromatography and enzymatic deglycosylation of virion glycoproteins were used to ascertain that the PrV UL1 product is O glycosylated. Therefore, we designated this protein PrV gL. Analysis of mutant PrV virions lacking gH showed that concomitantly with the absence of gH, gL was also missing in purified virions. In summary, we identified and characterized a novel structural PrV glycoprotein, gL, which represents the eighth PrV glycoprotein described. In addition, we show that virion location of PrV gL is dependent on the presence of PrV gH.     

The pseudorabies virus US3 protein is a component of primary and of mature virions

Herpesviruses acquire a primary envelope by budding of capsids at the inner leaflet of the nuclear membrane. They then traverse into the cytoplasm after fusion of the primary envelope with the outer leaflet of the nuclear membrane. In the alphaherpesvirus pseudorabies virus (PrV), the latter process is impaired when the US3 protein is absent. Acquisition of final tegument and envelope occurs in the cytoplasm. Besides the capsid components, only the UL31 and UL34 gene products of PrV have unequivocally been shown to be part of primary enveloped virions, whereas they lack several tegument proteins present in mature virions (reviewed by T. C. Mettenleiter, J. Virol. 76:1537-1547, 2002). Using immunoelectron microscopy, we show that the US3 protein is present in primary enveloped as well as in mature virions. It is also detectable in intracytoplasmic inclusions produced in the absence of other viral tegument components or envelope-associated glycoproteins. In particular, inclusions formed in the absence of the inner tegument protein UL37 contained the US3 protein. Thus, the US3 protein is a tegument component of both forms of enveloped alphaherpes virions. We hypothesize that US3 protein in primary virions modulates deenvelopment at the outer leaflet of the nuclear membrane and is either lost from primary virions during nuclear egress and subsequently reacquired early during tegumentation or is retained during transit of the nucleocapsid through the nuclear membrane.     

A pseudorabies virus recombinant simultaneously lacking the major tegument proteins encoded by the UL46, UL47, UL48, and UL49 genes is viable in cultured cells

The UL46, UL47, UL48, and UL49 genes, which encode major tegument proteins, are conserved in most alphaherpesvirus genomes. However, the relative importance of each of these proteins for replication of individual alphaherpesviruses appears to be different. Recently, we demonstrated that single deletions of UL47 or UL48 impair maturation and egress of pseudorabies virus (PrV) particles to different extents, whereas deletions of UL46 or UL49 have no significant effects on virus replication in cell culture (W. Fuchs, H. Granzow, B. G. Klupp, M. Kopp, and T. C. Mettenleiter, J. Virol. 76:6729-6742, 2002; M. Kopp, B. G. Klupp, H. Granzow, W. Fuchs, and T. C. Mettenleiter, J. Virol. 76:8820-8833, 2002). To test for possible functional redundancy between the four tegument proteins, a quadruple gene deletion mutant (PrV-DeltaUL46-49) was generated and characterized in vitro. Although plaque formation by this mutant was almost abolished and maximum titers were reduced more than 100-fold compared to those of parental wild-type virus, PrV-DeltaUL46-49 could be propagated and serially passaged in noncomplementing porcine and rabbit kidney cells. Electron-microscopic studies revealed that nucleocapsid formation and egress of PrV-DeltaUL46-49 from the host cell nucleus were not affected, but secondary envelopment of nucleocapsids in the cytoplasm was only rarely observed. The replication defect of PrV-DeltaUL46-49 could be fully corrected by reinsertion of the UL46-to-UL49 gene cluster. Plaque sizes and virus titers were only slightly increased after restoration of only UL47 expression, whereas repair of only UL48 resulted in a significant increase in replication capacity to the level of a UL47 deletion mutant. In conclusion, we show that none of the UL46 to UL49 tegument proteins is absolutely required for productive replication of PrV. Moreover, our data indicate that the UL47 and UL48 proteins function independently during cell-to-cell spread and virus egress.     

Physical interaction between envelope glycoproteins E and M of pseudorabies virus and the major tegument protein UL49

Envelope glycoprotein M (gM) and the complex formed by glycoproteins E (gE) and I (gI) are involved in the secondary envelopment of pseudorabies virus (PrV) particles in the cytoplasm of infected cells. In the absence of the gE-gI complex and gM, envelopment is blocked and capsids surrounded by tegument proteins accumulate in the cytoplasm (A. R. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Here we demonstrate by yeast two-hybrid analyses that the cytoplasmic domains of gE and gM specifically interact with the C-terminal part of the UL49 gene product of PrV, which represents a major tegument protein and which is homologous to VP22 of herpes simplex virus type 1. However, deletion of the UL49 gene from PrV had only minor effects on viral replication, and ultrastructural analyses of infected cells confirmed that virus maturation and egress, including secondary envelopment in the cytoplasm, were not detectably affected by the absence of UL49. Moreover, the UL49 gene product was shown to be dispensable for virion localization of gE and gM, and mutants lacking either gE or gM incorporated the UL49 protein efficiently into virus particles. In contrast, a PrV mutant with deletions of gE-gI and gM failed to incorporate the UL49 protein despite apparently unaltered intracytoplasmic UL49 expression. In summary, we describe specific interactions between herpesvirus envelope and tegument proteins which may play a role in secondary envelopment during herpesvirus virion maturation.     

The pseudorabies virus UL11 protein is a virion component involved in secondary envelopment in the cytoplasm

Homologs of the small tegument protein encoded by the UL11 gene of herpes simplex virus type 1 are conserved throughout all herpesvirus subfamilies. However, their function during viral replication has not yet been conclusively shown. Using a monospecific antiserum and an appropriate viral deletion and rescue mutant, we identified and functionally characterized the UL11 protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL11 encodes a protein with an apparent molecular mass of 10 to 13 kDa that is primarily detected at cytoplasmic membranes during viral replication. In the absence of the UL11 protein, viral titers were decreased approximately 10-fold and plaque sizes were reduced by 60% compared to wild-type virus. Intranuclear capsid maturation and nuclear egress resulting in translocation of DNA-containing capsids into the cytoplasm were not detectably affected. However, in the absence of the UL11 protein, intracytoplasmic membranes were distorted. Moreover, in PrV-DeltaUL11-infected cells, capsids accumulated in the cytoplasm and were often found associated with tegument in aggregated structures such as had previously been demonstrated in cells infected with a PrV triple-mutant virus lacking glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Thus, the PrV UL11 protein, like glycoproteins E, I, and M, appears to be involved in secondary envelopment.     

The UL20 gene product of pseudorabies virus functions in virus egress.

The UL20 open reading frame is positionally conserved in different alphaherpesvirus genomes and is predicted to encode an integral membrane protein. A previously described UL20- mutant of herpes simplex virus type 1 (HSV-1) exhibited a defect in egress correlating with retention of virions in the perinuclear space (J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman, J. Virol. 65:6414-6424, 1991). To analyze UL20 function in a related but different herpesvirus, we constructed a UL20- pseudorabies virus (PrV) mutant by insertional mutagenesis. Similar to HSV-1, UL20- PrV was found to be severely impaired in both cell-to-cell spread and release from cultured cells. The severity of this defect appeared to be cell type dependent, being more prominent in Vero than in human 143TK- cells. Surprisingly, electron microscopy revealed the retention of enveloped virus particles in cytoplasmic vesicles of Vero cells infected with UL20- PrV. This contrasts with the situation in the UL20- HSV-1 mutant, which accumulated virions in the perinuclear cisterna of Vero cells. Therefore, the UL20 gene products of PrV and HSV-1 appear to be involved in distinct steps of viral egress, acting in different intracellular compartments. This might be caused either by different functions of the UL20 proteins themselves or by generally different egress pathways of PrV and HSV-1 mediated by other viral gene products.     

Composition of pseudorabies virus particles lacking tegument protein US3, UL47, or UL49 or envelope glycoprotein E

Proteins located in the tegument layer of herpesvirus particles play important roles in the replicative cycle at both early and late times after infection. As major constituents of the virion, they execute important functions in particular during formation of progeny virions. These functions have mostly been elucidated by construction and analysis of mutant viruses deleted in single or multiple tegument protein-encoding genes (reviewed in the work of T. C. Mettenleiter, Virus Res. 106:167-180, 2004). However, since tegument proteins have been shown to be involved in numerous protein-protein interactions, the impact of single protein deletions on the composition of the virus particle is unknown, but they could impair correct interpretation of the results. To analyze how the absence of single virion constituents influences virion composition, we established a procedure to assay relative amounts of virion structural proteins in deletion mutants of the alphaherpesvirus Pseudorabies virus (PrV) in comparison to wild-type particles. The assay is based on the mass spectrometric quantitation of virion protein-derived peptides carrying stable isotope mass tags. After deletion of the US3, UL47, UL49, or glycoprotein E gene, relative amounts of a capsid protein (UL38), a capsid-associated protein (UL25), several tegument proteins (UL36 and UL47, if present), and glycoprotein H were unaffected, whereas the content of other tegument proteins (UL46, UL48, and UL49, if present) varied significantly. In the case of the UL48 gene product, a specific increase in incorporation of a smaller isoform was observed after deletion of the UL47 or UL49 gene, whereas a larger isoform remained unaffected. The cellular protein actin was enriched in virions of mutants deficient in any of the tegument proteins UL47, UL49, or US3. By two-dimensional gel electrophoresis multiple isoforms of host cell-derived heat shock protein 70 and annexins A1 and A2 were also identified as structural components of PrV virions.     

Influence of tegument proteins of pseudorabies virus on neuroinvasion and transneuronal spread in the nervous system of adult mice after intranasal inoculation

Pseudorabies virus (PrV) is a neurotropic alphaherpesvirus that, after intranasal infection of adult mice, enters peripheral neurons and propagates to the central nervous system. In recent years we have analyzed the contribution of virus-encoded glycoproteins to neuroinvasion and transneuronal spread (reviewed in T. C. Mettenleiter, Virus Res. 92:197-206, 2003). We now extend our studies to analyze the role of tegument proteins in these processes. To this end, PrV mutants unable to express the UL11, UL37, UL46, UL47, and UL48 tegument proteins, as well as the corresponding rescued viruses, were intranasally instilled into 6- to 8-week-old CD1 strain mice. First, mean survival times were determined which showed that mice infected with the UL46 deletion mutant succumbed to the disease as early as wild-type PrV-infected animals. Survival times increased in the order: PrV-DeltaUL47-, PrV-DeltaUL11-, and PrV-DeltaUL48-infected animals, a finding which parallels the growth phenotype of these viruses in cell culture. In contrast, none of the PrV-DeltaUL37-infected animals died. Upon closer histological examination, all viruses except PrV-DeltaUL37 were able to infect the nasal cavity and propagate to first- and second-order neurons as shown by two-color immunofluorescence. However, neuroinvasion was delayed in PrV-DeltaUL47, PrV-DeltaUL11, and PrV-DeltaUL48, a finding that correlated with the extended survival times. Surprisingly, whereas PrV-DeltaUL48 and PrV-DeltaUL37 replicated to similar titers in cell culture which were approximately 500-fold lower than those of wild-type virus, after intranasal infection of mice PrV-DeltaUL48 was able to infect areas of the brain like wild-type PrV, although only after a considerably longer time period. In contrast, PrV-DeltaUL37 was not able to enter neurons and was restricted to the infection of single cells in the nasal respiratory epithelium. Thus, our data demonstrate the importance of herpesviral tegument proteins in neuronal infection and show a different contribution of tegument proteins to the neuroinvasion phenotype of a neurotropic alphaherpesvirus.     

The herpes simplex virus type 1 UL20 protein modulates membrane fusion events during cytoplasmic virion morphogenesis and virus-induced cell fusion

The herpes simplex virus type 1 (HSV-1) UL20 protein is an important determinant for virion morphogenesis and virus-induced cell fusion. A precise deletion of the UL20 gene in the HSV-1 KOS strain was constructed without affecting the adjacent UL20.5 gene. The resultant KOS/UL20-null virus produced small plaques of 8 to 15 cells in Vero cells while it produced wild-type plaques on the complementing cell line G5. Electron microscopic examination of infected cells revealed that the KOS/UL20-null virions predominantly accumulated capsids in the cytoplasm while a small percentage of virions were found as enveloped virions within cytoplasmic vacuoles. Recently, it was shown that UL20 expression was necessary and sufficient for cell surface expression of gK (T. P. Foster, X. Alvarez, and K. G. Kousoulas, J. Virol. 77:499-510, 2003). Therefore, we investigated the effect of UL20 on virus-induced cell fusion caused by syncytial mutations in gB and gK by constructing recombinant viruses containing the gBsyn3 or gKsyn1 mutations in a UL20-null genetic background. Both recombinant viruses failed to cause virus-induced cell fusion in Vero cells while they readily caused fusion of UL20-null complementing G5 cells. Ultrastructural examination of UL20-null viruses carrying the gBsyn3 or gKsyn1 mutation revealed a similar distribution of virions as the KOS/UL20-null virus. However, cytoplasmic vacuoles contained aberrant virions having multiple capsids within a single envelope. These multicapsid virions may have been formed either by fusion of viral envelopes or by the concurrent reenvelopment of multiple capsids. These results suggest that the UL20 protein regulates membrane fusion phenomena involved in virion morphogenesis and virus-induced cell fusion.     

Inhibition of Virion Maturation by Simultaneous Deletion of Glycoproteins E, I, and M of Pseudorabies Virus

Glycoprotein M (gM), the product of the UL10 gene of pseudorabies virus (PrV), is one of the few nonessential glycoproteins conserved throughout the Herpesviridae. In contrast to wild-type PrV strains, the UL10 gene product of the attenuated PrV vaccine strain Bartha (PrV-Ba) is not modified by N-glycans due to a mutation in the DNA sequence encoding the consensus N-glycosylation motif. To assay function of the UL10 protein in PrV-Ba, a UL10-deletion mutant (PrV-Ba-UL10(-)) was isolated. Surprisingly, in contrast to gM-deleted wild-type PrV, PrV-Ba-UL10(-) was severely impaired in plaque formation, inducing only foci of very few infected RK13, Vero, and PSEK cells and tiny plaques on MDBK cells. Since this effect was significantly more dramatic than in wild-type PrV, additional mutations known to be present in PrV-Ba were analyzed for their contribution to this phenotype. trans-complementation of the mutated PrV-Ba UL21 or gC protein by the wild-type version had no influence on the observed phenotype. In contrast, complementation of the gE/gI deletion rescued the phenotype. The synergistic effect of deletions in gE/gI and gM on plaque size was verified by construction of a gE/I/M triple mutant derived from wild-type PrV which exhibited the same phenotype. The dramatic effect of deletion of gM on plaque size in a gE/I- virus background was mainly attributable to a function of gM, and not of the gM/gN complex, as shown by analysis of a gE/I/N triple mutant. Interestingly, despite the strong effect on plaque size, penetration was not significantly impaired. In noncomplementing cells infected with the gE/I/M triple mutant, electron microscopy showed absence of secondary envelopment in the cytoplasm but occurrence of intracytoplasmic accumulations of nucleocapsids in association with electron dense material, presumably tegument proteins. These structures were not observed after infection of cells expressing either gE/I or gM. We suggest that gE/I and gM are required for late stages in virion morphogenesis prior to final envelopment in the cytoplasm.     

Simultaneous deletion of pseudorabies virus tegument protein UL11 and glycoprotein M severely impairs secondary envelopment

The pseudorabies virus (PrV) proteins UL11, glycoprotein E (gE), and gM are involved in secondary envelopment of tegumented nucleocapsids in the cytoplasm. To assess the relative contributions of these proteins to the envelopment process, virus mutants with deletions of either UL11, gM, or gE as well as two newly constructed mutant viruses with simultaneous deletions of UL11 and gE or of UL11 and gM were analyzed in cell culture for their growth phenotype. We show here that simultaneous deletion of UL11 and gE reduced plaque size in an additive manner over the reduction observed by deletion of only UL11 or gE. However, one-step growth was not further impaired beyond the level of the UL11 deletion mutant. Moreover, in electron microscopic analyses PrV-DeltaUL11/gE exhibited a phenotype similar to that of the UL11 mutant virus. In contrast, plaque formation was virtually abolished by the simultaneous absence of UL11 and gM, and one-step growth was significantly reduced. Electron microscopy showed the presence of huge intracytoplasmic inclusions in PrV-DeltaUL11/gM-infected cells, with a size reaching 3 micro m and containing nucleocapsids embedded in tegument. We hypothesize that UL11 and gM are involved in different steps during secondary envelopment and that simultaneous deletion of both interrupts both processes, resulting in the observed drastic impairment of secondary envelopment.