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.     

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.     

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.     

The UL48 tegument protein of pseudorabies virus is critical for intracytoplasmic assembly of infectious virions

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-DeltaUL48) 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-DeltaUL48 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.     

Influence of pseudorabies virus proteins on neuroinvasion and neurovirulence in mice

Neurotropism is a distinctive feature of members of the Alphaherpesvirinae. However, its molecular basis remains enigmatic. In the past, research has been focused mainly on the role of viral envelope proteins in modulating herpesvirus neuroinvasion and neurovirulence (T. C. Mettenleiter, Virus Res. 92:192-206, 2003). To further analyze the molecular requirements for neuroinvasion of the alphaherpesvirus pseudorabies virus (PrV), adult mice were infected intranasally with a set of single- or multiple-deletion mutants lacking the UL3, UL4, UL7, UL11, UL13, UL16, UL17, UL21, UL31, UL34, UL37, UL41, UL43, UL46, UL47, UL48, UL51, US3, US9, glycoprotein E (gE), gM, UL11/US9, UL11/UL16, UL16/UL21, UL11/UL16/UL21, UL11/gE, UL11/gM, UL43/gK, UL43/gM, or UL43/gK/gM genes. Neurovirulence was evaluated by measuring mean survival times compared to that after wild-type virus infection. Furthermore, by immunohistochemical detection of infected neurons, the kinetics of viral spread in the murine central nervous system was investigated.     

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.     

Mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pUL36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo

Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein. Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C(26)S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C(26)S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.     

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.     

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.     

Pseudorabies virus UL37 gene product is involved in secondary envelopment

Herpesvirus envelopment is a two-step process which includes acquisition of a primary envelope resulting from budding of intranuclear capsids through the inner nuclear membrane. Fusion with the outer leaflet of the nuclear membrane releases nucleocapsids into the cytoplasm, which then gain their final envelope by budding into trans-Golgi vesicles. It has been shown that the UL34 gene product is required for primary envelopment of the alphaherpesvirus pseudorabies virus (PrV) (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063-10073, 2000). For secondary envelopment, several virus-encoded PrV proteins are necessary, including 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). We show here that the product of the UL37 gene of PrV, which is a constituent of mature virions, is involved in secondary envelopment. Replication of a UL37 deletion mutant, PrV-DeltaUL37, was impaired in normal cells; this defect could be complemented on cells stably expressing UL37. Ultrastructural analysis demonstrated that intranuclear capsid maturation and budding of capsids into and release from the perinuclear space were unimpaired. However, secondary envelopment was drastically reduced. Instead, apparently DNA-filled capsids accumulated in the cytoplasm in large aggregates similar to those observed in the absence of glycoproteins E/I and M but lacking the surrounding electron-dense tegument material. Although displaying an ordered structure, capsids did not contact each other directly. We postulate that the UL37 protein is necessary for correct addition of other tegument proteins, which are required for secondary envelopment. In the absence of the UL37 protein, capsids interact with each other through unknown components but do not acquire the electron-dense tegument which is normally found around wild-type capsids during and after secondary envelopment. Thus, apposition of the UL37 protein to cytoplasmic capsids may be crucial for the addition of other tegument proteins, which in turn are able to interact with viral glycoproteins to mediate secondary envelopment.     

Relevance of the interaction between alphaherpesvirus UL3.5 and UL48 proteins for virion maturation and neuroinvasion

The UL3.5 and UL48 genes, which are conserved in most alphaherpesvirus genomes, are important for maturation of pseudorabies virus (PrV) particles in the cytoplasm of infected cells (W. Fuchs, B. G. Klupp, H. J. Rziha, and T. C. Mettenleiter, J. Virol. 70:3517-3527, 1996; W. Fuchs, H. Granzow, B. G. Klupp, M. Kopp and T. C. Mettenleiter, J. Virol. 76:6729-6742, 2002). In bovine herpesvirus 1 (BoHV-1), the homologous gene products pUL3.5 and pUL48 have been demonstrated to interact physically (N. Lam and G. Letchworth, J. Virol. 74:2876-2884, 2000). Moreover, BoHV-1 pUL3.5 partially complemented a pUL3.5 defect in PrV (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886-8892, 1997). By using coimmunoprecipitation and yeast two-hybrid studies, we observed a similar interaction between pUL3.5 and pUL48 of PrV, as well as a heterologous interaction between the PrV and BoHV-1 gene products. The relevant domain could be confined to the first 43 amino acids of PrV pUL3.5. Unlike its BoHV-1 homologue, PrV pUL3.5 is processed by proteolytic cleavage, and only an abundant 14-kDa fragment consisting of amino acids 1 to >or=116 could be detected by peptide mass fingerprint analysis of purified wild-type PrV particles, which also contain the pUL48 tegument component. To determine the biological relevance of the protein-protein interaction, pUL3.5-, pUL48-, and double-negative PrV mutants were analyzed in parallel. All deletion mutants were replication competent but exhibited significantly reduced plaque sizes and virus titers in cultured rabbit kidney cells compared to wild-type and rescued viruses, which correlated with a delayed neuroinvasion in intranasally infected mice. Remarkably, the defects of the double-negative mutant were similar to those of pUL48-negative virus. Electron microscopy of cells infected with either deletion mutant revealed the retention of naked nucleocapsids in the cytoplasm and the absence of mature virus particles. In summary, our studies for the first time demonstrate the relevance of the pUL3.5-pUL48 interaction for secondary envelopment of an alphaherpesvirus, give a molecular basis for the observed trans-complementation between the PrV and BHV-1 pUL3.5 homologs, yield conclusive evidence for the incorporation of a proteolytically processed pUL3.5 into PrV virions, and demonstrate the importance of both proteins for neuroinvasion and neurovirulence of PrV.     

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.     

Pseudorabies virus UL3 gene codes for a nuclear protein which is dispensable for viral replication

Many of the products of the ca. 80 genes encoded by alphaherpesviruses have already been identified and, at least tentatively, functionally characterized. Among the least characterized proteins are the products of the genes homologous to herpes simplex virus UL3, which are present only in the subfamily Alphaherpesvirinae: To identify the UL3 protein of the porcine alphaherpesvirus pseudorabies virus (PrV), the complete PrV UL3 open reading frame was cloned, expressed in Escherichia coli as a glutathione S-transferase fusion protein, and used for immunization of a rabbit. In Western blots, the generated antiserum specifically detected a 34-kDa protein in PrV-infected cells, which was absent from purified virus preparations, indicating that PrV UL3 encodes a nonstructural protein. In indirect immunofluorescence analysis, the anti-UL3 serum produced predominantly nuclear staining in transfected as well as in infected cells, which was not altered in the absence of other virus-encoded nuclear proteins such as the UL31 and UL34 gene products. To investigate UL3 function, a deletion mutant, PrV-DeltaUL3F2, was constructed and characterized. This mutant replicated and formed plaques on noncomplementing cells indistinguishable from wild-type PrV, demonstrating that PrV UL3 is not required for virus propagation in cultured cells. Moreover, ultrastructural examinations revealed no impairment of capsid formation in the nucleus, nuclear egress of capsids, virion maturation in the cytoplasm, or virus release. Thus, the overall properties of PrV UL3 are similar to those described for the homologous herpes simplex virus proteins which may be indicative of a common, yet hitherto unknown, function in alphaherpesvirus replication. However, based on our studies, an involvement of the UL3 homologs in virion formation appears unlikely.     

Characterization of Marek's Disease Virus Serotype 1 (MDV-1) Deletion Mutants That Lack UL46 to UL49 Genes: MDV-1 UL49, Encoding VP22, Is Indispensable for Virus Growth

Experiments were conducted to investigate the roles of Marek's disease virus serotype 1 (MDV-1) major tegument proteins VP11/12, VP13/14, VP16, and VP22 in viral growth in cultured cells. Based on a bacterial artificial chromosome clone of MDV-1 (BAC20), mutant viruses were constructed in which the MDV-1 homologs of UL46, UL47, UL48, or UL49 were deleted alone and in various combinations. It could be demonstrated that the UL46, UL47, and UL48 genes are dispensable for MDV-1 growth in chicken embryonic skin and quail muscle QM7 cells, although the generated virus mutants exhibited reduced plaque sizes in all cell types investigated. In contrast, a UL49-negative MDV-1 (20 Delta 49) and a UL48-UL49 (20 Delta 48-49) doubly negative mutant were not able to produce MDV-1-specific plaques on either cell type. It was confirmed that this growth restriction is dependent on the absence of VP22 expression, because growth of these mutant viruses could be partially restored on cells that were cotransfected with a UL49 expression plasmid. In addition, we were able to demonstrate that cell-to-cell spread of MDV-1 conferred by VP22 is dependent on the expression of amino acids 37 to 187 of MDV-1 VP22, because expression plasmids containing MDV-1 UL49 mutant genes with deletions of amino acids 1 to 37 or 188 to 250 were still able to restore partial growth of the 20 Delta 49 and 20 Delta 48-49 viruses. These results demonstrate for the first time that an alphaherpesvirus UL49-homologous gene is essential for virus growth in cell culture.     

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.     

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.     

Determination of interactions between tegument proteins of herpes simplex virus type 1

The aim of this study was to elucidate protein-protein interactions between tegument proteins of herpes simplex virus type 1 (HSV-1). To do so, we have cloned and expressed in the LexA yeast (Saccharomyces cerevisiae) two-hybrid system, 13 of the 21 currently known tegument proteins of HSV-1. These included the tegument proteins essential for replication in cell lines, UL17, UL36, UL37, UL48, and UL49, and the nonessential tegument proteins US11, UL11, UL14, UL16, UL21, UL41, UL46, and UL47. A total of 104 combinations were screened in the yeast two-hybrid assay, with 9 interactions identified. These included: UL11-UL16, UL36-UL37, UL36-UL48, UL46-UL48, UL47-UL48, and UL48-UL49. The remaining interactions consisted of self-associations that were observed for US11, UL37, and UL49. The interactions UL36-UL37, UL36-UL48, UL37-UL37, UL46-UL48, and UL47-UL48 have not been previously reported for HSV-1. The interaction of UL46-UL48 was verified using an in vitro pull-down assay. The interactions of UL36-UL37 and UL37-UL37 were verified with a coimmunoprecipitation assay. Knowledge of HSV-1 tegument protein-protein interactions will provide insights into the pathways of tegument assembly, and the identified interactions are potential targets for new antiviral drugs.     

Identification of functional domains within the essential large tegument protein pUL36 of pseudorabies virus

Proteins of the capsid proximal tegument are involved in the transport of incoming capsids to the nucleus and secondary envelopment after nuclear egress. Homologs of the essential large capsid proximal tegument protein pUL36 are conserved within the Herpesviridae. They interact with another tegument component, pUL37, and contain a deubiquitinating activity in their N termini which, however, is not essential for virus replication. Whereas an internal deletion of 709 amino acids (aa) within the C-terminal half of the alphaherpesvirus pseudorabies virus (PrV) pUL36 does not impair its function (S. Böttcher, B. G. Klupp, H. Granzow, W. Fuchs, K. Michael, and T. C. Mettenleiter, J. Virol. 80:9910-9915, 2006), deletion of the very C terminus does (J. Lee, G. Luxton, and G. A. Smith, J. Virol. 80:12086-12094, 2006). For further characterization we deleted several predicted functional and structural motifs within PrV pUL36 and analyzed the resulting phenotypes in cell culture and a mouse infection model. Extension of the internal deletion to encompass aa 2087 to 2981 exerted only minor effects on virus replication but resulted in prolonged mean survival times of infected mice. Any additional extension did not yield viable virus. Deletion of an N-terminal region containing the deubiquitinating activity (aa 22 to 248) only slightly impaired viral replication in cell culture but slowed neuroinvasion in our mouse model, whereas a strong impairment of viral replication was observed after simultaneous removal of both nonessential domains. Absence of a region containing two predicted leucine zipper motifs (aa 748 to 991) also strongly impaired virus replication and spread. Thus, we identify several domains within the PrV UL36 protein, which, though not essential, are nevertheless important for virus replication.     

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.     

The pseudorabies virus VP1/2 tegument protein is required for intracellular capsid transport

Transport of capsids in cells is critical to alphaherpesvirus infection and pathogenesis; however, viral factors required for transport have yet to be identified. Here we provide a detailed examination of capsid dynamics during the egress phase of infection in Vero cells infected with pseudorabies virus. We demonstrate that the VP1/2 tegument protein is required for processive microtubule-based transport of capsids in the cytoplasm. A second tegument protein that binds to VP1/2, UL37, was necessary for wild-type transport but was not essential for this process. Both proteins were also required for efficient nuclear egress of capsids to the cytoplasm.