Identification of a precursor protein to the major glycoproteins of mouse mammary tumor virus.

Mouse mammary tumor virus-producing cultures of mouse mammary tumor cells synthesize a viral-related polypeptide of molecular weight of 73,000 (gp 73) which is rapidly labeled during a short pulse but disappears during the chase concomitantly with the appearance of label in the virion glycoproteins gp 49 and gp 37.5/33.5. The addition of the protein synthesis-inhibitor cycloheximide to the chase medium has little effect on this conversion. Treatment of the proposed precursor with alpha-chymotrypsin leads to the formation of a polypeptide of molecular weight 49,000, similar to the major virion glycoprotein. A comparison of tryptic digest maps of the glycoproteins involved supports the hypothesis that both the viral glycoproteins gp 49 and gp 37.5/33.5 are derived from gp 73.     

Identification of a Precursor Protein to the Major Glycoproteins of Mouse Mammary Tumor Virus

Mouse mammary tumor virus-producing cultures of mouse mammary tumor cells synthesize a viral-related polypeptide of molecular weight of 73,000 (gp 73) which is rapidly labeled during a short pulse but disappears during the chase concomitantly with the appearance of label in the virion glycoproteins gp 49 and gp 37.5/33.5. The addition of the protein synthesis-inhibitor cycloheximide to the chase medium has little effect on this conversion. Treatment of the proposed precursor with α-chymotrypsin leads to the formation of a polypeptide of molecular weight 49,000, similar to the major virion glycoprotein. A comparison of tryptic digest maps of the glycoproteins involved supports the hypothesis that both the viral glycoproteins gp 49 and gp 37.5/33.5 are derived from gp 73.     

Murine mammary tumor virus structural protein interactions: formation of oligomeric complexes with cleavable cross-linking agents

Murine mammary tumor virus protein interactions in the intact virion structure were studied with the use of the cleavable cross-linking reagents dithiobis(succinimidyl propionate) and methyl 4-mercaptobutyrimidate hydrochloride. Cross-linked oligomeric complexes of murine mammary tumor virus proteins were analyzed by two-dimensional gel electrophoresis. Among the complexes most consistently formed were a heterodimer of the two glycoproteins gp36 and gp52, the homodimer of gp36, and the homotrimer of gp52. A very prominent oligomer formed at higher concentrations of dithiobis(succinimidyl propionate) was a complex of about 230,000 molecular weight, made up of three molecules each of gp36 and gp52. A number of lines of evidence, including electron microscopic analysis, suggest that the 230,000-molecular-weight complex actually represents the murine mammary tumor virus spike structure. Of the murine mammary tumor virus core proteins, p14 forms homooligomers most readily. Upon cross-linking with methyl 4-mercaptobutyrimidate hydrochloride a small amount of what seems to be a heterodimer made up of the N-terminal gag protein p10 and the hydrophobic membrane glycoprotein gp36 can be observed.     

Biochemical and Immunological Characterization of the Major Envelope Glycoprotein gp69/71 and Degradation Fragments from Rauscher Leukemia Virus

Analysis of the proteins of Rauscher murine oncornavirus by immunoprecipitation showed that antiserum to the purified envelope glycoprotein of approximately 69,000 and 71,000 daltons (gp69/71) reacted as well with a number of other components of several murine oncornaviruses of approximately 45,000, 32,000, and 15,000 daltons. Polypeptides of similar size were also produced by limited proteolysis of purified gp69/71; these degradation fragments were shown to contain carbohydrate by the incorporation of (3)H from sodium boro[(3)H]hydride after neuraminidase and galactose oxidase treatment. Each of these glycoproteins was isolated by preparative polyacrylamide gel electrophoresis and was analyzed by tryptic peptide mapping. The major virion components of 69,000 and 71,000 daltons were nearly identical, as were the primary degradation fragments. Analysis of the immunological properties of the glycoproteins showed that the 71,000-, 69,000-, and 32,000-dalton glycoproteins behaved similarly with respect to type and group-specific antigenic determinants. In contrast, the 45,000-dalton glycoprotein lacked detectable interspecies and some of the group-specific reactivity. Components of about 45,000 and 32,000 daltons isolated directly from virions were also identified as constituents of the major envelope glycoprotein by immune precipitation and tryptic peptide mapping. These results indicate that all of the examined virion glycoproteins of approximately 71,000, 69,000, 45,000, and 32,000 daltons are derived from the same viral gene and that these lower-molecular-weight glycoproteins can readily be produced from the major envelope glycoprotein.     

The purification and characterization of a major glycoprotein of the murine mammary tumor virus.

Affinity chromatography of solubilized murine mammary tumor virus on concanavalin A-Sepharose was clearly affected by different mixtures of detergent present in the elution buffer: A complex consisting of a glycoprotein of 52,000 daltons (gp52), and a glycoprotein of 36,000 daltons (gp36), besides free gp52 were isolated. The gp36 could be purified by gel filtration of the complex in the presence of a high concentration of sodium deoxycholate. The elution of gp36 in the void volume of the Sephadex column and the results obtained with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed strong hydrophobic interactions within the molecule. The glycoprotein was immunochemically characterized by competitive radioimmunoassay and immunoelectrophoresis. No cross-reactivity of gp36 with gp52 or two nonglycosylated viral polypeptides was observed.     

Radioimmunoassays for the 36,000-dalton glycoprotein of murine mammary tumor viruses demonstrate type, group, and interspecies determinants.

Mouse mammary tumor viruses (MMTVs) contain distinct membrane glycoproteins of 52,000 daltons (gp52) and 36,000 daltons (gp36). We report here the development of new radioimmunoassays for gp36, using gp36 purified by hydrophobic chromatography and gel filtration. These assays demonstrate that gp36 has both type-specific and group-specific antigenic determinants. The virus-coded nature of these determinants was shown by utilizing different MMTVs grown in the same feline cell line. Interspecies determinants on gp36 were demonstrated by the observations that (i) MC-MTV (a virus isolate from the Asian rodent Mus cervicolor, and morphologically identical to MMTVs) competed, with an altered slope, in the gp36 radioimmunoassay, and (ii) antisera raised against MC-MTV immunopreciptitated 125I-labeled gp36. The detection of gp36 in spontaneous mammary tumors of several strains of mice also facilitates further studies on the replication of MMTVs and the host's immune response to MMTV-mediated oncogenesis.     

Origin of the minor glycoproteins of murine leukemia viruses.

Polyacrylamide gel electrophoretic analysis and immunoprecipitation were used to study glycoproteins from purified Rauscher murine leukemia virus (R-MuLV) and from AKR thymic lymphoblastoid cell membranes. In addition to gp70, a minor glycoprotein of approximately 52,000 daltons (gp52) was demonstrated in purified R-MuLV preparations, which was antigenically related to gp70. Analysis of R-MuLV glycopeptides obtained after exhaustive Pronase digestion showed that gp70 has at least two different glycopeptide size classes with molecular weights of 5,100 and 2,900, respectively. gp52, however, contained only a single glycopeptide size class of approximately 5,100 daltons, indicating that the two glycoproteins contain distinct carbohydrate components. Trypsin treatment of R-MuLV converted gp70 into a product with a molecular mass of approximately 52,000 daltons as well as a 45,000-dalton minor product, with little effect on virus infectivity. Similarly, trypsin treatment of 125I-labeled glycoproteins derived from AKR mouse lymphoblastoid cell membranes generated fragments antigenically related to gp70 and similar in size to those obtained by trypsin treatment of R-MuLV. In both cases, the appearance of cleavage products was accompanied by a decrease in gp70 during trypsin treatment. The occurrence of glycosylated components antigenically related to gp70 in AKR membrane glycoprotein preparations and in purified R-MuLV preparations which were similar to those generated by trypsin treatment supports the concept that these minor components arise from proteolytic cleavage of gp70.     

Antibody-independent detection of virus-specific glycoprotein synthesis is oncornavirus-infected cells.

A method is described for detecting the synthesis of avian and murine oncornavirus-specific glycoproteins without the use of antibodies raised against viral structural proteins. As applied to cells infected with avian tumor virus, the method served to resolve pr90, the precursor of the major envelope glycoprotein. A virus-specific glycoprotein of about 85,000 daltons, which has several properties expected to a precursor to gp69/71, was detected in cells infected with murine leukemia virus.     

Immunoenzyme Western blotting analysis of antibody specificity in Aleutian disease of mink, a parvovirus infection.

Aleutian disease virus (ADV), an autonomous parvovirus, persistently infects mink and induces very high levels of virus-specific antibody. All strains of ADV infect all mink, but only highly virulent strains cause progressive disease in non-Aleutian mink. The development of antibody to individual ADV proteins was evaluated by Western blotting by using the sera of 22 uninfected mink and 163 naturally or experimentally infected mink. ADV has virion proteins of 86,000 and 78,000 daltons that are closely related. A new, possibly nonvirion protein of 143,000 daltons was observed, as well as a known nonvirion protein of 71,000 daltons. Sera from mink experimentally or naturally infected with ADV of high or low virulence generally reacted about equally with all four proteins. The only exceptions noted were that 8 of 15 sera of mink infected transplacentally preferentially reacted with the two virion proteins and sera from mink with the monoclonal gammopathy of Aleutian disease reacted preferentially with either virion (10 of 12) or nonvirion (2 of 12) proteins.     

Mason-Pfizer monkey virus: analysis and localization of virion proteins and glycoproteins.

The polypeptide composition of Mason-Pfizer monkey virus was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six major polypeptides of molecular weights 68,000, 27,000, 20,000, 14,000, 12,000, and 10,000 were resolved regardless of the cell type (i.e., two human and two rhesus) in which the virus was grown. Protein gp68 (68,000) represented the major virus glycoprotein and protein gp20 (20,000) represented a minor glycoprotein of the virion, again regardless of the cell type of origin of the virus. Protein gp68 appears to be located on the outer surface of the viral envelope, as demonstrated by lactoperoxidase catalyzed iodination of intact virions. Additional glycoproteins were shown to be virion associated; their presence depended, however, on the cell type in which the virus was propagated.     

Changes in the transmembrane region of the human immunodeficiency virus type 1 gp41 envelope glycoprotein affect membrane fusion.

The charged amino acids near or within the membrane-spanning region of the human immunodeficiency virus type 1 gp41 envelope glycoprotein were altered. Two mutants were defective for syncytium formation and virus replication even though levels of envelope glycoproteins on the cell or virion surface and CD4 binding were comparable to those of the wild-type proteins. Thus, in addition to anchoring the envelope glycoproteins, sequences proximal to the membrane-spanning gp41 region are important for the membrane fusion process.     

Transmembrane envelope glycoproteins of human immunodeficiency virus type 2 and simian immunodeficiency virus SIV-mac exist as homodimers.

An 80-kilodalton glycoprotein (gp80) was produced in human immunodeficiency virus type 2 (HIV-2)-infected cells along with three envelope glycoproteins that we have recently reported: the extracellular glycoprotein (gp125), the envelope glycoprotein precursor (gp140), and the transient dimeric form of the precursor (gp300). gp125 and gp80 were detectable after the synthesis of gp140 and the formation of gp300. Using a specific monoclonal antibody, we showed here that gp80 is a dimeric form of the transmembrane glycoprotein gp36 of HIV-2. Dimerization of the envelope glycoprotein precursor and dimeric forms of the transmembrane glycoproteins were also observed in cells infected with simian immunodeficiency virus (SIV-mac), a virus closely related to HIV-2. Under routine conditions of our experiments (i.e., extraction by 1% Triton X-100 before polyacrylamide gel electrophoresis in sodium dodecyl sulfate [SDS]), monomeric forms of the transmembrane glycoprotein of HIV-2 and SIV-mac were only seldomly observed. Dimeric forms of the envelope precursors and the transmembrane glycoproteins are probably stabilized by extraction in the nonionic detergent Triton X-100 since such dimeric forms resist dissociation during subsequent electrophoresis in the presence of the ionic detergent SDS. However, the dissociation of these dimeric forms might occur when samples are prepared by extraction directly in 1% SDS or by incubation of the purified dimers at acidic pH. Dimerization of the envelope precursor might be required for its processing to give the mature envelope proteins, whereas the transmembrane dimer might be essential for optimal structure of the virion and thus its infectivity.     

Modifications that stabilize human immunodeficiency virus envelope glycoprotein trimers in solution

The functional unit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins is a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modifications that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: disruption of the proteolytic cleavage site between gp120 and gp41, introduction of cysteines that form intersubunit disulfide bonds, and addition of GCN4 trimeric helices. Characterization of these secreted glycoproteins by immunologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing the trimers, the formation of intersubunit disulfide bonds between appropriately placed cysteines, and the ability of the trimers to interact with a helical, C-terminal gp41 peptide (DP178) support a model in which the N-terminal gp41 coiled coil exists in the envelope glycoprotein precursor and contributes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progress in understanding the structure and function of the virion envelope glycoprotein spikes.     

Two small virus-specific polypeptides are produced during infection with Sindbis virus.

We have identified and characterized two small virus-specific polypeptides which are produced during infection of cells with Sindbis virus, but which are not incorporated into the mature virion. The larger of these is a glycoprotein with an approximate molecular weight of 9,800 and is found predominantly in the medium of infected cells. Three independent lines of evidence demonstrate conclusively that this 9,800-dalton glycoprotein is produced during the proteolytic conversion of the precursor polypeptide, PE2, to the virion glycoprotein E2. This small glycoprotein is therefore analogous to the virion glycoprotein E3 of the very closely related alphavirus, Semliki Forest virus. The 9,800-dalton glycoprotein of Sindbis virus, unlike the E3 glycoprotein of Semliki Forest virus, is not, however, present in the viral particle. The other virus-specific polypeptide is 4,200 daltons in size, does not appear to be a glycoprotein, and is neither incorporated into the mature virus nor released into the culture medium. The gene for this small polypeptide is present in the viral 26S mRNA (the mRNA which encodes all the viral structural polypeptides) and appears to be located in the portion of the mRNA which encodes the two viral glycoproteins. The possibility that this 4,200-dalton polypeptide functions as a signal peptide during the synthesis of the viral membrane glycoproteins is discussed.     

Analysis of the envelope of Rauscher murine oncornavirus: in vitro labeling of glycopeptides.

The identity and localization of the oligosaccharides of Rauscher murine type C viral glycoproteins have been examined by techniques of in vitro labeling. Terminal sialic acid was labeled with tritium by borohydride reduction after selective periodate oxidation, and galactose was labeled by borohydride reduction after specific enzymatic oxidation of the nonreducing terminal of the sugar. The results were compared with those of protein surface labeling with pyridoxal phosphate or lactoperoxidase catalyzed radioiodination. Examination of the labeled reaction products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that in every case the major component labeled was a glycoprotein of about 70,000 daltons. The identity of this glycoprotein as the virion envelope component was confirmed by immunoprecipitation with mono-specific antiserum prepared against purified Rauscher virus glycopeptides of 69,000 and 71,000 daltons. No other protein or glycoprotein on the surface of the virion was detected, and disruption of virions-before labeling did not reveal additional distinctive glycoproteins. There was minor labeling of sugar residues of other components, but these remain to be characterized and are not now identified as other viral proteins. Studies of the structural organization of virion proteins using the cross-linking reagent methyl-4-mercaptobutyrimidate showed only linkage of the virion envelope or core proteins to themselves. These results indicate that most, if not all, of the oligosaccharides at the surface of Rauscher virus are entities of the 69,000- and 71,000-dalton glycopeptides and that they contain a terminal sialic acid and galactose and a subterminal galactose.     

Type-specific antigenic determinants on the major external glycoprotein of high- and low-oncogenic murine mammary tumor viruses.

We recently showed that the 52,000-dalton external glycoprotein (gp52) of the highly oncogenic mouse mammary tumor viruses (MMTVs) of RIII, GR, and C3H mice contains both type- and group-specific antigenic determinants. This was demonstrated by using a competition radioimmunoassay with 125I-externally labeled virions and antisera to the gp52 of MMTV from RIII mice (Proc. Natl. Acad. Sci. U.S.A. 74:3564-3568, 1977). We report here that we were able to distinguish between the gp52's of the high-oncogenic MMTV of C3H mice [MMTV(C3H)] and the low-oncogenic MMTV of that same mouse strain [MMTV(C3Hf)]. This was accomplished by use of a competition radioimmunoassay with 125I-externally labeled virions of MMTV(C3H) and antisera prepared against MMTV(C3H). A comparison of the intact virion and purified gp52 radioimmunoassays showed that MMTV type-specific differences were enhanced with the intact virion radioimmunoassay. These differences were further magnified with appropriately absorbed antisera. These findings thus allow an immunological distinction between the surface glycoproteins of a low-oncogenic endogenous and a high-oncogenic exogenous MMTV of the same mouse strain.     

Polypeptides of mumps virus.

Mumps virus was propagated in the extra-embryonic fluids of embryonated chicken eggs and was labeled by cionjection of radioactively labeled amino acids. The virus was purified by density gradient centrifugation, and its polypeptides were analyzed by polyarylamide gel electrophoresis. The virus was found to be composed of six polypeptides, ranging in size from 40,000 to 64,000 daltons. Viral proteins 1 and 3 were the glycoproteins of the virons. When the virus particle was treated with noniontic detergents, a small fraction of these glycoproteins could be released into the supernatant. After treatment with nonionic detergents in high salt and alkaline conditions, more of the surface glycoproteins were removed. This treatment also released the smallest viral polypeptide from the virion. The glycoproteins were separated using an affinity chromatographic column of agarose-fetuin. The heavier glycoprotein, viral protein 1, was found to contain both the neuraminidase and hemagglutinating activity. The two glycoproteins were tested for their ability to react in complement-fixing tests with mumps antisera. Only the heavier glycoprotein reacted with antisera possessing both anti-S and anti-V activity. Neither glycoprotein reacted with antisera specific for the S antigen. Thus, it was concluded that this glycoprotein corresponds to the classical V antigen of mumps virus.     

Protein-coding potential of mouse mammary tumor virus genome RNA as examined by in vitro translation.

The protein-coding capacity of the mouse mammary tumor virus genome has been examined by in vitro translation of genome length and polyadenylated subgenomic fragments of viral RNA. Intact genome RNA of about 35S programmed synthesis of the Pr77gag, Pr110gag and Pr160gag/pol precursors seen in infected cells in vivo. Polyadenylated RNA fragments of 18 to 28S encoded products whose tryptic peptide maps resembled those of the nonglycosylated precursor to the envelope glycoproteins, confirming the gene order 5'-gag-pol-env-3'. Translation of polyadenylated RNA fragments smaller than 18S yielded a series of related proteins whose peptide maps bore no resemblance to any of the virion structural proteins. Thus, a region of the mouse mammary tumor virus genome distal to the env gene appears to have an open reading frame sufficient to encode at least 36,000 daltons of protein as of yet unknown function.     

Alterations of a mouse mammary tumor virus glycoprotein with interferon treatment.

The effects of exogenous mouse interferon on the MJY-alpha mammary tumor cell line chronically infected with mouse mammary tumor virus (MMTV) were examined. Interferon at concentrations of 25 to 2,000 IU/ml in culture medium did not alter the growth rate or morphology of the cell layers. Electron microscopic examination of interferon-treated cells indicated a decrease in the numbers of A-type and budding B-type particles of MMTV. However, the levels of extracellular MMTV virions in the culture supernatants were not significantly reduced. Profiles of MMTV glycoproteins and nonglycosylated polypeptides obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virions purified from interferon-treated cultures revealed increases in the relative levels of the 60,000-dalton glycoprotein, gp60.     

N-butyldeoxynojirimycin-mediated inhibition of human immunodeficiency virus entry correlates with impaired gp120 shedding and gp41 exposure.

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although an NB-DNJ-mediated change in viral envelope N-glycan composition inhibits HIV entry at the level of post-CD4 binding, the exact mechanism of inhibition remains to be established. In this study we have examined the effects of NB-DNJ on virion envelope composition and CD4-induced gp120 shedding and gp41 exposure. Virion composition analysis revealed an NB-DNJ-mediated reduction of 15% in overall virion envelope glycoprotein content and a reduction of 26% in the proteolytic maturation of virion gp160. Taken together, these two effects resulted in a reduction of approximately 40% in virion gp120 content. CD4-induced shedding of gp120 from the surfaces of envelope-transfected Cos cells was undetectable when gp120 was expressed in the presence of NB-DNJ. Similarly, the shedding of virion-associated gp120 was reduced 7.4-fold. CD4-induced exposure of cryptic gp41 epitopes on the surfaces of HIV-expressing ACH-2 cells was also greatly impaired, and the exposure of virion-associated gp41 epitopes was reduced 4.0-fold. Finally, CD4-induced increases in the binding of antibodies to the V3 loop of ACH-2-cell-expressed envelope glycoproteins were reduced 25-fold when the glycoproteins were expressed in the presence of NB-DNJ. These results suggest that the NB-DNJ-mediated retention of glycosylated N-glycans inhibits HIV entry by a combined effect of a reduction in virion gp120 content and a qualitative defect within the remaining gp120, preventing it from undergoing conformational changes after CD4 binding.