Spontaneous fusions to prv43 can suppress the export defect of pseudorabies virus gIII signal peptide mutants.

We have devised an enrichment scheme for the isolation of export-competent derivatives of pseudorabies virus glycoprotein gIII signal peptide mutants. Enrichment is based upon a growth advantage imparted upon gIII-containing virions compared with virions lacking the glycoprotein. Each of identified derivatives suppressed the gIII signal peptide defect by fusing the gIII gene in frame to the prv43 gene that lay immediately upstream; the result was the synthesis of a Prv43-gIII hybrid protein. The deduced Prv43 protein is predicted to span a membrane multiple times, and it appeared that the gIII portion of each hybrid used a hydrophobic domain of Prv43 protein to initiate its export. For at least two of the isolates, the hybrid protein was efficiently translocated across the endoplasmic reticulum membrane but appeared to be poorly exported out of the endoplasmic reticulum. Nonetheless, the prv43-gIII fusions encoded a gIII species that was localized to the virus envelope. Because the gIII portion of each hybrid protein must be exposed on the virion surface to provide a growth advantage, our results also suggest a preliminary membrane topology for wild-type Prv43 protein.     

Construction of an infectious pseudorabies virus recombinant expressing a glycoprotein gIII-β-galactosidase fusion protein

An infectious herpesvirus mutant has been constructed in which a major structural envelope glycoprotein gene was replaced by a hybrid gene encoding a novel fusion protein consisting of the N-terminus of the viral glycoprotein joined to Escherichia coli β-galactosidase (ßGal). Specifically, we fused DNA encoding the first 157 amino acids of the structural glycoprotein gIII from pseudorabies virus strain Becker to the E. coli lacZ gene in a bacterial expression vector. The resulting hybrid gene was then used to replace the wild-type gIII gene in the virus by cotransfection of plasmid and viral DNA. The desired viral recombinants were identified by their inability to react with specific monoclonal antibodies that recognized only wild-type gIII protein. One such mutant virus, PRV-Z1, was chosen for further analysis. PRV-Z1 expressed a glycosylated gIII-ßGal fusion protein after infection of PK15 cells. The fusion protein has no demonstrable ßGal activity and, although glycosylated, remains sensitive to the enzyme endo-β-N-acetylglucosaminidase H, unlike the mature gIII gene product, indicating that the fusion protein was incompletely processed.     

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.     

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.     

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.     

Expression of glycoprotein gIII-human decay-accelerating factor chimera on the bovine herpesvirus 1 virion via a glycosyl phosphatidylinositol-based membrane anchor.

Mutants of bovine herpesvirus 1 that express a truncated envelope glycoprotein gIII or a gIII-human decay-accelerating factor (hDAF) chimeric protein (gIII.hDAF) were employed to evaluate the function of the transmembrane and cytoplasmic domains of the gIII molecule. Truncated gIII (i.e., lacking the transmembrane and cytoplasmic region) was readily released from infected cells and was not detected on mature virus particles. In contrast, replacement of the transmembrane and cytoplasmic domains with the carboxyl-terminal portion of hDAF restored the expression of gIII on the membranes of infected cells as well as on virion surfaces. The presence of the gIII.hDAF chimera on virus particles was also associated with normal gIII function, i.e., the mediation of virus attachment and penetration. The gIII-hDAF chimera, which is present on both infected cell surfaces and virions, could be cleaved by a phosphatidylinositol-specific phospholipase C, indicating that it was anchored in the membrane via glycosyl phosphatidylinositol. Our results from this study suggest that the transmembrane and cytoplasmic regions of the gIII molecule serve as a general membrane anchor, but they do not contain structural signals required for the specific assembly of envelope proteins into mature virions.     

Pseudorabies virus gIII and bovine herpesvirus 1 gIII share complementary functions.

The gIII glycoproteins of bovine herpesvirus 1 (BHV-1) and of pseudorabies virus (PRV) are structurally homologous. Both proteins also play preeminent roles in mediating virus attachment to permissive cells. To directly compare the functional relation between these glycoproteins, we constructed a recombinant BHV-1 in which the BHV-1 gIII coding sequence was replaced by the PRV gene homolog. The resultant recombinant virus efficiently expressed PRV gIII and then incorporated it into its envelope. The levels of PRV gIII expression and incorporation were equivalent to those achieved by the wild-type virus for BHV-1 gIII. The recombinant virus was fully susceptible to neutralization by anti-PRV gIII neutralizing antibody. In addition, the virus attachment and penetration functions, as well as the virus replication efficiency, which were lost by deleting the BHV-1 gIII gene, were restored by expressing the PRV gIII homolog in its place. These results demonstrated that PRV gIII and BHV-1 gIII share complementary functions.     

Effects of deletions in the carboxy-terminal hydrophobic region of herpes simplex virus glycoprotein gB on intracellular transport and membrane anchoring.

The gB glycoprotein of herpes simplex virus type 1 is involved in viral entry and fusion and contains a predicted membrane-anchoring sequence of 69 hydrophobic amino acids, which can span the membrane three times, near the carboxy terminus. To define the membrane-anchoring sequence and the role of this hydrophobic stretch, we have constructed deletion mutants of gB-1, lacking one, two, or three predicted membrane-spanning segments within the 69 amino acids. Expression of the wild-type and mutant glycoproteins in COS-1 cells show that mutant glycoproteins lacking segment 3 (amino acids 774 to 795 of the gB-1 protein) were secreted from the cells. Protease digestion and alkaline extraction of microsomes containing labeled mutant proteins further showed that segment 3 was sufficient for stable membrane anchoring of the glycoproteins, indicating that this segment may specify the transmembrane domain of the gB glycoprotein. Also, the mutant glycoproteins containing segment 3 were localized in the nuclear envelop, which is the site of virus budding. Deletion of any of the hydrophobic segments, however, affected the intracellular transport and processing of the mutant glycoproteins. The mutant glycoproteins, although localized in the nuclear envelope, failed to complement the gB-null virus (K082). These results suggest that the carboxy-terminal hydrophobic region contains essential structural determinants of the functional gB glycoprotein.     

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.     

Cytoplasmic domain requirement for incorporation of a foreign envelope protein into vesicular stomatitis virus.

Incorporation of human immunodeficiency virus type 1 (HIV-1) envelope proteins into vesicular stomatitis virus (VSV) particles was studied in a system that allows expressed envelope proteins to rescue phenotypically a temperature-sensitive mutant of VSV (tsO45). This mutant exhibits defective transport of its own envelope glycoprotein (G) and can be rescued by simultaneous expression of wild-type G protein from cDNA. We report here that a hybrid HIV-1-VSV protein containing the extracellular and transmembrane domains of the HIV-1 envelope protein fused to the cytoplasmic domain of VSV G protein was able to rescue the tsO45 mutant lacking the G protein, while the wild-type HIV-1 envelope protein was not. The VSV(HIV) pseudotypes obtained infected only CD4+ cells and were neutralized specifically by anti-HIV-1 sera. Our results indicate that the cytoplasmic tail of the VSV glycoprotein contains an independent signal capable of directing a foreign protein into VSV particles. The VSV(HIV) pseudotypes generated here were prepared in the absence of HIV-1 and should be useful for identifying molecules that block HIV-1 entry.     

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.     

Defective mutant of Sindbis virus with a smaller-molecular-weight form of the E1 glycoprotein.

We have isolated from a single plaque a mutant of Sindbis virus characterized by an E1 glycoprotein with higher electrophoretic mobility. This higher mobility is not attributable to a different extent of glycosylation of the protein nor to an altered proteolytic maturation pathway of the polypeptide precursor, but is the result of a deletion occurring during the replication of the viral RNA. The 26S RNA (the messenger for the Sindbis structural proteins) extracted from cells infected with the mutant is about 0.75 x 10(5) daltons smaller than the 26S RNA from the parental strain. As a consequence, in cells infected with the mutant, an E1 glycoprotein is synthesized with a polypeptide chain about 70 amino acids shorter. The biological relevance of this naturally occurring deletion of the viral genome is discussed.     

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.     

DNA sequence of the herpes simplex virus type 1 gene encoding glycoprotein gH, and identification of homologues in the genomes of varicella-zoster virus and Epstein-Barr virus.

We have determined the sequence of herpes simplex virus type 1 DNA around the previously mapped location of sequences encoding an epitope of glycoprotein gH, and have deduced the structure of the gH gene and the amino acid sequence of gH. The unprocessed polypeptide is predicted to contain 838 amino acids, and to possess an N-terminal signal sequence and a C-terminal transmembrane sequence. Temperature-sensitive mutant tsQ26 maps within the predicted gH coding sequence. Homologous genes were identified in the genomes of two other herpesviruses, namely varicella-zoster virus and Epstein-Barr virus.     

Origin of unenveloped capsids in the cytoplasm of cells infected with herpes simplex virus 1.

In cells infected with herpes simplex viruses the capsids acquire an envelope at the nuclear membrane and are usually found in the cytoplasm in structures bound by membranes. Infected cells also accumulate unenveloped capsids alone or juxtaposed to cytoplasmic membranes. The juxtaposed capsids have been variously interpreted as either undergoing terminal deenvelopment resulting from fusion of the envelope with the membrane of the cytoplasmic vesicles or undergoing sequential envelopment and deenvelopment as capsids transit the cytoplasm into the extracellular space. Recent reports have shown that (i) wild-type virus attaches to but does not penetrate cells expressing glycoprotein D (G. Campadelli-Fiume, M. Arsenakis, F. Farabegoli, and B. Roizman, J. Virol. 62:159-167, 1988) and that (ii) a mutation in glycoprotein D enables the mutant virus to productively infect cells expressing the wild-type glycoprotein (G. Campadelli-Fiume, S. Qi, E. Avitabile, L. Foa-Tomasi, R. Brandimarti, and B. Roizman, J. Virol. 64:6070-6079, 1990). If the unenveloped capsids in the cytoplasm result from fusion of the cytoplasmic membranes with the envelopes of viruses transiting the cytoplasm, cells infected with virus carrying the mutation in glycoprotein D should contain many more unenveloped capsids in the cytoplasm inasmuch as there would be little or no restriction in the fusion of the envelope with cytoplasmic membranes. Comparison of thin sections of baby hamster kidney cells infected with wild-type and mutant viruses indicated that this was the case. Moreover, in contrast to the wild-type parent, the mutant virus was not released efficiently from infected cells. The conclusion that the unenveloped capsids are arrested forms of deenveloped capsids is supported by the observation that the unenveloped capsids were unstable in that they exhibited partially extruded DNA.     

Basic amino acids as modulators of an O-linked glycosylation signal of the herpes simplex virus type 1 glycoprotein gC: functional roles in viral infectivity

The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1 is engaged both in viral attachment and viral immune evasion mechanisms in the infected host. Besides several N-linked glycans, gC-1 contains numerous O-linked glycans, mainly localized in two pronase-resistant clusters in the N-terminal domain of gC-1. In the present study we construct and characterize one gC-1 mutant virus, in which two basic amino acids (114K and 117R) in a putative O-glycosylation sequon were changed to alanine. We found that this modification did not modify the N-linked glycosylation but increased the content of O-linked glycans considerably. Analysis of the O-glycosylation capacity of wild-type and mutant gC-1 was performed by in vitro glycosylation assays with synthetic peptides derived from the mutant region predicted to present new O-glycosylation sites. Thus the mutant peptide region served as a better substrate for polypeptide GalNAc-transferase 2 than the wild-type peptide, resulting in increased rate and number of O-glycan attachment sites. The predicted increase in O-linked glycosylation resulted in two modifications of the biological properties of mutant virus-that is, an impaired binding to cells expressing chondroitin sulfate but not heparan sulfate on the cell surface and a significantly reduced plaque size in cultured cells. The results suggested that basic amino acids present within O-glycosylation signals may down-regulate the amount of O-linked glycans attached to a protein and that substitution of such amino acid residues may have functional consequences for a viral glycoprotein involving virus attachment to permissive cells as well as viral cell-to-cell spread.     

Mutational analysis of hepatitis B surface antigen particle assembly and secretion.

Cells infected with hepatitis B virus produce both virions and 20-nm subviral (surface antigen or HBsAg) particles; the latter are composed of viral envelope proteins and host-derived lipid. Although hepatitis B virus encodes three envelope proteins (L, M, and S), all of the information required to produce an HBsAg particle resides within the S protein. This polypeptide spans the bilayer at least twice and contains three hydrophobic regions, two of which are known to harbor topogenic signal sequences that direct this transmembrane orientation. We have examined the effects of mutations in these and other regions of the S protein on particle assembly and export. Lesions in the N terminal signal sequence (signal I) can still insert into the endoplasmic reticulum bilayer but do not participate in any of the subsequent steps in assembly. Deletion of the major internal signal (signal II) completely destabilizes the chain. Deletion of the C-terminal hydrophobic domain results in a stable, glycosylated, but nonsecreted chain. However, when coexpressed with wild-type S protein this mutant polypeptide can be incorporated into particles and secreted, indicating that the chain is still competent for some of the distal steps in particle assembly. The correct transmembrane disposition of the N terminus of the molecule is important for particle formation: addition of a heterologous (globin) domain to this region impairs secretion, but the defect can be corrected by provision of an N-terminal signal sequence that restores the proper topology of this region. The resulting chimeric chain is assembled into subviral particles that are secreted with normal efficiency.