An amino-terminal deletion mutation of pseudorabies virus glycoprotein gIII affects protein localization and RNA accumulation.

We have constructed a pseudorabies virus mutant that contains virtually a complete deletion of the predicted signal sequence coding region for a nonessential envelope glycoprotein, gIII. No signal sequence mutants have been reported previously for a herpesvirus glycoprotein. Through endoglycosidase treatments and pulse-chase analysis, we have determined that the mutant gIII protein is not posttranslationally modified like the wild-type polypeptide, but rather is present as a single, stable species within the infected cell. The mutant polypeptide cannot be detected in the virus envelope, nor is it aberrantly localized to the tissue culture medium. Immunofluorescence studies have indicated that the mutant protein also is not localized to the surfaces of infected cells. In addition, Northern (RNA) and slot blot analyses, as well as in vitro translation experiments using infected-cell cytoplasmic RNA, have indicated that the mutant gIII allele is expressed at lower levels than the wild-type gene is. This is despite the fact that no alterations have been made upstream of the gIII coding sequence. From these results, it appears that the first 22 amino acids of the wild-type gIII protein define a necessary signal peptide that is responsible for at least the correct initiation of translocation and subsequent glycosylation of the gIII envelope glycoprotein within infected cells.     

Genome location and identification of functions defective in the Bartha vaccine strain of pseudorabies virus.

We have shown previously (Lomniczi et al., J. Virol. 52:198-205, 1984) that the Bartha vaccine strain of pseudorabies virus has a deletion in the short unique (Us) region of its genome--a deletion that is related to the absence of virus virulence. This strain is, however, also defective in other genes involved in virulence. We show here that virulence can be restored by marker rescue of the Bartha strain to which an intact Us has been restored (but not to the parental Bartha strain) by sequences derived from approximate map units 0.460 and 0.505 of the wild-type virus genome. No difference in the ability to grow in cell culture was observed between parental Bartha, Bartha 43/25a (Bartha to which an intact Us has been restored), or the doubly rescued Bartha strains. However, only the doubly rescued Bartha strain was virulent for both chickens and pigs and replicated to high titers when inoculated directly into the brains of chickens. The sequences that could restore virulence to the Bartha 43/25a strain encode four genes, all of which are involved in processes leading to the assembly of nucleocapsids. Since these sequences rescue virulence, it appears that a function that plays a role in nucleocapsid assembly is defective in the Bartha strain and that this defect contributes to the lack of virulence of this virus.     

Glycoprotein gIII of pseudorabies virus is multifunctional.

One of the major glycoproteins of pseudorabies virus, gIII, is nonessential for growth in cell culture. Mutants defective in gIII, however, consistently yield lower titers of infectious virus (3- to 20-fold) than does wild-type virus. The interactions of gIII- mutants with their host cells were compared with those of wild-type virus in an attempt to uncover the functions of gIII. We show that gIII plays a major role in the stable adsorption of the virus to its host cell; in the absence of gIII, the rate of adsorption is reduced and adsorption is easily reversed by washing. Thus, adsorption of pseudorabies virus can be said to occur in at least the following two ways: (i) a gIII-mediated rapid adsorption or (ii) a slower and more labile adsorption that is independent of gIII. After virions have been complexed with monoclonal antibodies against gIII (but not some monoclonal antibodies against other glycoproteins), both modes of adsorption were inhibited. Glycoprotein gIII affects virus stability and virus release, as well as adsorption. The effect on virus release is marked when the virus is defective in additional functions. Thus, although we found no obvious difference in the release of virus from gIII- or wild-type virus-infected rabbit kidney cells, release of a gIII-/gI- double mutant from the cells occurred less readily than did release of a gI- mutant. The gIII-/gI- and gIII- mutants, however, adsorbed to cells at a similar rate, indicating that the effects of gIII on adsorption and virus release constitute separate functions. The Bartha vaccine strain of pseudorabies virus has a defective gIII gene and is released poorly from rabbit kidney cells. After the resident Bartha gIII gene was replaced by the gIII gene of wild-type virus, virus release was enhanced considerably. Since inactivation of gIII in wild-type pseudorabies virus did not significantly affect virus release, the Bartha strain must be defective in another function which, in conjunction with gIII, significantly affects virus release. These results indicate again that gIII affects virus release in conjunction with other functions. Also, although the Bartha strain was functionally defective in virus release, it adsorbed to cells as well as wild-type virus did, showing that the effects of gIII on virus adsorption and release constitute separate functions. We conclude that gIII is a multifunctional glycoprotein.     

The putative cytoplasmic domain of the pseudorabies virus envelope protein gIII, the herpes simplex virus type 1 glycoprotein C homolog, is not required for normal export and localization.

Glycoprotein gIII of pseudorabies virus is a member of a conserved gene family found in at least seven diverse herpesviruses. We report here that the putative cytoplasmic domain of gIII is not required for transport to the cell surface and, unlike the prototype domain from herpes simplex virus type 1 glycoprotein C, is not required for stable membrane anchoring. Furthermore, this domain does not appear to be essential for incorporation of the glycoprotein into virions.     

Role of glycoprotein gIII of pseudorabies virus in virulence.

Deletion mutants of pseudorabies virus unable to express glycoprotein gIII, gI, or gp63 or double and triple mutants defective in these glycoproteins were constructed, and their virulence for day-old chickens inoculated intracerebrally was determined. Mutants of wild-type pseudorabies virus defective in glycoprotein gIII, gI, or gp63 were only slightly less virulent (at most, fivefold) for chickens than was the wild-type virus. However, mutants defective in both gIII and gI or gIII and gp63 were avirulent for chickens, despite their ability to grow in cell culture in vitro to about the same extent as mutants defective in gIII alone (which were virulent). These results show that gIII plays a role in virulence and does so in conjunction with gI or gp63. The effect of gIII on virulence was also shown when the resident gIII gene of variants of the Bartha vaccine strain (which codes for gIIIB) was replaced with a gIII gene derived from a virulent wild-type strain (which codes for gIIIKa); gIIIKa significantly enhanced the virulence of a variant of the Bartha strain to which partial virulence had been previously restored by marker rescue. Our results show that viral functions that play a role in the virulence of the virus (as measured by intracerebral inoculation of chickens) may act synergistically to affect the expression of virulence and that the ability of the virus to grow in cell culture is not necessarily correlated with virulence.     

The UL49.5 gene of pseudorabies virus codes for an O-glycosylated structural protein of the viral envelope.

Sequence analysis of BamHI fragment 1 of the pseudorabies virus (PrV) genome identified a novel PrV gene located upstream of the UL50 gene encoding PrV dUTPase. The deduced protein product displayed homology to the product of the herpes simplex virus type 1 UL49.5 protein. The predicted PrV UL49.5 protein consists of 98 amino acids with a calculated molecular mass of 10,155 Da. It contains putative signal peptide and transmembrane domains but lacks a consensus sequence for N glycosylation. PrV UL49.5 was expressed as a fusion protein with glutathione S-transferase in Escherichia coli, and a rabbit antiserum was generated. In Western blots (immunoblots) of purified virions, the antiserum detected a protein with an apparent molecular mass of 14 kDa. After fractionation of the virions, the 14-kDa protein was detected in the envelope fraction. Localization of the UL49.5 protein in the viral envelope was confirmed by immunoelectron microscopy. The treatment of purified virions with glycosidases led to a reduction of the apparent molecular mass in Western blots by approximately 2 kDa following digestion with neuraminidase and O-glycosidase. Our results demonstrate that the PrV UL49.5 protein is an O-glycosylated structural component of the viral envelope. It represents the 10th PrV glycoprotein described. According to the unified nomenclature for alphaherpesvirus glycoproteins, we propose to designate it glycoprotein N (gN).     

The attenuated pseudorabies virus strain Bartha fails to package the tegument proteins Us3 and VP22

The Bartha strain of pseudorabies virus has several recognized mutations, including a deletion in the unique short region encompassing the glycoprotein I (gI), gE, Us9, and Us2 genes and point mutations in the gC, gM, and UL21 genes. We have determined that Bartha has mutations in the serine/threonine kinase encoded by the Us3 gene relative to the wild-type Becker strain. Our analysis revealed that Becker virions contain the Us3 protein, whereas Bartha virions do not. To test whether the mutations in the Bartha Us3 protein were responsible for this observation, we constructed a recombinant Bartha strain, PRV632, which expresses the Becker Us3 protein. PRV632 failed to package Us3 into the tegument, indicating that mutations other than those in the Us3 primary amino acid sequence were responsible for the failure of Bartha to package its Us3 protein. A recombinant Becker strain, PRV634, which expresses the Bartha Us3 protein, was constructed to test whether it was capable of being packaged into virions. The Bartha Us3 protein was not incorporated into PRV634 virions efficiently, suggesting that the primary sequence of the Bartha Us3 protein affects packaging into the tegument. To determine whether the packaging of other tegument proteins was affected in the Bartha strain, we examined VP22. Whereas Becker packaged VP22 into virions, Bartha had a severe deficiency in VP22 incorporation. Analysis of VP22 expression in Bartha-infected cells revealed that Bartha VP22 had a slower mobility on sodium dodecyl sulfate-polyacrylamide gels, indicating either primary sequence differences and/or different posttranslational modifications relative to Becker VP22. Taken together, these data indicate that, while the primary sequence of the Us3 protein does affect its incorporation into the tegument, other factors are involved. Furthermore, our data suggest that one or more of the gI, gE, Us9, or Us2 genes influences the localization of the Us3 protein in infected cells, and this effect may be important for the proper incorporation of Us3 into virions.     

The gIII glycoprotein of pseudorabies virus is involved in two distinct steps of virus attachment.

The entry of herpesviruses into cells involves two distinct stages: attachment or adsorption to the cell surface followed by internalization. The virus envelope glycoproteins have been implicated in both stages. Pseudorabies virus attaches to cells by an early interaction that involves the viral glycoprotein gIII and a cellular heparinlike substance. We examined the role of gIII in the attachment process by analysis of a set of viruses carrying defined gIII mutations. The initial attachment of gIII mutants with an internal deletion of 134 amino acids (PrV2) to MDBK cells was indistinguishable from that of wild-type virus. The adsorption of these mutants was, however, much more sensitive than that of wild-type virus to competing heparin. Furthermore, while attachment of wild-type virus to MDBK cells led to a rapid loss of sensitivity to heparin, this was not the case with PrV2, which could be displaced from the cell surface by heparin after it had attached to the cells. We conclude that glycoprotein gIII is involved in two distinct steps of virus attachment and that the second of these steps but not the first is defective in PrV2.     

Mutations in the C-terminal hydrophobic domain of pseudorabies virus gIII affect both membrane anchoring and protein export.

The transmembrane and anchor region of pseudorabies virus gIII is postulated to be in the 35 hydrophobic amino acids (residues 436 to 470) found near the carboxy terminus of the 479-amino-acid envelope protein. In this study, we used a genetic approach to localize the functional gIII membrane anchor between amino acids 443 and 466. Mutant gIII proteins lacking the membrane anchor were not associated with virus particles, indicating that membrane retention is a prerequisite for virion localization. Unexpectedly, the specific hydrophobic gIII sequence defined by these deletions was not required for membrane anchor function since the entire region could be replaced with leucine residues without affecting gIII membrane retention, export, or virion localization. The hydrophobic region appears to encode more than the membrane anchor domain since both efficiency of posttranslational processing and localization to virions are affected by mutations in this region. We speculate that the composition of the hydrophobic domain influences the overall conformation of gIII, which in turn effects the efficiency of gIII export and processing. The virion localization phenotype is probably indirect and reflects the efficiency of protein processing. This conclusion provides insight into the mechanism of glycoprotein incorporation into virions.     

Interaction of glycoprotein gIII with a cellular heparinlike substance mediates adsorption of pseudorabies virus.

Glycoprotein gIII is one of the major envelope glycoproteins of pseudorabies virus (PrV) (Suid herpesvirus 1). Although it is dispensable for viral growth, it has been shown to play a prominent role in the attachment of the virus to target cells, since gIII- deletion mutants are severely impaired in adsorption (C. Schreurs, T. C. Mettenleiter, F. Zuckermann, N. Sugg, and T. Ben-Porat, J. Virol. 62:2251-2257, 1988). We show here that during the process of adsorption of PrV, the viral glycoprotein gIII interacts with a cellular heparinlike receptor. This conclusion is based on the following findings. (i) Heparin inhibits plaque formation of PrV by preventing the adsorption of wild-type virions to target cells. However, heparin does not interfere with the plaque formation of PrV mutants that lack glycoprotein gIII. (ii) Wild-type virions readily adsorb to matrix-bound heparin, whereas gIII- mutants do not. (iii) Pretreatment of cells with heparinase reduces considerably the ability of wild-type PrV to adsorb to these cells and to form plaques but does not negatively affect gIII- mutants. (iv) Glycoprotein gIII binds to heparin and appears to do so in conjunction with glycoprotein gII. Although heparin significantly reduces the adsorption of wild-type virus to all cell types tested, quantitative differences in the degree of inhibition of virus adsorption by heparin to different cell types were observed. Different cell types also retain their abilities to adsorb wild-type PrV to a different extent after treatment with heparinase and differ somewhat in their relative abilities to adsorb gIII- mutants. Our results show that while the primary pathway of adsorption of wild-type PrV to cells occurs via the interaction of viral glycoprotein gIII with a cellular heparinlike receptor, an alternative mode of adsorption, which is not dependent on either component, exists. Furthermore, the relative abilities of different cell types to adsorb PrV by the gIII-dependent or the alternative mode vary to some extent.     

Characterization of the envelope proteins of pseudorabies virus.

Previously we have reported that among the proteins of purified pseudorabies virions there are four major glycoproteins (T. Ben-Porat and A. S. Kaplan, Virology 41:265-273, 1970). Several minor glycoproteins can also be identified by two-dimensional gel electrophoresis. Removal of the viral envelope with Triton X-100 selectively removes from the virions all of the glycoproteins as well as several non-glycosylated proteins. Sedimentation analysis or chromatography of these proteins reveals that several are complexed with one another, some being covalently linked via disulfide bridges. Analysis of the proteins by immunoprecipitation with monoclonal antibodies reactive with the membrane proteins showed also that three of the four major virus glycoproteins (125K, 74K, and 58K; gIIa, gIIb, and gIIc, respectively) are linked covalently by disulfide bridges. Furthermore, all three share extensive sequence homology as indicated by the identity of their antigenic determinants and by partial peptide mapping; they probably originate from a single protein precursor. The fourth major glycoprotein (98K; gIII) is not complexed to any other protein. Three minor glycoproteins (130K [gI], 98K [gIV], and 62K [gV]), which form a noncovalently linked complex with a 115K nonglycosylated protein, have also been identified. Of the monoclonal antibodies used in this study, only those reactive with the major 98K glycoprotein (gIII) inhibit virus adsorption and neutralize virus infectivity in the absence of complement. However, all react with surface components of the virion, indicating that the proteins with which they react are exposed on the surface of the virions. A nomenclature for the pseudorabies virus glycoproteins is proposed.     

Analysis of pseudorabies virus glycoprotein gIII localization and modification by using novel infectious viral mutants carrying unique EcoRI sites.

We have constructed two pseudorabies virus (PRV) mutants, each with a unique EcoRI restriction site in the nonessential gIII envelope glycoprotein gene. Since no natural PRV isolate has been reported to contain EcoRI sites, the isolation and single-step growth curve analysis of these mutants established that PRV can carry such a site with little ill effect in tissue culture. Virus carrying these defined mutations produced novel gIII proteins that enabled us to begin functional assignment of protein localization information within the gIII gene. Specifically, one viral mutant contained an in-frame synthetic EcoRI linker sequence that was flanked on one side by the first one-third of the gIII gene and on the other side by the last one-third of the gene. The resulting protein lacked the middle one-third of the parental species, including five of eight putative N-linked glycosylation signals, but was still glycosylated and found in enveloped virions; it was not secreted into the medium. A second viral mutant contained an in-frame synthetic EcoRI linker sequence that additionally specified a nonsense codon at position 158, producing a gIII protein that was glycosylated and secreted into the medium; the fragment was not found in enveloped virions. By endoglycosidase and pulse-chase analyses, we established a precursor-product relationship between the various forms of gIII expressed in the parental and mutant strains, and perhaps determined certain features of the gIII protein that are required for its efficient export within the cell.     

Replacement of the pseudorabies virus glycoprotein gIII gene with its postulated homolog, the glycoprotein gC gene of herpes simplex virus type 1.

gIII, the major envelope glycoprotein of pseudorabies virus (PRV), shares approximately 20% amino acid similarity with glycoprotein gC of herpes simplex virus type 1 (HSV-1) and HSV-2. We describe here our first experiments on the potential conservation of function between these two genes and gene products. We constructed PRV recombinants in which the gIII gene and regulatory sequences have been replaced with the entire HSV-1 gC gene and its regulatory sequences. The gC promoter functions in the PRV genome, and authentic HSV-1 gC protein is produced, albeit at a low level, in infected cells. The gC protein is present at the cell surface but cannot be detected in the PRV envelope.     

The amino-terminal one-third of pseudorabies virus glycoprotein gIII contains a functional attachment domain, but this domain is not required for the efficient penetration of Vero cells.

We have examined the attachment and penetration phenotypes of several glycoprotein gIII mutants of pseudorabies virus (PRV) and have identified the first one-third of gIII as a region that mediates efficient virus attachment to PK15 and Vero cells. This portion of gIII, amino acids 25 through 157 of the wild-type sequence, appeared to support attachment by binding to heparinlike molecules on cell surfaces. Virions containing the first one-third of gIII were sensitive to heparin competition and showed greatly reduced infectivity on cells treated with heparinase. PRV virions lacking the first one-third of the mature glycoprotein exhibited only residual binding to cells if challenged by vigorous washing with phosphate-buffered saline at 2 h postinfection at 4 degrees C. This residual binding was resistant to heparin competition, and strains lacking the first one-third of gIII were able to infect cells treated with heparinase as effectively as untreated cells. When we determined the penetration phenotypes for each strain, we found that gIII-mediated virus attachment was necessary for timely penetration of PK15 cells but remarkably was not required for efficient virus penetration of Vero cells. Moreover, wild-type PRV was actually prohibited from rapid penetration of Vero cells by a gIII-heparan sulfate interaction. Our results indicate that initial virus binding to heparan sulfate via glycoprotein gIII is not required for efficient PRV infection of all cell types and may in fact be detrimental in some instances.     

The herpes simplex virus 1 gene encoding a protease also contains within its coding domain the gene encoding the more abundant substrate.

The herpes simplex virus 1 open reading frames UL26 and UL26.5 are 3' coterminal. The larger, UL26 open reading frame encodes a protein approximately 80,000 in apparent molecular weight and contains the promoter and coding sequence of the UL26.5 gene, which specifies a capsid protein designated infected cell protein 35. The larger product contains in its entirety the amino acid sequence of the smaller protein. We report that the UL26 gene encodes a protease which catalyzes its own cleavage and that of the more abundant product of UL26.5. By inserting the coding sequence of an epitope to a cytomegalovirus monoclonal antibody and homologs of the immunoglobulin G binding domain of staphylococcal protein A into the 3' termini of the coding domains of the two open reading frames, we identified both products of the cleavage and determined that the cleavage site is approximately 20 amino acids from the carboxyl termini of both proteins.     

Intracellular trafficking and localization of the pseudorabies virus Us9 type II envelope protein to host and viral membranes

The Us9 protein is a phosphorylated membrane protein present in the lipid envelope of pseudorabies virus (PRV) particles in a unique tail-anchored type II membrane topology. In this report, we demonstrate that the steady-state residence of the Us9 protein is in a cellular compartment in or near the trans-Golgi network (TGN). Through internalization assays with an enhanced green fluorescent protein epitope-tagged Us9 protein, we demonstrate that the maintenance of Us9 to the TGN region is a dynamic process involving retrieval of molecules from the cell surface. Deletion analysis of the cytoplasmic tail reveals that an acidic cluster containing putative phosphorylation sites is necessary for the recycling of Us9 from the plasma membrane. The absence of this cluster results in the relocalization of Us9 to the plasma membrane due to a defect in endocytosis. The acidic motif, however, does not contain signals needed to direct the incorporation of Us9 into viral envelopes. In this study, we also investigate the role of a dileucine endocytosis signal in the Us9 cytoplasmic tail in the recycling and retention of Us9 to the TGN region. Site-directed mutagenesis of the dileucine motif results in an increase in Us9 plasma membrane staining and a partial internalization defect.     

Early interactions of pseudorabies virus with host cells: functions of glycoprotein gIII.

Adsorption of mutants of pseudorabies virus (PrV) lacking glycoprotein gIII is slower and less efficient than is that of wild-type virus (C. Schreurs, T. C. Mettenleiter, F. Zuckermann, N. Snugg, and T. Ben-Porat, J. Virol. 62:2251-2257, 1988). To ascertain the functions of gIII in the early interactions of PrV with its host cells, we compared the effect on wild-type virus and gIII- mutants of antibodies specific for various PrV proteins. Although adsorption of wild-type virus was inhibited by polyvalent antisera against PrV as well as by sera against gIII and gp50 (but not sera against gII), adsorption of the gIII- mutants was not inhibited by any of these antisera. These results suggest that, in contrast to adsorption of wild-type PrV, the initial interactions of the gIII- mutants with their host cells are not mediated by specific viral proteins. Furthermore, competition experiments showed that wild-type Prv and the gIII- mutants do not compete for attachment to the same cellular components. These findings show that the initial attachment of PrV to its host cells can occur by a least two different modes--one mediated by glycoprotein gIII and the other unspecific. gIII- mutants not only did not adsorb as readily to cells as did wild-type virus but also did not penetrate cells as rapidly as did wild-type virus after having adsorbed. Antibodies against gIII did not inhibit the penetration of adsorbed virus (wild type or gIII-), whereas antibodies against gII and gp50 did. It is unlikely, therefore, that gIII functions directly in virus penetration. Our results support the premises that efficient adsorption of PrV to host cell components is mediated either directly or indirectly by gIII (or a complex of viral proteins for which the presence of gIII is functionally essential) and that this pathway of adsorption promotes the interactions of other viral membrane proteins with the appropriate cellular proteins, leading to the rapid penetration of the virus into the cells. The slower penetration of the gIII- mutants than of wild-type PrV appears to be related to the slower and less efficient alternative mode of adsorption of PrV that occurs in the absence of glycoprotein gIII.     

Structural determinants for nuclear envelope localization and function of pseudorabies virus pUL34

Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies virus (PrV) pUL34 is a type II membrane protein of 262 amino acids (aa). The transmembrane region (TM) is predicted to be located between aa 245 and 261, leaving only one amino acid in the C terminus that probably extends into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins, pointing to intrinsic localization motifs, and shows structural similarity to cellular INM proteins like lamina-associated polypeptide (Lap) 2ß and Emerin. To investigate which domains of pUL34 are relevant for localization and function, we constructed chimeric proteins by replacing parts of pUL34 with regions of cellular INM proteins. First the 18 C-terminal amino acids encompassing the TM were exchanged with TM regions and C-terminal domains of Lap2ß and Emerin or with the first TM region of the polytopic lamin B receptor (LBR), including the nine following amino acids. All resulting chimeric proteins complemented the replication defect of PrV-ΔUL34, demonstrating that the substitution of the TM and the extension of the C-terminal domain does not interfere with the function of pUL34. Complementation was reduced but not abolished when the C-terminal 50 aa were replaced by corresponding Lap2ß sequences (pUL34-LapCT50). However, replacing the C-terminal 100 aa (pUL34-LapCT100) resulted in a nonfunctional protein despite continuing pUL31 binding, pointing to an important functional role of this region. The replacement of the N-terminal 100 aa (pUL34-LapNT100) had no effect on nuclear envelope localization but abrogated pUL31 binding and function.