Mutations within the proteolytic cleavage site of the Rous sarcoma virus glycoprotein that block processing to gp85 and gp37.

We have investigated the specificity of the proteolytic cleavage of the Rous sarcoma virus glycoprotein precursor by introducing two mutations into the putative cleavage region (Arg-Arg-Lys-Arg). We show that neither a deletion of the cleavage sequence nor a glutamic acid for lysine substitution altered intracellular transport or surface expression of the env gene products. However, both the four-amino-acid deletion and the glutamic acid substitution block processing of the env precursor. Susceptibility of the glutamic acid-substituted env precursor to proteases indicated that tertiary protein structure was unaffected. While inhibitor experiments suggested that more than one endopeptidase might be capable of mediating the proteolytic cleavage, the results presented here point to the presence in the Golgi apparatus of a novel endopeptidase, required for retroviral glycoprotein cleavage, that has a high specificity for lysine-containing peptides.     

A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus.

Epstein-Barr virus (EBV) codes for at least three glycoproteins, gp350, gp220, and gp85. The two largest glycoproteins are thought to be involved in the attachment of the virus to its receptor on B cells, but despite the fact that gp85 induces neutralizing antibody, no function has been attributed to it. As an indirect approach to understanding the role of gp85 in the initiation of infection, we determined the point at which a neutralizing, monoclonal antibody that reacted with the glycoprotein interfered with virus replication. The antibody had no effect on virus binding. To examine the effect of the antibody on later stages of infection, the fusion assay of Hoekstra and colleagues (D. Hoekstra, T. de Boer, K. Klappe, and J. Wilshaut, Biochemistry 23:5675-5681, 1984) was adapted for use with EBV. The virus was labeled with a fluorescent amphiphile that was self-quenched at the high concentration obtained in the virus membrane. When the virus and cell membrane fused, there was a measurable relief of self-quenching that could be monitored kinetically. Labeling had no effect on virus binding or infectivity. The assay could be used to monitor virus fusion with lymphoblastoid lines or normal B cells, and its validity was confirmed by the use of fixed cells and the Molt 4 cell line, which binds but does not internalize the virus. The monoclonal antibody to gp85 that neutralized virus infectivity, but not a second nonneutralizing antibody to the same molecule, inhibited the relief of self-quenching in a dose-dependent manner. This finding suggests that gp85 may play an active role in the fusion of EBV with B-cell membranes.     

Mutational analysis of the cleavage sequence of the human immunodeficiency virus type 1 envelope glycoprotein precursor gp160.

The envelope glycoproteins of the human immunodeficiency virus (HIV) type 1 are synthesized as a precursor molecule, gp160, which is cleaved to generate the two mature envelope glycoproteins, gp120 and gp41. The cleavage reaction, which is mediated by a host protease, occurs at a sequence highly conserved in retroviral envelope glycoprotein precursors. We have investigated the sequence requirements for this cleavage reaction by introducing four single-amino-acid changes into the glutamic acid-lysine-arginine sequence immediately amino terminal to the site of cleavage. We have also examined the effects of these mutations on the syncytium formation induced by HIV envelope glycoproteins. Our results indicate that a glutamic acid to glycine change at gp120 amino acid 516, a lysine to isoleucine change at amino acid 517, and an arginine to lysine change at amino acid 518 affect neither gp160 cleavage nor syncytium formation. The results obtained with the arginine to lysine change at amino acid 518 differ significantly from the results obtained with the same mutation at the envelope precursor cleavage site of a murine leukemia virus (E. O. Freed, and R. Risser, J. Virol. 61:2852-2856, 1987). An arginine to threonine mutation at gp120 amino acid 518, the terminal residue of gp120, abolishes both gp160 cleavage and syncytium formation. These findings demonstrate that despite its highly conserved nature, the basic pair of amino acids at the site of gp160 cleavage is not absolutely required for proper envelope glycoprotein processing. This report also supports the idea that cleavage of gp160 is required for activation of the HIV envelope fusion function.     

gp120-independent fusion mediated by the human immunodeficiency virus type 1 gp41 envelope glycoprotein: a reassessment.

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.     

Envelope glycoprotein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice.

Phenotypically complemented pseudorabies virus gp50 null mutants are able to produce plaques on noncomplementing cell lines despite the fact that progeny virions are noninfectious. To determine whether gp50 null mutants and gp50+gp63 null mutants are also able to replicate and spread in animals, mice were infected subcutaneously or intraperitoneally. Surprisingly, both gp50 mutants and gp50+gp63 double mutants proved to be lethal for mice. In comparison with the wild-type virus, gp50 mutants were still highly virulent, whereas the virulence of gp50+gp63 mutants was significantly reduced. Severe signs of neurological disorders, notably pruritus, were apparent in animals infected with the wild-type virus or a gp50 mutant but were much less pronounced in animals infected with a gp50+gp63 or gp63 mutant. Immunohistochemical examination of infected animals showed that all viruses were able to reach, and replicate in, the brain. Examination of visceral organs of intraperitoneally infected animals showed that viral antigen was predominantly present in peripheral nerves, suggesting that the viruses reached the central nervous system by means of retrograde axonal transport. Infectious virus could not be recovered from the brains and organs of animals infected with gp50 or gp50+gp63 mutants, indicating that progeny virions produced in vivo are noninfectious. Virions that lacked gp50 in their envelopes, and a phenotypically complemented pseudorabies virus gII mutant (which is unable to produce plaques in tissue culture cells), proved to be nonvirulent for mice. Together, these results show that gp50 is required for the primary infection but not for subsequent replication and viral spread in vivo. These results furthermore indicate that transsynaptic transport of the virus is independent of gp50. Since progeny virions produced by gp50 mutants are noninfectious, they are unable to spread from one animal to another. Therefore, such mutants may be used for the development of a new generation of safer (carrier) vaccines.     

Processing of the herpes simplex virus type 2 glycoprotein gG-2 results in secretion of a 34,000-Mr cleavage product.

Herpes simplex virus type 2 glycoprotein gG-2 undergoes a cleavage event during its synthesis and processing. The focus of this report is on the detection and fate of the small-molecular-weight component of gG-2, designated the 34K component. In cultures containing the inhibitor monensin, a 31K component accumulated within infected cells. In contrast, the intracellular accumulation of this 31K precursor was not detected in cultures grown in the absence of the inhibitor. However, the 34K component of gG-2 was found in the extracellular culture fluid. The data suggest that the 31K high-mannose cleavage product of gG-2 is further glycosylated and rapidly secreted from herpes simplex virus type 2-infected cells; however, if glycosylation is perturbed, the 31K high-mannose form remains cell associated.     

Expression of herpes simplex virus type 1 glycoprotein L (gL) in transfected mammalian cells: evidence that gL is not independently anchored to cell membranes.

We expressed herpes simplex virus type 1 glycoprotein L (gL) in transfected cells to investigate whether it is independently anchored to plasma membranes or is membrane associated as a result of complex formation with gH. gL was detected by immunofluorescence microscopy at the surfaces of cotransfected cells when it was expressed with gH but not when it was expressed in the absence of gH or with a truncated form of gH, gHTrunc(792), which lacks the membrane-spanning region and terminates at amino acid 792. Immunoprecipitation studies of transfected-cell culture media revealed that gL was secreted from cells when expressed in the absence of gH and was secreted from cotransfected cells complexed with gHTrunc(792). These observations demonstrate that gL is not independently anchored to plasma membranes but is membrane associated as a result of complex formation with gH.     

Analysis of the cleavage site of the human immunodeficiency virus type 1 glycoprotein: requirement of precursor cleavage for glycoprotein incorporation.

Endoproteolytic cleavage of the glycoprotein precursor to the mature SU and TM proteins is an essential step in the maturation of retroviral glycoproteins. Cleavage of the precursor polyprotein occurs at a conserved, basic tetrapeptide sequence and is carried out by a cellular protease. The glycoprotein of the human immunodeficiency virus type 1 contains two potential cleavage sequences immediately preceding the N terminus of the TM protein. To determine the functional significance of these two potential cleavage sites, a series of mutations has been constructed in each site individually, as well as in combinations that altered both sites simultaneously. A majority of the mutations in either potential cleavage site continued to allow efficient cleavage when present alone but abrogated cleavage of the precursor when combined. Despite being transported efficiently to the cell surface, these cleavage-defective glycoproteins were unable to initiate cell-cell fusion and viruses containing them were not infectious. Viruses that contained glycoproteins with a single mutation, and that retained the ability to be processed, were capable of mediating a productive infection, although infectivity was impaired in several of these mutants. Protein analyses indicated that uncleaved glycoprotein precursors were inefficiently incorporated into virions, suggesting that cleavage of the glycoprotein may be a prerequisite to incorporation into virions. The substitution of a glutamic acid residue for a highly conserved lysine residue in the primary cleavage site (residue 510) had no effect on glycoprotein cleavage or function, even though it removed the only dibasic amino acid pair in this site. Peptide sequencing of the N terminus of gp41 produced from this mutant glycoprotein demonstrated that cleavage continued to take place at this site. These results, demonstrating that normal cleavage of the human immunodeficiency virus type 1 glycoprotein can occur when no dibasic sequence is present at the cleavage site, raise questions about the specificity of the cellular protease that mediates this cleavage and suggest that cleavage of the glycoprotein is required for efficient incorporation of the glycoprotein into virions.     

Herpes simplex virus type 1 glycoprotein E is not indispensable for viral infectivity.

A mutant of the herpes simplex virus type 1 Angelotti was isolated in which 87% of the coding region of glycoprotein E (gE) was deleted and replaced by a functional neomycin resistance gene of the Tn5 transposon. The mutant was characterized by restriction enzyme analyses and Southern blotting. Western blotting of proteins and immunofluorescence assays revealed that gE was completely absent and that the Fc receptor was not expressed in cells infected with the mutant. The fact that this mutant was viable and that it replicated to a slightly lower titer than did the wild-type virus suggests that the presence of gE is not a prerequisite of viral infectivity in tissue culture.     

Physical and chemical properties of the major envelope glycoprotein gp85 from avian myeloblastosis virus

The major envelope glycoprotein gp85 of avian myeloblastosis virus, observed by electron microscopy as nearly spherical knobs projecting from the virus surface, was purified to homogeneity by gel filtration in 6 M guanidinium chloride followed by ion-exchange chromatography. The purified glycoprotein has a molecular weight of 80 000 from sedimentation equilibrium analysis. Glycoprotein gp85 contains approx. 45% carbohydrate including 25% N-acetylglucosamine, while the remaining weight consists of a polypeptide chain of approx. 45 000 daltons. Based on the oligosaccharide chain molecular weight data of Lai and Duesberg (Lai, M.M.C. and Duesberg, P.H. (1972) Virology 50, 359-372), the carbohydrate is calculated to be distributed between seven to nine oligosaccharide side chains. No self-association of gp85 was observed up to 2.0 mg/ml in dilute salt solution. The hydrodynamic properties of gp85 in dilute salt solution indicate a highly elongated molecule with an axial ratio of 7. One structural model which reconciles the hydrodynamic properties of gp85 with the nearly spherical architecture observed by electron microscopy requires the organization of the polypeptide chain and approx. 50% of the carbohydrate into a globular form. The remaining covalently linked oligosaccharides would by necessity extend outwardly from the globular structure as randomly oriented chains.     

Identification of a site on herpes simplex virus type 1 glycoprotein D that is essential for infectivity.

Herpes simplex virus glycoprotein D (gD) plays an essential role during penetration of the virus into cells. There is evidence that it recognizes a specific receptor after initial attachment of virions to cell surface heparan sulfate and also that gD-1, gD-2, and gp50 (the pseudorabies virus gD homolog) bind to the same receptor. Although the antigenic structure of gD has been studied intensively, little is known about functional regions of the protein. Antigenic site I is a major target for neutralizing antibodies and has been partially mapped by using deletion mutants and neutralization-resistant viruses. Working on the assumption that such a site may overlap with a functional region of gD, we showed previously that combining two or more amino acid substitutions within site I prevents gD-1 from functioning and is therefore lethal. We have now used a complementation assay to measure the functional activity of a panel of deletion mutants and compared the results with an antigenic analysis. Several mutations cause gross changes in protein folding and destroy functional activity, whereas deletions at the N and C termini have little or no effect on either. In contrast, deletion of residues 234 to 244 has only localized effects on antigenicity but completely abolishes functional activity. This region, which is part of antigenic site Ib, is therefore essential for gD-1 function. The complementation assay was also used to show that a gD-negative type 1 virus can be rescued by gD-2 and by two gD-1-gD-2 hybrids but not by gp50, providing some support for the existence of a common receptor for herpes simplex virus types 1 and 2 but not pseudorabies virus. Alternatively, gp50 may lack a signal for incorporation into herpes simplex virions.     

Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.     

Characterization of a Borna disease virus glycoprotein, gp18.

Borna disease virus is a nonsegmented negative-strand RNA virus that causes neurologic disease in a wide variety of animal hosts. Here we describe identification and characterization of the first glycoprotein in this viral system. The 18-kDa glycoprotein, gp18, has been purified from infected rat brain. Isolation and microsequencing of this protein allowed identification of a 16.2-kDa open reading frame in the viral antigenome. Lectin binding and endoglycosidase sensitivity assays indicate that gp18 is an unusual N-linked glycoprotein.     

The UL45 gene product is required for herpes simplex virus type 1 glycoprotein B-induced fusion.

Herpes simplex virus type 1 (HSV-1) syncytial (syn) mutants cause formation of giant polykaryocytes and have been utilized to identify genes promoting or suppressing cell fusion. We previously described an HSV-1 recombinant, F1 (J.L. Goodman, M. L. Cook, F. Sederati, K. Izumi, and J. G. Stevens, J. Virol. 63:1153-1161, 1989), which has unique virulence properties and a syn mutation in the carboxy terminus of glycoprotein B (gB). We attempted to replace this single-base-pair syn mutation through cotransfection with a 379-bp PCR-generated fragment of wild-type gB. The nonsyncytial viruses isolated were shown by DNA sequencing not to have acquired the expected wild-type gB sequence. Instead, they had lost their cell-cell fusion properties because of alterations mapping to the UL45 gene. The mutant UL45 gene is one nonsyncytial derivative of F1, A4B, was found to have a deletion of a C at UL45 nucleotide 230, resulting in a predicted frame shift and termination at 92 rather than 172 amino acids. Northern (RNA) analysis showed that the mutant UL45 gene was normally transcribed. However, Western immunoblotting showed no detectable UL45 gene product from A4B or from another similarly isolated nonsyncytial F1 derivative, A61B, while another such virus, 1ACSS, expressed reduced amounts of UL45. When A4B was cotransfected with the wild-type UL45 gene, restoration of UL45 expression correlated with restoration of syncytium formation. Conversely, cloned DNA fragments containing the mutant A4B UL45 gene transferred the loss of cell-cell fusion to other gB syn mutants, rendering them UL45 negative and nonsyncytial. We conclude that normal UL45 expression is required to allow cell fusion induced by gB syn mutants and that the nonessential UL45 protein may play an important role as a mediator of fusion events during HSV-1 infection.     

gp160 of HIV-I synthesized by persistently infected Molt-3 cells is terminally glycosylated: evidence that cleavage of gp160 occurs subsequent to oligosaccharide processing

The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent Mr 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV IIIB). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetyl-galactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [35S]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a half-time of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment.     

The membrane glycoprotein of Friend spleen focus-forming virus: evidence that the cell surface component is required for pathogenesis and that it binds to a receptor.

The leukemogenic membrane glycoprotein of Friend spleen focus-forming virus (SFFV) has an apparent Mr of 55,000 (gp55), is encoded by a recombinant env gene, and occurs on cell surfaces and in intracellular organelles. There is evidence that the amino-terminal region of gp55 forms a dualtropic-specific domain that is connected to the remainder of the glycoprotein by a proline-rich linker (C. Machida, R. Bestwick, B. Boswell, and D. Kabat, Virology 144:158-172, 1985). Using the colinear form of a cloned polycythemic strain of SFFV proviral DNA, we constructed seven in-phase env mutants by insertion of linkers and by a deletion. The mutagenized SFFVs were transfected into fibroblasts and were rescued by superinfection with a helper murine leukemia virus. Four of the mutants cause erythroblastosis. These include one with a 6-base-pair (bp) insert in the ecotropic-related sequence near the 3' end of the gene, two with a 12- or 18-bp insert in the region that encodes the proline-rich linker, and one with a 6-bp insert in the dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific sequences that are highly conserved among strains of SFFV. A pathogenic revertant (RI-rev) was isolated from one mouse that developed erythroblastosis 3 weeks after infection with RI. RI-rev contains a second-site env mutation that affects the same domain as the primary mutation does and that increases the size of the encoded glycoprotein. All pathogenic SFFVs encode glycoproteins that are expressed on cell surfaces, whereas the nonpathogenic glycoproteins are exclusively intracellular. The pathogenic SFFVs also specifically cause a weak interference to superinfection by dualtropic MuLVs. These results are compatible with the multidomain model for the structure of gp55 and suggest that processing of gp55 to plasma membranes is required for pathogenesis. The amino-terminal region of gp55 binds to dualtropic murine leukemia virus receptors, and this interaction is preserved in the SFFV mutants that cause erythroblastosis.     

The product of the avian erythroblastosis virus erbB locus is a glycoprotein

Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds. A locus, v-erbB, within the viral genome has been implicated in AEV-mediated oncogenesis. We report here the detection and partial characterization of the protein product of the v-erbB oncogene in AEV-transformed cells. We obtained the antisera necessary for our analysis by expressing a portion of the molecularly cloned v-erbB locus in Escherichia coli and immunizing rabbits with the resulting bacterial erbB polypeptide. Antisera directed against the bacterial polypeptide reacted with v-erbB proteins obtained from virus-infected avian cells. By three criteria—tunicamycin inhibition, lectin binding and metabolic labeling with radioactive sugar precursors—the product of the v-erbB gene appears to be a glycoprotein.     

Evidence that type II 5'-deiodinase is not a selenoprotein.

Brain type II 5'-iodothyronine deiodinase and liver type I 5'-iodothyronine deiodinase activities are decreased in rats fed a Se(2+)-deficient diet suggesting that both enzymes are Se(2+)-dependent proteins. Since serum thyroxine (T4) concentrations are twice normal in the Se(2+)-deficient animals, it is unclear whether the Se2+ deficiency or the increased circulating T4 account for the decrease in the brain enzyme. In order to separate these two possibilities, the effects of Se2+ on 5'-deiodinase in glial cells (type II) and LLC-PK1 cells (type I) were examined. LLC-PK1 and glial cells were grown in serum-free defined medium containing 0, 1 pM, 10 nM, and 40 nM Se2+ for 3-5 days or in medium containing 75Se2+ for 24 h. Deiodinase isozymes were determined by measuring catalytic activity and by quantification of the BrAc[125I]T4 affinity-labeled substrate binding subunits. Se2+ deficiency was confirmed by measuring the activity of the selenoprotein, glutathione peroxidase. Se2+ caused a concentration-dependent increase in glutathione peroxidase activity in both cell types, as well as in the type I enzyme, but had no effect on the type II enzyme. LLC-PK1 cells contained multiple 75Se(2+)-labeled proteins including the 27-kDa substrate binding subunit of the type I 5'-deiodinase. Glial cells contained seven 75Se(2+)-labeled proteins ranging in size from 12 to 62 kDa, none of which corresponded to the type II substrate binding subunit. these data show that, unlike the type I enzyme, the type II enzyme does not contain a selenocysteine or selenomethionine, further emphasizing the differences between these two isozymes.