No glycolipid anchors are added to Thy-1 glycoprotein in Thy-1-negative mutant thymoma cells of four different complementation classes.

Recent evidence shows that the mature Thy-1 surface glycoprotein lacks the C-terminal amino acids 113 to 143 predicted from the cDNA sequence and is anchored in the plasma membrane by a complex, phosphatidylinositol-containing glycolipid attached to the alpha-carboxyl group of amino acid 112. Here we studied the biosynthesis of Thy-1 in two previously described and two newly isolated Thy-1-deficient mutant cell lines. Somatic cell hybridization indicated that their mutations affected some processing step rather than the Thy-1 structural gene. The Thy-1 made by mutants of classes C, F, and H bound detergent but, in contrast to wild-type Thy-1, their detergent-binding moieties could not be removed by phospholipase C. In addition, tryptophan, which only occurs in position 124, was incorporated into Thy-1 of these mutants but not of wild-type cells. Last, the Thy-1 of wild-type but not mutant cells could be radiolabeled with [3H]palmitic acid. Together, these findings strongly suggest that mutants of classes C, F, and H accumulate a biosynthetic intermediate of Thy-1 which retains at least part of the hydrophobic C-terminal peptide. The Thy-1 of these mutants remained endoglycosidase H sensitive, suggesting that it accumulated in the rough endoplasmic reticulum or the Cis-Golgi. A different Thy-1 intermediate was found in a class B mutant cell line: the Thy-1 of this mutant was 2 kilodaltons smaller than the Thy-1 of other cell lines, did not bind detergent, and was rapidly secreted via a normal secretory pathway.     

The phenotype of five classes of T lymphoma mutants. Defective glycophospholipid anchoring, rapid degradation, and secretion of Thy-1 glycoprotein

Thy-1 glycoprotein is a member of a class of proteins which are anchored to the plasma membrane via a covalently bound glycophospholipid. The biosynthesis and anchoring of Thy-1 were investigated in a family of wild-type and mutant (complementation groups A, B, C, E, and F) T lymphomas. The mutants all synthesize Thy-1 but fail to express it on the cell surface. Analysis of the size of D-[2-3H]mannose-labeled dolichol-linked oligosaccharides showed that the class E mutant is the only cell line which does not synthesize dolichol-P-P-Glc3Man9GlcNAc2. Turnover and possible secretion of Thy-1 by mutant T lymphoma cells were documented in D-[2-3H]mannose pulse-chase experiments. The turnover of [3H]Thy-1 for all wild-type cells is considerably slower than for the mutant cells. Class B and E cells release appreciably more [3H]Thy-1 than wild-type cells. Additional experiments were performed to determine the electrophoretic mobility and hydrophobicity of cell-associated and released forms of Thy-1 labeled overnight with [3H]mannose. All wild-type and class A, C, E, and F mutant cells contain a major Triton X-114 binding species of cell-associated [3H]Thy-1. All extracellular [3H]Thy-1 was almost exclusively hydrophilic. The presence of two Thy-1 anchor components, ethanolamine and palmitate, was investigated. Biosynthetic labeling with [3H]palmitic acid showed that all of the wild-type cells but none of the mutants incorporated this anchor precursor into Thy-1. In [3H]ethanolamine-labeling experiments, incorporation was detected in the Thy-1 of all wild-type cells and in two mutants, S1A-b and T1M1-c. Based on the above studies, the phenotype of Thy-1 negative T lymphoma mutants can be re-evaluated. In classes A and F, dolichol-linked oligosaccharides appear normal and no anchor is detected. In class B, dolichol-linked oligosaccharides appear normal, a partial anchor may be present, and a substantial amount of Thy-1 is released. In class C, dolichol-linked oligosaccharides appear normal and a partial anchor may be present. In class E, truncated dolichol-linked oligosaccharides are formed, no anchor is detected, but a substantial amount of newly synthesized Thy-1 is released. These observations are discussed with reference to the possibility that the lesions which characterize the mutants pertain to the biosynthesis of the glycophospholipid moiety of Thy-1.     

Cell surface molecules of friend erythroleukemias: Decrease in T200 glycoprotein expression after induction

Monoclonal antibodies against the Thy-1 and T200 glycoproteins were used to study the expression of cell surface molecules on mouse hematopoietic cell lines. Friend erythroleukemias express T200 glycoprotein but do not express significant amounts of Thy-1 glycoprotein on their cell surface. The rate of T200 glycoprotein synthesis in maximally-induced Friend erythroleukemia 745.6 cells is less than 10% that in noninduced cells, although total protein synthesis shows only a twofold decline and induced cells express 2-6-fold less T200 glycoprotein on their surface compared to noninduced cells. T200 glycoprotein expression is reduced in a variant cell line obtained by selection for growth in dimethylsulfoxide, showing that the reduction in T200 glycoprotein synthesis characteristic of induced cells is an event that can be dissociated from commitment and hemoglobin synthesis. Analysis of T200 glycoprotein negative cell lines, isolated by cytotoxic immunoselection against T200 glycoprotein, indicates that the presence of T200 glycoprotein on the cell surface is not necessary for induction of hemoglobin synthesis and terminal differentiation of Friend erythroleukemias.     

Genetic and phenotypic analysis of herpes simplex virus type 1 mutants conditionally resistant to immune cytolysis

Nine temperature-sensitive (ts) mutants of herpes simplex virus type 1 selected for their inability to render cells susceptible to immune cytolysis after infection at the nonpermissive temperature have been characterized genetically and phenotypically. The mutations in four mutants were mapped physically by marker rescue and assigned to functional groups by complementation analysis. In an effort to determine the molecular basis for cytolysis resistance, cells infected with each of the nine mutants were monitored for the synthesis of viral glycoprotein in total cell extracts and for the presence of these glycoproteins in plasma membranes. The four mutants whose ts mutations were mapped were selected with polypeptide-specific antiserum to glycoproteins gA and gB; however, three of the four mutations mapped to DNA sequences outside the limits of the structural gene specifying these glycoproteins. Combined complementation and phenotypic analysis indicates that the fourth mutation also lies elsewhere. The ts mutations in five additional cytolysis-resistant mutants could not be rescued with single cloned DNA fragments representing the entire herpes simplex virus type 1 genome, suggesting that these mutants may possess multiple mutations. Complementation tests with the four mutants whose ts lesions had been mapped physically demonstrated that each represents a new viral gene. Examination of mutant-infected cells at the nonpermissive temperature for the presence of viral glycoproteins in total cell extracts and in membranes at the cell surface demonstrated that (i) none of the five major viral glycoproteins was detected in extracts of cells infected with one mutant, suggesting that this mutant is defective in a very early function; (ii) cells infected with six of the nine mutants exhibited greatly reduced levels of all the major viral glycoproteins at the infected cell surface, indicating that these mutants possess defects in the synthesis or processing of viral glycoproteins; and (iii) in cells infected with one mutant, all viral glycoproteins were precipitable at the surface of the infected cell, despite the resistance of these cells to cytolysis. This mutant is most likely mutated in a gene affecting a late stage in glycoprotein processing, leading to altered presentation of glycoproteins at the plasma membrane. The finding that the synthesis of both gB and gC was affected coordinately in cells infected with six of the nine mutants suggests that synthesis of these two glycoproteins, their transport to the cell surface, or their insertion into plasma membranes is coordinately regulated.     

Isolation of protein glycosylation mutants in the fission yeast Schizosaccharomyces pombe.

We have isolated mutants in the fission yeast Schizosaccharomyces pombe that are defective in protein glycosylation. A collection of osmotically sensitive mutants was prepared and screened for glycosylation defects using lectin staining as an assay. Mutants singly defective in four glycoprotein synthesis genes (gps1-4) were isolated, all of which bind less galactose-specific lectin. Acid phosphatase and other glycoproteins from the gps mutants have increased electrophoretic mobility, suggesting that these mutants make glycans of reduced size. N-linked glycan analysis revealed that terminal oligosaccharide modification is defective in the gps1 and gps2 mutants. Both mutants synthesize the Man9GlcNAc2 core glycan but have reduced amounts of larger structures. Modified core glycans from gps1 cells have normal amounts of galactose (Gal) residues, but reduced amounts of Man, consistent with a defect in a Golgi mannosyltransferase in this mutant. In contrast, N-linked oligosaccharides from gps2 mutants have much less Gal than wild type, because of reduced levels of the Gal donor, UDP-Gal. This reduction is caused by decreased activity of UDP-glucose 4-epimerase, which synthesizes UDP-Gal. Neither the gps1 or gps2 mutations are lethal, although the cells grow at reduced rates. These findings suggest that S. pombe cells can survive with incompletely glycosylated cell wall glycoproteins. In particular, these results suggest that Gal, which comprises approximately 30% by weight of cell wall glycoprotein glycans, is not crucial for cell growth or survival.     

Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains.

The spike glycoprotein G of vesicular stomatitis virus (VSV) induces membrane fusion at low pH. We used linker insertion mutagenesis to characterize the domain(s) of G glycoprotein involved in low-pH-induced membrane fusion. Two or three amino acids were inserted in frame into various positions in the extracellular domain of G, and 14 mutants were isolated. All of the mutants expressed fully glycosylated proteins in COS cells. However, only seven mutant G glycoproteins were transported to the cell surface. Two of these mutants, D1 and A6, showed wild-type fusogenic properties. The mutant A2 had a temperature-sensitive defect in the transport of the mutant G glycoprotein to the cell surface. The other four mutants, H2, H5, H10, and A4, although present in cell surface, failed to induce cell fusion when cells expressing these mutant glycoproteins were exposed to acidic pH. These four mutant G proteins could form trimers, indicating that the defect in fusion was not due to defective oligomerization. One of these mutations, H2, is within a region of conserved, uncharged amino acids that has been proposed as a possible fusogenic sequence. The mutation in H5 was about 70 amino acids downstream of the mutation in H2, while mutations in H10 and A4 were about 300 amino acids downstream of the mutation in H2. Conserved sequences were also noted in the H10 and A4 segment. The results suggest that in the case of VSV G glycoprotein, the fusogenic activity may involve several spatially separated regions in the extracellular domain of the protein.     

Mutant analysis of herpes simplex virus-induced cell surface antigens: resistance to complement-mediated immune cytolysis.

BHK-21 cells infected with temperature-sensitive mutants of herpes simplex virus type 1 strain KOS representing 16 complementation groups were tested for susceptibility to complement-mediated immune cytolysis at permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures. Only cells infected by mutants in complementation group E were resistant to immune cytolysis in a temperature-sensitive manner compared with wild-type infections. The expression of group E mutant cell surface antigens during infections at 34 and 39 degrees C was characterized by a combination of cell surface radioiodination, specific immunoprecipitation, and gel electrophoretic analysis of immunoprecipitates. Resistance to immune lysis at 39 degrees C correlated with the absence of viral antigens exposed at the cell surface. Intrinsic radiolabeling of group E mutant infections with [14C]glucosamine revealed that normal glycoproteins were produced at 34 degrees C but none were synthesized at 39 degrees C. The effect of 2-deoxy-D-glucose on glycosylation of group E mutants at 39 degrees C suggested that the viral glycoprotein precursors were not synthesized. The complementation group E mutants failed to complement herpes simplex virus type 1 mutants isolated by other workers. These included the group B mutants of strain KOS, the temperature-sensitive group D mutants of strain 17, and the LB2 mutant of strain HFEM. These mutants should be considered members of herpes simplex virus type 1 complementation group 1.2, in keeping with the new herpes simplex virus type 1 nomenclature.     

Immunogenicity of herpes simplex virus glycoproteins gC and gB and their role in protective immunity.

The relative antigenicity of the individual herpes simplex virus type 1 (KOS) glycoproteins gC and gB was analyzed in BALB/c mice by using KOS mutants altered in their ability to present these antigens on cell surface membranes during infection. The mutants employed were as follows: syn LD70 , a non-temperature-sensitive mutant defective in the synthesis of cell surface membrane gC; tsF13 , a temperature-sensitive mutant defective in the processing of the precursor form of gB to the mature cell surface form at 39 degrees C; and ts606 , an immediate early temperature-sensitive mutant defective in the production of all early and late proteins including the glycoproteins. By comparing the relative susceptibility to immunolysis of mouse 3T3 cells infected at 39 degrees C with wild-type virus, presenting the full complement of the glycoprotein antigens, gC, gB, and gD, with target cells infected with mutants presenting only subsets of these antigens, we determined that a major portion of cytolytic antibody contained in hyperimmune anti-herpes simplex virus type 1 (KOS) mouse antiserum was directed against glycoproteins gC and gB. The relative immunogenicity of wild-type and mutant virus-infected cells also was compared in BALB/c mice. Immunogen lacking the mature form of gB induced a cytolytic antibody titer comparable to that of the wild-type virus, whereas that lacking the mature form of gC showed a 70% reduction in titer. The absence of the mature cell surface forms of gB and gC in immunogen preparations resulted in a 4- to 15-fold reduction in in virus neutralizing titer. Animals immunized with ts606 -infected cells (39 degrees C) induced relatively little virus-specific cytolytic and neutralizing antibody. Analysis of the glycoprotein specificities of these antisera by radioimmunoprecipitation showed that the antigens immunoprecipitated reflected the viral plasma membrane glycoprotein profiles of the immunogens. The absence of the mature forms of gC or gB in the immunizing preparation did not appreciably affect the immunoprecipitating antibody response to other antigens. Mice immunized with wild-type and mutant virus-infected cells were tested for their resistance to intracranial and intraperitoneal challenge with the highly virulent WAL strain of herpes simplex virus type 1. Despite the observed alterations in serum virus-specific antibody induced with the individual immunogens, all animals survived an intraperitoneal challenge of 10 50% lethal doses. However, differences in the survival of animals were obtained upon intracranial challenge.(ABSTRACT TRUNCATED AT 400 WORDS)     

A mutant lymphoma cell line with a defectiveThy-1 glycoprotein gene

We characterized a mutant T -cell lymphoma line selected for the inability to express the Thy-1 glycoprotein. This cell line is a member of the D complementation class of Thy-1^– somatic cell mutants, and it lacks detectable cell-surface Thy-1.1 glycoprotein and detectable cytoplasmic Thy-1 mRNA. Southern blot analysis using a number of probes isolated from the clonedThy-1.2 gene demonstrated that, in the mutant, one copy of theThy-1 gene is absent from the genome and the other has undergone rearrangement. This rearrangement results from a deletion of the 5 portion of the gene removing the first two alternate exons and promoters and a portion of the second intron. The deletion breakpoint within the mutantThy-1 gene was localized to within 400 nucleotides by Southern blot analysis. The breakpoint is near two classes of mouse repetitive elements-a mouse B1-family repetitive element and a simple repetitive sequence-suggesting a mechanism of rearrangement leading to the mutation. Southern blot analysis demonstrated that two closely linked molecular markers on chromosome 9 are unaltered, demonstrating that the deletion in this mutant cell line is subchromosomal.     

A Thy-1 − mutant defining a gene acting in trans position to regulate cell-surface Thy-1 glycoprotein expression and Thy-1 messenger RNA content

The Thy-1 glycoprotein is a differentiation antigen which exhibits tissue-specific regulation. A mutant of a Thy-1.1⁺ T-cell lymphoma has been isolated which does not express Thy-1 glycoprotein on the cell surface and does not accumulate Thy-1 mRNA in the cytoplasm. Hybrids between the mutant and a Thy-1.2⁺ T-cell lymphoma express 20–30-fold lower levels of Thy-1 glycoprotein on their cell surface compared to wild-type T-cell lymphomas, and they have correspondingly low levels of cytoplasmic Thy-1 mRNA. A revertant of one hybrid was isolated which expressed wild-type levels of both Thy-1 alleles on its surface and contained correspondingly increased levels of Thy-1 mRNA. A Thy-1⁺ revertant of the Thy-1 ^– mutant was isolated by cell sorting. A second generation Thy-1 ^– mutant could be isolated from this revertant which also did not accumulate Thy-1 mRNA and which behaved in a way similar to the first generation mutant when hybridized to a Thy-1.2⁺ lymphoma. No changes in the structure or copy number of the Thy-1 structural gene could be detected in this series of mutants and revertants. These properties are consistent with a mutation in one (or more) gene(s) which acts in trans position to regulate Thy-1 glycoprotein expression.     

N-linked glycosylation of rabies virus glycoprotein. Individual sequons differ in their glycosylation efficiencies and influence on cell surface expression.

Many eukaryotic proteins are modified by N-linked glycosylation, a process in which oligosaccharides are added to asparagine residues in the sequon Asn-X-Ser/Thr. However, not all such sequons are glycosylated. For example, rabies virus glycoprotein (RGP) contains three sequons, only two of which appear to be glycosylated in virions. To examine further the signals in proteins which regulate N-linked core glycosylation, the glycosylation efficiencies of each of the three sequons in the antigenic domain of RGP were compared. For these studies, mutants were generated in which one or more sequons were deleted by site-directed mutagenesis. Core glycosylation of these mutants was studied using two independent systems: 1) in vitro translation in rabbit reticulocyte lysate supplemented with dog pancreatic microsomes, and 2) transfection into glycosylation-deficient Chinese hamster ovary cells. Parallel results were obtained with both systems, demonstrating that the sequon at Asn37 is inefficiently glycosylated, the sequons at Asn247 and Asn319 are efficiently glycosylated, and the glycosylation efficiency of each sequon is not influenced by glycosylation at other sequons in this protein. High levels of cell surface expression of RGP in Chinese hamster ovary cells are seen with any mutant containing an intact sequon at Asn247 or Asn319, whereas low levels of cell surface expression are seen when the sequon at Asn37 is present alone; deletion of all three sequons completely blocks RGP cell surface expression. Thus, although core glycosylation at Asn37 is inefficient, it is still sufficient to support a biological function, cell surface expression. Future studies using mutagenesis of this model protein and its expression in these two well defined systems will aim to begin to unravel the rules governing core glycosylation of glycoproteins.     

Synthesis and processing of viral glycoproteins in two nonconditional mutants of Rous sarcoma virus.

We have studied the pattern of glycoprotein synthesis in two nonconditional mutants of Rous sarcoma virus. One mutant, SE33, produces no viral particles but synthesizes Pr92env, which is cleaved intracellularly to mature glycoproteins. The second mutant, SE521, encodes a gPr92env which is not cleaved to gp85 or gp37 and therefore produces virions with the phenotype of Bryan RSV(-) or NY8. Neither of these mutants have detectable genomic deletions. The study of these mutants has led to the following conclusions. (i) In the absence of particle production or p15 synthesis, gPr92env can be cleaved to the mature glycoprotein which is found on the cell surface. (ii) Noncleaved gPr92env is not packaged into virions but is found on the cell surface. (iii) gPr92env alone can account for subgroup specific viral interference. (iv) gPr92env is probably transported to the cell surface before additional glycosylation or cleavage to mature virion glycoprotein. The nonprocessed precursor of SE521 appears to be glycosylated normally, and thus far we have been unable to determine the basis for the defect in this mutant.     

Thy-1 variants of mouse lymphomas: biochemical characterization of the genetic defect

The surface proteins of Thy-1 positive mouse lymphomas and Thy-1 negative variants were labeled by lactoperoxidase-catalyzed iodination and characterized by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and by immunological studies. The loss of the serologically defined Thy-1 antigenic determinant correlated with the absence of a radioactive band corresponding to a T lymphocyte-specific surface glycoprotein, T25, on autoradiographs of the iodinated proteins of Thy-1 negative variants. Reexpression of Thy-1 on hybrid cells derived from fusions of complementary Thy-1 negative variants correlated with the reappearance of T25. Quantitative absorption studies using an antiserum which specifically recognized T25 confirmed that the Thy-1 negative variants have no detectable T25 (<0.03 of that of the Thy-1 positive lines) on their surface.Biosynthetic labeling studies revealed that none of the Thy-1 negative variants synthesized T25. However, synthesis of an immunologically cross-reactive molecule could be detected in two variants. On the basis of these results, we propose a model which describes a possible structure of the Thy-1 antigenic determinant and explains the biochemical nature of the genetic lesions leading to the loss of Thy-1 in these variants.     

Identification of a glycosylphosphatidylinositol anchor-modifying β1-3 N-acetylglucosaminyl transferase in Trypanosoma brucei

Trypanosoma brucei expresses complex glycoproteins throughout its life cycle. A review of its repertoire of glycosidic linkages suggests a minimum of 38 glycosyltransferase activities. Of these, five have been experimentally related to specific genes and a further nine can be associated with candidate genes. The remaining linkages have no obvious candidate glycosyltransferase genes; however, the T. brucei genome contains a family of 21 putative UDP sugar-dependent glycosyltransferases of unknown function. One representative, TbGT8 , was used to establish a functional characterization workflow. Bloodstream and procyclic-form TbGT8 null mutants were created and both exhibited normal growth. The major surface glycoprotein of the procyclic form, the procyclin, exhibited a marked reduction in molecular weight due to changes in the procyclin glycosylphosphatidylinositol (GPI) anchor side-chains. Structural analysis of the mutant procyclin GPI anchors indicated that TbGT8 encodes a UDP-GlcNAc: β-Gal-GPI β1-3 GlcNAc transferase. This is only the second GPI-modifying glycosyltransferase to have been identified from any organism. The glycosylation of the major glycoprotein of bloodstream-form T. brucei , the variant surface glycoprotein, was unaffected in the TbGT8 mutant. However, changes in the lectin binding of other glycoproteins suggest that TbGT8 influences the processing of the poly N-acetyllactosamine-containing asparagine-linked glycans of this life cycle stage.     

Alterations in expression and glycosylation pattern of the Thy-1 glycoprotein during maturation and transformation of mouse T lymphocytes

The mouse Thy-1 glycoprotein of normal and transformed lymphoid cells was studied with regard to amount per cell, apparent m.w., and glycosylation characteristics. Thy-1 was measured by a solid-phase radioimmunoassay calibrated with pure mouse brain Thy-1. Thymocytes were shown to contain five times the amount of Thy-1 found in lymph node cells (1 X 10(6) vs 2 X 10(5) molecules per cell), whereas the T cell lymphomas studied (P52-127-166, RBL-5, YWA, Y191, Y274, YAC-1, RL male 1, and BW5147) varied in their Thy-1 content. The apparent m.w. of Thy-1, as determined by SDS-PAGE, was in all cases 25,000 to 30,000. However, the appearance of the Thy-1 bands revealed a size heterogeneity that was less pronounced with material from lymph node cells than from thymocytes. This band broadening seemed to be inversely correlated to the affinity for lentil lectin. Whereas half the Thy-1 molecules from thymocytes were bound to the lectin, lymph nodes Thy-1 showed 75% binding. All T lymphomas but one (BW5147) contained Thy-1 also heterogeneous in lentil lectin binding. The charge, previously shown to be dependent on the sialic acid content, was shifted to more acidic forms for lymph node Thy-1 compared to thymocytes. The T lymphomas possessed Thy-1 with charge properties similar to those of the thymocytes; the only exception was BW5147, which showed more basic forms. These results show that the expression and the glycosylation of Thy-1 is altered when thymocytes mature into immunocompetent cells and after malignant transformation of lymphocytes.     

Use of carbohydrate probes in conjunction with fluorescence-activated cell sorting to select mutant cell lines of Chlamydomonas with defects in cell surface glycoproteins

Two carbohydrate-binding probes (the lectin concanavalin A and the anti-carbohydrate monoclonal antibody FMG-1) have been utilized in conjunction with fluorescence-activated cell sorting to select cell lines of Chlamydomonas reinhardtii that contain defects in cell surface-exposed glycoproteins. Two very different selection strategies (sorting cells with the lowest binding for the FMG-1 monoclonal antibody or the highest binding of concanavalin A) yield a class of mutant cells that exhibit a total lack of binding of the monoclonal antibody to cell wall and plasma membrane glycoproteins along with an increased affinity for concanavalin A. Detailed characterization of one such mutant cell line, designated L-23, is provided. The subtle glycosylation defect exhibited by this cell line does not alter the ability of the affected glycoproteins to be targeted to the flagellar membrane and does not affect the expression of flagellar surface motility, a phenomenon that appears to involve the major concanavalin A-binding glycoprotein of the flagellar membrane. This approach has general applicability for dissecting the role of carbohydrate epitopes in the targeting and function of any cell surface glycoprotein for which suitable carbohydrate probes are available.     

Somatic genetic analysis of the expression of cell surface molecules

Many mutations that affect the biosynthesis and expression of cell surface molecules are potentially lethal in vivo. Somatic cell genetics provides a means of isolating novel mutants and studying their effects. This approach has been applied to study the genes important in mediating the cell surface expression of the murine Thy-1 glycoprotein, a molecule present on subsets of cells within the hematopoietic system. Nine classes of mutants with no known in vivo counterpart have been identified and studied.     

Effects of 2-deoxyglucose, glucosamine, and mannose on cell fusion and the glycoproteins of herpes simplex virus.

2-Deoxyglucose and glucosamine were found to inhibit cell fusion caused by a syncytial mutant of herpes simplex virus and to inhibit the glycosylation of viral glycoproteins in the infected cells. The inhibition of fusion and the inhibition of glycosylation caused by 2-deoxyglucose were substantially prevented when mannose was also present during infection. When glycosylation was inhibited, three new bands were found in major glycoprotein region on sodium dodecyl sulfate-polyacrylamide gels. These bands may be precursors to the normal glycoproteins. The correlation between fusion and glycosylation in the presence of 2-deoxyglucose, glucosamine, and mannose suggests that the cells cannot fuse if their glycoproteins have a considerably reduced carbohydrate content.