Polyprotein Precursors to Mouse Mammary Tumor Virus Proteins

Mouse mammary tumor virus (MMTV) derived from the culture medium of GR cells contained seven proteins, identified as gp55, gp33, p25, pp20, p16, p12, and p10. The major viral phosphoprotein was the 20,000-molecular-weight protein, pp20. Immunoprecipitation of cytoplasmic extracts from pulse-labeled GR cells identified three MMTV gag-specific proteins, termed Pr78(gag), Pr110(gag), and Pr180(gag+). These intracellular polyproteins were precipitable from cytoplasmic extracts by antisera to virions p25 and p12 but not by antisera to gp55. The major intracellular gag-specific precursor polyprotein, Pr78(gag), contained antigenic determinants and tryptic peptides characteristic of p25, p12, p10, and presumably pp20. This precursor is presumably derived from nascent chain cleavage or rapid posttranslational cleavage of the larger intracellular precursor-like protein, designated Pr110(gag). Pr110(gag) contained all but one of the leucine-containing tryptic peptides of Pr78(gag), plus several additional peptides. In addition to Pr78(gag) and Pr110(gag), monospecific antisera to virion p12 and p25 were also capable of precipitating from pulse-labeled cells a small amount of a 180,000-molecular-weight precursor-like protein, designated Pr180(gag+). This large polyprotein contained nearly all of the leucine-containing tryptic peptides of Pr78(gag) and Pr110(gag) plus several additional peptides. By analogy to type C viral systems, Pr180(gag+) is presumed to represent a gag-pol common precursor which is the major pathway for synthesis of MMTV polymerase. Immunoprecipitation of cytoplasmic extracts from pulse-labeled cells with antisera to gp55 identified two env-specific proteins, designated gPr76(env) and gP79(env). The major env precursor, gPr76(env), could be labeled with radioactive glucosamine and was shown to contain antigenic determinants and tryptic peptides characteristic of gp55 and gp33. A minor glycoprotein, gP79(env), contained both fucose and glucosamine and was precipitable from cytoplasmic extracts with monospecific serum to gp55. It is suggested that gP79(env) represents fucosylated gPr76(env) which is transiently synthesized and cleaved rapidly into gp55 and gp33.     

Transformation of NIH 3T3 cells by DNA of the MCF-7 human mammary carcinoma cell line induces expression of an endogenous murine leukemia provirus.

Previous studies identified two glycoproteins of 86 (gp86) and 72 (gp72) kilodaltons and two nonglycosylated proteins of 70 (p70) and 19 (p19) kilodaltons which were specifically expressed in NIH cells transformed by DNA of the MCF-7 human mammary carcinoma cell line. Pulse-chase experiments and the use of tunicamycin to inhibit glycosylation suggested that gp86, gp72, and p19 were related as precursor products. Characteristics of the four transformation-associated proteins resembled those of murine leukemia virus (MuLV) proteins. Sera raised against disrupted MuLV immunoprecipitated the same four proteins in extracts of NIH(MCF-7) cells and MuLV-infected NIH 3T3 cells. In addition, a monoclonal antibody against MuLV gp70 immunoprecipitated proteins gp86 and gp72, whereas a monoclonal antibody against MuLV p15(E) immunoprecipitated gp86 and p19. These results indicate that proteins gp86, gp72, and p19 expressed in NIH(MCF-7) transformants correspond to MuLV envelope proteins gp80env, gp70, and p15(E), respectively. The transformation-associated protein p70 appears to be a non-envelope MuLV protein, most likely p65gag. Northern blot analysis confirmed that transformation of NIH cells by MCF-7 mammary carcinoma DNA led to the induction of an endogenous MuLV provirus.     

Functional role of the glycan cluster of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) ectodomain.

To examine the role of the glycans of human immunodeficiency virus type 1 transmembrane glycoprotein gp41, conserved glycosylation sites within the env sequence (Asn-621, Asn-630, and Asn-642) were mutated to Gln. The mutated and control wild-type env genes were introduced into recombinant vaccinia virus and used to infect BHK-21 or CD4+ CEM cells. Mutated gp41 appeared as a 35-kDa band in a Western blot (immunoblot), and it comigrated with the deglycosylated form of wild-type gp41. Proteolytic cleavage of the recombinant wild-type and mutant forms of the gp160 envelope glycoprotein precursor was analyzed by pulse-chase experiments and enzyme-linked immunosorbent assay: gp160 synthesis was similar whether cells were infected with control or mutated env-expressing recombinant vaccinia virus, but about 10-fold less cleaved gp120 and gp41 was produced by the mutated construct than the control construct. The rates of gp120-gp41 cleavage at each of the two potential sites appeared to be comparable in the two constructs. By using a panel of antibodies specific for gp41 and gp120 epitopes, it was shown that the overall immunoreactivities of control and mutated gp41 proteins were similar but that reactivity to epitopes at the C and N termini of gp120, as present on gp160 produced by the mutated construct, was enhanced. This was no longer observed for cleaved gp120 in supernatants. Both gp120 proteins, from control and mutated env, were expressed on the cell surface under a cleaved form and could bind to membrane CD4, as determined by quantitative immunofluorescence assay. In contrast, and despite sufficient expression of env products at the cell membrane, gp41 produced by the mutated construct was unable to induce membrane fusion. Therefore, while contradictory results reported in the literature suggest that gp41 individual glycosylation sites are dispensable for the bioactivity and conformation of env products, it appears that such is not the case when the whole gp41 glycan cluster is removed.     

A unique glycoprotein containing GR-mouse mammary tumor virus peptides and additional peptides unrelated to viral structural proteins

A glycoprotein of molecular weight 130,000 (gP130) has been precipitated from the cytoplasm of GR-strain mouse mammary tumor (GR-MMT) cells by a rabbit antiserum (anti-MMTV) to GR-strain mouse mammary tumor virus (GR-MMTV). This protein was not precipitated by antisera specific for detergent-disrupted C3H-strain MMTV (C3H-MMTV); C3H-MMTV glycoproteins; C3H-MMTV nonglycosylated proteins; GR-MMTV p25 or p12; RIII strain (milk) MMTV proteins; or Rauscher murine leukemia virus (R-MuLV) proteins; nor was it precipitated by normal rabbit serum. Two-dimensional thin layer analysis of 35S-methionine-containing tryptic peptides revealed that five of nine gp33 peptides and one of seven gp55 peptides are shared by gP130 and gPr76env. The envelope protein precursor, gPr76env, contains all of the gp33 peptides and six of seven gp55 peptides. One peptide in gPr76env, possibly a gp55-gp33 junction peptide, is also apparently present in gP130. Six of ten p25 peptides and four more gag-related peptides are shared by PR78gag and gP130. Protein gP130 also contains several tryptic peptides not found in gPr76env or in the core protein precursors Pr78gag, Pr110gag or Pr180gag-pol. Radioimmunoprecipitation experiments showed that gP130 could be precipitated from extracts of GR-MMTV cells with anti-MMTV serum even after antibodies to the known MMTV structural proteins had been removed from the serum by absorption. Both gP130 and a second protein, p30, were found in immunoprecipitates of detergent-disrupted isotopically labeled GR-MMTV treated with the absorbed anti-MMTV serum. These results suggest that antibodies to gP130 in the anti-MMTV serum are capable of recognizing those protein sequences unique to gP130; that is, those protein sequences which are not related to viral structural proteins. In light of these data and data published previously, gP130 is apparently a polyprotein containing juxtaposed components translated from the 5' and 3' end of the MMTV genome and protein components not previously identified as virus-specific.     

Inhibition of Rous sarcoma virus replication by 2-deoxyglucose and tunicamycin: identification of an unglycosylated env gene product.

Two inhibitors of glycosylation, 2-deoxyglucose and tunicamycin, depressed the synthesis of infectious Rous sarcoma virus greater than 100-fold. Under the same conditions only a two- to threefold decrease in the production of virus particles was observed. The noninfectious particles had a lower density (1.145 g/ml) in isopycnic sucrose gradients and lacked the two virion glycoproteins, gp85 and gp37, found on infectious virions. The four internal structural proteins of the virus, p27, p19, p15, and p12, appeared to be assembled normally into the noninfectious virus. Polypeptides related to the Rous sarcoma virus glycoproteins were immunoprecipitated from pulse-labeled Rous sarcoma virus (Prague strain, subgroup B)-transformed cells. pr95gp, the polyprotein precursor to gp85 and gp37, was the major protein precipitated from untreated cells. PR95GP, THE POLYPROTEIN PRECURSOR TO GP85 AND GP37, WAS THE MAJOR PROTEIN PRECIPITATED FROM UNTREATED CELLS. This was absent in both tunicamycin- and 2-deoxyglucose-treatec ells, and a new polypeptide of molecular weight 57,000 to 58,000 was the major species precipitated. In tunicamycin-treated cells this product was unstable and was degraded during a 2-h chase; in 2-deoxyglucose-treated cells, on the other hand, the polypeptide appeared to be more stable and underwent partial glycosylation. The synthesis and processing of pr76, the polyprotein precursor to the internal structural proteins of the virion, occurred normally in both treated and untreated cells. It is concluded that the unglycosylated env gene product is a polypeptide of molecular weight 57,000 to 58,000.     

Evaluation of envelope vaccines derived from the South African subtype C human immunodeficiency virus type 1 TV1 strain

Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1DeltaV2), followed by boosting with oligomeric protein (o-gp140TV1DeltaV2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.     

Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents

Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.     

Characterization of monoclonal antibodies to human immunodefiency virus type 1 gp41 by HIV-1 polypeptides expressed in Escherichia coli

Abstract Two monoclonal antibodies (MAbs) were produced in Balb/c mice by immunization with recombinant gp41 derived from expression of λ-BH10 cDNA of the human immunowdeficiency virus-1 (HIV-1) in the prokaryotic expression vector pEX-41 [1, 2]. Characterization of the epitopes recognized by these MAbs was done with HIV-1 envelope (env) fusion proteins expressed in Escherochia coli encoding ten distinct segments of the env proteins [3]. In comparison, another mouse MAb, M25 [4], a human MAb directed against gp41, which was produced by the xeno hydridoma line 3D6 [5, 6] and a pool of human patient sera containing antibodies to HIV-1 were tested. We were able to demonstrate that the epitopes recognized by our MAbs are located betweeni arg732 and ser759 [7] of the HIV-1 env glycoprotein gp160 of HTLV-III strain B. M25 reacted with epitopes between ser647 and pro731, which includes the hydrophobic transmembrane region of gp41 [4]. The human MAb against gp41, 3D6 [5, 6] reacts with epitopes between ile474 and trp646, a polypeptide stretch consisting of gp120 and gp41 specific amino acids. The human serum pool, positive for HIV-1 antibodies, reacted predominantly with antigenic determinants locatedp between ile474 and leu863. The recombinant env fusion proteins were initially produced to test the immunoreactivity with patient sera and to characterize epitopes which are relevant for immunodiagnostic purposes [3]. In this study, we showed that the set of recombinant evr proteins is also a simple and accurate tool for the characterization of MAbs directed to the HIV envelope proteins.     

Human foamy virus polypeptides: identification of env and bel gene products.

Human foamy virus (HFV) proteins were identified in human cells cultured in vitro by immunoprecipitation and immunoblotting with specific antisera. Among several viral polypeptides, four glycoproteins of approximately 160, 130, 70, and 48 kDa were identified in HFV-infected cells. gp130 was shown to represent the intracellular env precursor, and gp70 and gp48 were shown to represent the external and transmembrane env proteins, respectively. The nature of gp160, which shares sequences with the env, bel1, and bel2 proteins, is not yet resolved. In addition, a p62 identified with bel1- and bel2-specific antisera likely corresponds to the bet gene product.     

The errors in assembly of MuLV in interferon treated cells

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of noninfectious particles (interferon virions) containing the structural proteins of env and gag genes as well as additional viral polypeptides. In the control virions the major glycoprotein detected is gp71, interferon virions contain in addition to gp71 and 85k dalton (gp85) glucosamine-containing, fucose-deficient glycoprotein which is recognized by antiserum to MuLV but not by the gp71 antiserum. The surface iodination of the intact virions indicates that both gp71 and gp85 are the major components of the external virions envelope. However, unlike the control virions in which gp71 associates with p15E (gp90), the gp71-p15E complex was not detected in interferon virions. The analysis of the iodinated proteins of the disrupted interferon virions revealed the presence of 85k and 65k dalton polypeptides preciptable with antiserum against MuLV, which are not present in the control virions. The difference in the polypeptide pattern of virions produced in the presence of interferon does not seem to be a consequence of the slowdown in the synthesis of viral proteins or their processing in the interferon-treated cells. Both the structural proteins of env and gag genes seem to be synthesized and processed at a comparable rate in the interferon-treated and -untreated cells. These results indicate an alteration of virus assembly in the presence of interferon.     

Immunohistochemical localization of retroviral-related antigens expressed in normal baboon placental villous tissue

Endogenous retroviral particles (ERVs) have been detected in the genome of all eukaryotes. They are generally non-pathogenic except in mice where they have been found to induce tumors and immunological disorders. The ERVs have morphological features consistent with type-C retroviral particles and are commonly expressed in normal placental villous tissues. ERVs may have a role in the regulation of placental gene expression, syncytiotrophoblast formation, or pregnancy-related immunosuppression. In this study, well-characterized antibodies (monoclonal and polyclonal antibodies) raised against retroviral proteins (anti-HIV and anti-SIV) and endogenous retroviral (ERV) particles were assessed for their cross-reactivity (by using immunohistochemistry) with normal baboon placental and other adult tissues. The monoclonal antibodies to exogenous retroviral proteins (anti-HIV-2 gp120, anti-HIV-1 gp41, anti-SIVmac p27, anti-HIV-1 RT, and anti-HIV-2 core protein) showed specific immunohistochemical reactivity with the syncytiotrophoblast. Antibodies to endogenous retroviral gene products (anti-ERV3 env, anti-HERV-K RT, and anti-HERV-K env) also reacted in a similar manner and did not cross-react with other adult tissues. These studies have shown that retroviral-cross-reactive proteins are expressed in baboon placental syncytiotrophoblast and may have a role to play at the feto-maternal interface.     

Processing and amino acid sequence analysis of the mouse mammary tumor virus env gene product.

The envelope proteins of mouse mammary tumor virus (MMTV) are synthesized from a subgenomic 24S mRNA as a 75,000-dalton glycosylated precursor polyprotein which is eventually processed to the mature glycoproteins gp52 and gp36. In vivo synthesis of this env precursor in the presence of the core glycosylation inhibitor tunicamycin yielded a precursor of approximately 61,000 daltons (P61env). However, a 67,000-dalton protein (P67env) was obtained from cell-free translation with the MMTV 24S mRNA as the template. To determine whether the portion of the protein cleaved from P67env to give P61env was removed from the NH2-terminal end of P67env and as such would represent a leader sequence, the NH2-terminal amino acid sequence of the terminal peptide gp52 was determined. Glutamic acid, and not methionine, was found to be the amino-terminal residue of gp52, indicating that the cleaved portion was derived from the NH2-terminal end of P67env. The NH2-terminal amino acid sequences of gp52's from endogenous and exogenous C3H MMTVs were determined though 46 residues and found to be identical. However, amino acid composition and type-specific gp52 radioimmunoassays from MMTVs grown in heterologous cells indicated primary structure differences between gp52's of the two viruses. The nucleic acid sequence of cloned MMTV DNA fragments (J. Majors and H. E. Varmus, personal communication) in conjunction with the NH2-terminal sequence of gp52 allowed localization of the env gene in the MMTV genome. Nucleotides coding for the NH2 terminus of gp52 begin approximately 0.8 kilobase to the 3' side of the single EcoRI cleavage site. Localization of the env gene at that point agrees with the proposed gene order -gag-pol-env- and also allows sufficient coding potential for the glycoprotein precursor without extending into the long terminal repeat.     

Antigenic specificity of antibody-dependent cell-mediated cytotoxicity directed against human immunodeficiency virus in antibody-positive sera.

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for human immunodeficiency virus (HIV) has been described for HIV-infected individuals. To determine the antigenic specificity of this immune response and to define its relationship to the disease state, an ADCC assay was developed using Epstein-Barr virus-transformed lymphoblastoid cell line targets infected with vaccinia virus vectors expressing HIV proteins. The vaccinia virus vectors induced appropriate HIV proteins (envelope glycoproteins gp160, gp120, and gp41 or gag proteins p55, p40, p24, and p17) in infected lymphoblastoid cell lines as demonstrated by radioimmunoprecipitation and syncytia formation with c8166 cells. Killer cell-mediated, HIV-specific ADCC was found in sera from HIV-seropositive but not HIV-seronegative hemophiliacs. This HIV-specific response was directed against envelope glycoprotein but was completely absent against target cells expressing the HIV gag proteins. The ADCC directed against gp160 was present at serum dilutions up to 1/316,000. There was no correlation between serum ADCC titer and the stage of HIV-related illness as determined by T-helper-cell numbers. These experiments clearly implicated gp160 as the target antigen of HIV-specific ADCC activity following natural infection. Vaccines which stimulate antibodies directed against gp160, which are capable of mediating ADCC against infected cells, could be important for protection against infection by cell-associated virus.     

Characterization of neutralization epitopes in the V2 region of human immunodeficiency virus type 1 gp120: role of glycosylation in the correct folding of the V1/V2 domain.

A number of monoclonal antibodies (MAbs) with various levels of neutralizing activity that recognize epitopes in the V1/V2 domain of LAI-related gp120s have been described. These include rodent antibodies directed against linear and conformational epitopes and a chimpanzee MAb, C108G, with extremely potent neutralizing activity directed against a glycan-dependent epitope. A fusion glycoprotein expression system that expressed the isolated V1/V2 domain of gp120 in native form was used to analyze the structural characteristics of these epitopes. A number of MAbs (C108G, G3-4, 684-238, SC258, 11/68b, 38/66a, 38/66c, 38/62c, and CRA3) that did not bind with high affinity to peptides immunoprecipitated a fusion glycoprotein expressing the V1/V2 domain of HXB2 gp120 in the absence of other human immunodeficiency virus sequences, establishing that their epitopes were fully specified within this region. Biochemical analyses indicated that in the majority of V1/V2 fusion molecules only five of the six glycosylation signals in the V1/V2 domain were utilized, and the glycoforms were found to be differentially recognized by particular MAbs. Both C108G and MAbs directed against conformational epitopes reacted with large fractions of the fully glycosylated molecules but with only small fractions of the incompletely glycosylated molecules. Mutational analysis of the V1 and V2 glycosylation signals indicated that in most cases the unutilized site was located either at position 156 or at position 160, suggesting the occurrence of competition for glycan addition at these neighboring positions. Mutation of glycosylation site 160 destroyed the C108G epitope but increased the fraction of the molecules that presented the conformational epitopes, while mutation of the highly conserved glycosylation site at position 156 greatly diminished the expression of the conformational epitopes and increased expression of the C108G epitope. Similar heterogeneity in glycosylation was also observed when the HXB2 V1/V2 fusion glycoprotein was expressed without most of the gp70 carrier protein, and thus, this appeared to be an intrinsic property of the V1/V2 domain. Heterogeneity in expression of conformational and glycan-dependent epitopes was also observed for the natural viral env precursor, gPr160, but not for gp120. These results suggested that the closely spaced glycosylation sites 156 and 160 are often alternatively utilized and that the pattern of glycosylation at these positions affects the formation of the conformational structures needed for both expression of native epitopes in this region and processing of gPr160 to mature env products.     

Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products.

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus.     

Mutational analysis of N-linked glycosylation sites of Friend murine leukemia virus envelope protein.

The roles played by the N-linked glycans of the Friend murine leukemia virus envelope proteins were investigated by site-specific mutagenesis. The surface protein gp70 has eight potential attachment sites for N-linked glycan; each signal asparagine was converted to aspartate, and mutant viruses were tested for the ability to grow in NIH 3T3 fibroblasts. Seven of the mutations did not affect virus infectivity, whereas mutation of the fourth glycosylation signal from the amino terminus (gs4) resulted in a noninfectious phenotype. Characterization of mutant gene products by radioimmunoprecipitation confirmed that glycosylation occurs at all eight consensus signals in gp70 and that gs2 carries an endoglycosidase H-sensitive glycan. Elimination of gs2 did not cause retention of an endoglycosidase H-sensitive glycan at a different site, demonstrating that this structure does not play an essential role in envelope protein function. The gs3- mutation affected a second posttranslational modification of unknown type, which was manifested as production of gp70 that remained smaller than wild-type gp70 after removal of all N-linked glycans by peptide N-glycosidase F. The gs4- mutation decreased processing of gPr80 to gPr90, completely inhibited proteolytic processing of gPr90 to gp70 and Pr15(E), and prevented incorporation of envelope products into virus particles. Brefeldin A-induced mixing of the endoplasmic reticulum and parts of the Golgi apparatus allowed proteolytic processing of wild-type gPr90 to occur in the absence of protein transport, but it did not overcome the cleavage defect of the gs4- precursor, indicating that gs4- gPr90 is resistant to the processing protease. The work reported here demonstrates that the gs4 region is important for env precursor processing and suggests that gs4 may be a critical target in the disruption of murine leukemia virus env product processing by inhibitors of N-linked glycosylation.     

MCF-specific murine monoclonal antibodies made against AKR-247 MCF virus recognize a unique determinant associated with the gp70-p-15(E) complex.

Hybridomas obtained from (NFS X AKR)F mice immunized with syngeneic cells infected with AKR-247 MCF virus produced antibodies specific for only AKR-247 or closely related MCF viruses which hare a previously defined MCF antigen (MCFA-3). These monoclonal antibodies recognized a new type of viral antigenic determinant which appeared to be a conformational determinant associated with the env precursor polyprotein (pr80env) or its disulfide-linked gp70-p15(E) complex (gp80) but not with free gp70 or p15(E) or any other virion or virus-induced protein.     

Erythropoietin receptor (EpoR)-dependent mitogenicity of spleen focus-forming virus correlates with viral pathogenicity and processing of env protein but not with formation of gp52-EpoR complexes in the endoplasmic reticulum.

Recent evidence suggests that interactions between spleen focus-forming virus (SFFV) env products and the erythropoietin receptor (EpoR) are responsible for viral pathogenicity. Infection of factor-dependent cell lines expressing epoR (the cloned gene for EpoR) with SFFVP is mitogenic, generating cell lines that are no longer dependent on added growth factor, and an immunoprecipitable complex between EpoR and immature env protein in the endoplasmic reticulum has been identified. The dependence of these in vitro activities on env protein processing and their relationship to pathogenicity of SFFV were explored by using glycosylation site mutants of SFFV env. Mutants carrying Asn-->Asp mutations at each of the two consensus signals for N-linked glycosylation in the N-terminal domain of SFFVAP-L env (gs1 and gs2), the gs1-2- double mutant, and the gs0 quadruple mutant (mutated at all four signals utilized for N-linked glycosylation in SFFVAP-L env) were made. The primary translation products (gp52) of single-site mutant envs were processed into more highly glycosylated forms, and the corresponding viruses induced splenomegaly in susceptible mice, whereas the gs1-2- and gs0 proteins were not processed, and these viruses were not pathogenic. Unprocessed env proteins of both pathogenic and nonpathogenic mutants coprecipitated with EpoR. In the BaF3 cell assay for epoR-dependent mitogenicity, the pathogenic single mutants induced factor-independent growth efficiently whereas the nonpathogenic gs1-2- and gs0 mutants did not. These data demonstrate that the ability of gp52 to form complexes with EpoR in the endoplasmic reticulum is not sufficient for either mitogenicity in cell culture or induction of splenomegaly in mice while supporting the hypothesis that pathogenicity and mitogenicity of SFFV both result from an interaction between EpoR and SFFV env protein.     

The genetic bottleneck in vertical transmission of subtype C HIV-1 is not driven by selection of especially neutralization-resistant virus from the maternal viral population

Subtype C human immunodeficiency virus type 1 (HIV-1C) continues to cause the majority of new cases of mother-to-child transmission (MTCT), and yet there are limited data on HIV-1C transmission. We amplified env from plasma RNA for 19 HIV-1C MTCT pairs, 10 transmitting in utero (IU) and 9 transmitting intrapartum (IP). There was a strong genetic bottleneck between all mother-infant pairs, with a majority of transmission events involving the transmission of a single virus. env genes of viruses transmitted to infants IP, but not IU, encoded Env proteins that were shorter and had fewer putative N-linked glycosylation sites in the V1-V5 region than matched maternal sequences. Viruses pseudotyped with env clones representative of each maternal and infant population were tested for neutralization sensitivity. The 50% inhibitory concentration of autologous serum was similar against both transmitted (infant) and nontransmitted (maternal) viruses in a paired analysis. Mother and infant Env proteins were also similar in sensitivity to soluble CD4, to a panel of monoclonal antibodies, and to heterologous HIV-1C sera. In addition, there was no difference in the breadth or potency of neutralizing antibodies between sera from 50 nontransmitting and 23 IU and 23 IP transmitting HIV-1C-infected women against four Env proteins from heterologous viruses. Thus, while a strong genetic bottleneck was detected during MCTC, with viruses of shorter and fewer glycosylation sites in env present in IP transmission, our data do not support this bottleneck being driven by selective resistance to antibodies.