Bovine herpesvirus 1 UL49.5 homolog gene encodes a novel viral envelope protein that forms a disulfide-linked complex with a second virion structural protein.

We previously reported that the genome of bovine herpesvirus 1 (BHV-1) contains an open reading frame (ORF) homologous to the herpes simplex virus UL49.5 ORF, and as with the herpes simplex virus UL49.5 ORF, the deduced amino acid sequence of the BHV-1 UL49.5 homolog (UL49.5h) contains features characteristic of an integral membrane protein, implying that it may constitute a functional gene encoding a novel viral envelope protein. This communication reports on the identification of the BHV-1 UL49.5h gene product. By employing an antibody against a synthetic BHV-1 UL49.5h peptide and an UL49.5h gene deletion mutant, the primary product of BHV-UL49.5h gene was identified as a polypeptide with a size of approximately 9 kDa; in both infected cells and isolated virions, the UL49.5h products were found to exist in three forms; monomer, disulfide-linked homodimer, and disulfide-linked heterodimer containing a second viral protein with a size of about 39 kDa. O-Glycosidase digestion and [3H]glucosamine labelling experiments showed that the UL49.5h protein is not glycosylated. Although the deduced amino acid sequence contains putative sites for myristylation and phosphorylation, we were unable to detect either modification. Surface labelling and trypsin digestion protection experiments showed that the BHV-1 UL49.5h protein was present on the surface of infected cells and on the surface of mature virions. Nonionic detergent partition of isolated virions revealed that the UL49.5h protein is more tightly associated with the virion tegument-nucleocapsid structure than envelope protein gD. The results from this study demonstrate that the BHV-1 UL49.5h gene encodes a nonglycosylated virion envelope protein which may associate with virion internal structures by forming a complex with the 39-kDa virion structural protein.     

Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region.

The membrane orientation of the NB protein of influenza B virus, a small (Mr, approximately 18,000) glycoprotein with a single internal hydrophobic domain, was investigated by biochemical and genetic means. Cell fractionation and protein solubility studies indicate NB is an integral membrane protein, and NB has been shown to be a dimer under nonreducing conditions. Treatment of infected-cell surfaces with proteinase K and endoglycosidase F and immunoprecipitation with a site-specific antibody suggests that the 18-amino-acid NH2-terminal region of NB is exposed at the cell surface. Oligonucleotide-directed mutagenesis to eliminate each of the four potential sites of N-linked glycosylation and expression of the mutant NB proteins in eucaryotic cells suggest that the two sites adjacent to the NH2 terminus are glycosylated. This provides further evidence that NB, which lacks a cleavable NH2-terminal signal sequence, has an exposed NH2 terminus at the cell surface.     

Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene.

With the aid of three monoclonal antibodies (MAbs), a glycoprotein specifically localized to the outer envelope of vaccinia virus was shown to be encoded by the A33R gene. These MAbs reacted with a glycosylated protein that migrated as 23- to 28-kDa and 55-kDa species under reducing and nonreducing conditions, respectively. The protein recognized by the three MAbs was synthesized by all 11 orthopoxviruses tested: eight strains of vaccinia virus (including modified vaccinia virus Ankara) and one strain each of cowpox, rabbitpox, and ectromelia viruses. The observation that the protein synthesized by ectromelia virus-infected cells reacted with only one of the three MAbs provided a means of mapping the gene encoding the glycoprotein. By transfecting vaccinia virus DNA into cells infected with ectromelia virus and assaying for MAb reactivity, we mapped the glycoprotein to the A33R open reading frame. The amino acid sequence and hydrophilicity plot predicted that the A33R gene product is a type II membrane protein with two asparagine-linked glycosylation sites. Triton X-114 partitioning experiments indicated that the A33R gene product is an integral membrane protein. The ectromelia virus homolog of the vaccinia virus A33R gene was sequenced, revealing 90% predicted amino acid identity. The vaccinia and variola virus homolog sequences predict 94% identical amino acids, the latter having one fewer internal amino acid. Electron microscopy revealed that the A33R gene product is expressed on the surface of extracellular enveloped virions but not on the intracellular mature form of virus. The conservation of this protein and its specific incorporation into viral envelopes suggest that it is important for virus dissemination.     

Function of the respiratory syncytial virus small hydrophobic protein

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, encodes a small hydrophobic (SH) protein of unknown function. Parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, also encodes an SH protein, which inhibits tumor necrosis factor alpha (TNF-α) signaling. In this study, recombinant PIV5 viruses without their own SH but containing RSV SH (from RSV strain A2 or B1) in its place (PIV5ΔSH-RSV SH) and RSV lacking its own SH (RSVΔSH) were generated and analyzed. The results indicate that the SH protein of RSV has a function similar to that of PIV5 SH and that it can inhibit TNF-α signaling.     

Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus.

The transmembrane (TM) envelope protein of lentiviruses, including equine infectious anemia virus (EIAV), is significantly larger than that of other retroviruses and may extend in the C-terminal direction 100 to 200 amino acids beyond the TM domain. This size difference suggests a lentivirus-specific function for the long C-terminal extension. We have investigated the synthesis and processing of the EIAV TM protein by immune precipitation and immunoblotting experiments, by using several envelope-specific peptide antisera. We show that the TM protein in EIAV particles is cleaved by proteolysis to an N-terminal glycosylated 32- to 35-kilodalton (kDa) segment and a C-terminal nonglycosylated 20-kDa segment. The 20-kDa fragment was isolated from virus fractionated by high-pressure liquid chromatography, and its N-terminal amino acid sequence was determined for 13 residues. Together with the known nucleotide sequence, this fixes the cleavage site at a His-Leu bond located 240 amino acids from the N terminus of the TM protein. Since the 32- to 35-kDa fragment and the 20-kDa fragment are not detectable in infected cells, we assume that cleavage occurs in the virus particle and that the viral protease may be responsible. We have also found that some cells producing a tissue-culture-adapted strain of EIAV synthesize a truncated envelope precursor polyprotein. The point of truncation differs slightly in the two cases we have observed but lies just downstream from the membrane-spanning domain, close to the cleavage point described above. In one case, virus producing the truncated envelope protein appeared to be much more infectious than virus producing the full-size protein, suggesting that host cell factors can select for virus on the basis of the C-terminal domain of the TM protein.     

Processing and localization of the african swine fever virus CD2v transmembrane protein

The African swine fever virus (ASFV)-encoded CD2v transmembrane protein is required for the hemadsorption of red blood cells around infected cells and is also required for the inhibition of bystander lymphocyte proliferation in response to mitogens. We studied the expression of CD2v by expressing the gene with a V5 tag downstream from the signal peptide near the N terminus and a hemagglutinin (HA) tag at the C terminus. In ASFV-infected cells, a full-length glycosylated form of the CD2v protein, which migrated mainly as a 89-kDa product, was detected, as well as an N-terminal glycosylated fragment of 63 kDa and a C-terminal nonglycosylated fragment of 26 kDa. All of these forms of the protein were localized in the membrane fraction of cells. The 26-kDa C-terminal fragment was also produced in infected cells treated with brefeldin A. These data indicate that the CD2v protein is cleaved within the luminal domain and that this occurs in the endoplasmic reticulum or Golgi compartments. Confocal microscopy showed that most of the expressed CD2v protein was localized within cells rather than at the cell surface. Comparison of the localization of full-length CD2v with that of a deletion mutant lacking all of the cytoplasmic tail apart from the 12 membrane-proximal amino acids indicated that signals within the cytoplasmic tail are responsible for the predominant localization of the full-length and C-terminal 26-kDa fragment within membranes around the virus factories, which contain markers for the Golgi compartment. Processing of the CD2v protein was not observed in uninfected cells, indicating that it is induced by ASFV infection.     

Structural properties of a soluble bioactive precursor for transforming growth factor-alpha

The precursor for transforming growth factor-alpha, proTGF-alpha, is synthesized as an integral membrane glycoprotein with the mature TGF-alpha sequence located in the extracellular domain. Retrovirally transformed rat embryo fibroblasts (FeSV-Fre cells) expressing the endogenous proTGF-alpha gene release and accumulate in the medium mature TGF-alpha as well as a heterogeneous (17-19 kDa) group of soluble, bioactive TGF-alpha precursor forms. These precursors correspond to the heterogeneously glycosylated extracellular domain of proTGF-alpha which is released from the membrane by proteolytic cleavage. They are designated mesoTGF-alpha to denote their intermediate position in the proTGF-alpha processing pathway. The nature of the carbohydrate linked to mesoTGF-alpha has been examined by treatment with glycosidases and the use of metabolic inhibitors of glycosylation. The results indicate that the TGF-alpha precursors from FeSV-Fre cells contain O-linked carbohydrate as well as sialylated N-linked carbohydrate. Heterogeneous N-linked glycosylation of an 11-kDa core polypeptide accounts for the heterogeneous nature of mesoTGF-alpha. MesoTGF-alpha released by cells treated with inhibitors of N-linked carbohydrate processing appears as a 17-kDa species. Treatment with these inhibitors does not alter significantly the production of mesoTGF-alpha or mature TGF-alpha by the cells. However, treatment of cells with an inhibitor of co-translational N-linked glycosylation, tunicamycin, reduces the accumulation of mesoTGF-alpha in the medium and blocks the production of mature TGF-alpha under conditions in which overall protein synthesis is only minimally affected. These findings suggest that the proTGF-alpha processing activity is limiting in FeSV-Fre cells and other transformed cells that accumulate mesoTGF-alpha in the medium and that proTGF-alpha processing depends on a component whose function may require N-linked glycosylation.     

Characterization of glycosylated Gag expressed by a neurovirulent murine leukemia virus: identification of differences in processing in vitro and in vivo.

The neuroinvasiveness of a chimeric murine retrovirus, CasFrKP (KP), is dependent on the expression of glycosylated Gag (gp85gag). This viral protein is the product of alternate translation initiation 88 codons upstream of and in frame with the initiation codon of pr65gag, the precursor of the viral core proteins. Although expression of glycosylated Gag affects virus spread in the spleen, it appears not to affect virus spread in vitro in fibroblast cell lines (J. L. Portis et al., J. Virol. 68:3879-3887, 1994). The differential effects of this protein in vitro and in vivo have not been explained, and its function is unknown. We have here compared the in vitro processing of this molecule with that expressed in spleens of infected mice. In vitro, gp85gag was cleaved near the middle of the molecule, releasing the C-terminal half (containing capsid and nucleocapsid domains of pr65gag) as a secreted glycoprotein. The N-terminal half of the protein was associated with the plasma membrane as a approximately 55-kDa glycoprotein bearing the matrix domain of pr65gag as well as the N-terminal 88 residue L domain. This processing scheme was also observed in vivo, although two differences were seen. There were differences in N-linked glycosylation of the secreted form of the protein expressed in the spleen. In addition, whereas the membrane-associated species assumed the orientation of a type II integral membrane protein (N(cyto) C(exo)) in fibroblasts in vitro, a subpopulation of spleen cells was detected in which the N terminus of the protein was exposed at the cell surface. These results suggest that the differential effects of glycosylated Gag expression in vivo and in vitro may be related to differences in posttranslational processing of the protein.     

Sequence and expression of a bovine herpesvirus-1 gene homologous to the glycoprotein K-encoding gene of herpes simplex virus-1

In the bovine herpesvirus-1 (BHV-1) genome, a gene equivalent to the glycoprotein K (gK)-encoding gene of other herpesviruses was identified and sequenced. The primary translation product is predicted to comprise 338 amino acids (aa) and to exhibit a molecular mass of 37.5 kDa. It possesses characteristics typical for membrane glycoproteins including a potential cleavable signal sequence, three transmembrane domains and two potential N-linked glycosylation sites. Comparison to the gK proteins of other herpesviruses revealed aa sequence homologies of 46, 44, 53, 43 and 46% with the gK counterparts of herpes simplex viruses-1 and 2 (HSV-1 and 2), equine herpesvirus-1 (EHV-1), Marek's disease virus (MDV) and varicella zoster virus (VZV), respectively. A 30-kDa primary translation product was identified following in vitro translation of in vitro transcribed mRNA. When canine microsomal membranes were added to the translation reaction, a 38-kDa glycosylated protein was detected. Treatment with endoglycosidase For H (endo For H) removed the glycosyl groups and reduced the apparent molecular mass of the 38-kDa glycoprotein.     

Secretion of glycosylated alpha-lactalbumin in yeast Pichia pastoris

The secretion of N-linked glycosylated alpha-lactalbumin was much higher in the expression system of yeast Pichia pastoris carrying goat alpha-lactalbumin cDNA than in mammalian milk. This is possibly because of the presence of N-linked glycosylation signal sequences, Asn(45)-Asp(46)-Ser(47) and Asn(74)-Ile(75)-Ser(76), in wild-type alpha-lactalbumin. Attempts to elucidate the mechanism of the higher secretion of glycosylated alpha-lactalbumin in P. pastoris were made. Mutant N45D that deleted the N-linked glycosylation signal sequence at position 45 predominantly secreted nonglycosylated protein. On the other hand, mutant D46N with another N-glycosylation signal site at position 46 only secreted N-linked glycosylated alpha-lactalbumin, i.e. not the nonglycosylated protein. The total secreted amount of mutant N45D was greatly enhanced, while the secreted amounts of the wild-type and mutant D46N were very low, suggesting that the increase in the number of glycosylation sites greatly reduced the secretion of alpha-lactalbumin. It seems likely that the glycosylated alpha-lactalbumin may be degraded by the quality control system.     

N-glycosylation of HIV-gp120 may constrain recognition by T lymphocytes.

The HIV envelope protein gp120 is heavily glycosylated, having 55% of its molecular mass contributed by N-linked carbohydrates. We investigated the role of N-glycosylation in presentation of HIV-gp120 to T cells. T cell clones obtained from humans immunized with a recombinant nonglycosylated form of HIV-gp120 (env 2-3) were studied for their ability to recognize both env 2-3 and glycosylated gp120. We found that 20% of CD4+ T cell clones specific for env 2-3 fail to respond to glycosylated gp120 of the same HIV isolate. Using synthetic peptides, we mapped one of the epitopes recognized by such clones to the sequence 292-300 (NESVAINCT), which contains two asparagines that are glycosylated in the native gp120. These findings suggest that N-linked carbohydrates within an epitope can function as hindering structures that limit Ag recognition by T lymphocytes.     

Identification and characterization of three immunodominant structural proteins of fowlpox virus

Genes encoding fowlpox virus (FWPV) structural proteins have been identified mainly by sequence homology with those from vaccinia virus (VACV), but little is known about the encoded proteins. Production of monoclonal antibodies (MAbs) against Poxine and HP1-440 (Munich) clone FP9 allowed the identification of three immunodominant FWPV proteins: the 39-kDa core protein (encoded by FPV168, homologous to VACV A4L), a 30- and 35-kDa protein doublet, and an abundant 63-kDa protein. The 30- and 35-kDa proteins are nonglycosylated, antigenically related proteins present in the intracellular mature virus membrane and localizing closely with the viral factories. N-terminal sequencing identified the 35-kDa protein as encoded by FPV140 (the FWPV homolog of VACV H3L). The 63-kDa protein forms covalently linked dimers and oligomers. It remained mainly insoluble upon detergent treatment of purified virus but did not localize closely with the viral factory. N-terminal sequencing was unsuccessful, suggesting N-terminal blocking. CNBr digestion generated a peptide encoded by FPV191, predicted to encode one of two FWPV A-type inclusion (ATI) proteins. The characteristics of the 63-kDa protein were inconsistent with published observations on cowpox or VACV ATI proteins (it appears to be essential). The 63-kDa protein, however, shares characteristics with both VACV p4c virus occlusion and 14-kDa fusion proteins. Gene assignment at the poxvirus ATI locus (between VACV A24R and A28L) is complicated by sequence redundancies and variations, often due to deletions and multiple frameshift mutations. The identity of FPV191 in relation to genes at this locus is discussed.     

LRIG1 protein in human cells and tissues

We have recently cloned the human LRIG1 gene (formerly LIG1). LRIG1 is a predicted integral cell-surface protein showing similarities to Kekkon-1, the Drosophila melanogaster epidermal growth-factor-receptor antagonist. A specific peptide antibody, LRIG1-151, was raised in rabbits and used to study the LRIG1 protein. LRIG1 migrated in denaturing polyacrylamide gel electrophoresis under reducing conditions as two species with apparent molecular weights of 143 kDa and 134 kDa, and as two fragments corresponding to an N-terminal 111-kDa species and a C-terminal 32-kDa species. Under non-reducing conditions, both apparent monomers and apparent higher molecular weight complexes were evident. Immunoblotting analysis of cell-surface-biotinylated lysates and confocal microscopy revealed that LRIG1 was localized to the cell surface in ZR-75 cells expressing endogenous LRIG1 and in COS-7 cells expressing a synthetic LRIG1-GFP fusion protein. Immunohistochemical analysis of normal human tissues showed staining for LRIG1 in epithelia in various organs, scattered neurons, and muscles. Immunoblotting demonstrated LRIG1 protein in tissue lysates from normal human prostate, mammary epithelial cells, ileum, stomach, lung, and cerebral cortex. These results demonstrate that LRIG1 is an integral cell-surface membrane protein that is expressed by specific cells in various human tissues and that its 143-kDa form might be cleaved into 111-kDa and 32-kDa fragments.     

Characterization of severe acute respiratory syndrome coronavirus membrane protein

The coronavirus membrane protein (M) is the key player in the assembly of virions at intracellular membranes between endoplasmic-reticulum and Golgi-complex. Using a newly established human monoclonal anti-M antibody we detected glycosylated and nonglycosylated membrane-associated M in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infected cells and in purified virions. Further analyses revealed that M contained a single N-glycosylation site at asparagine 4. Recombinant M was transported to the plasma membrane and gained complex-type N-glycosylation. In SARS-CoV infected cells and in purified virions, however, N-glycosylation of M remained endoglycosidase H-sensitive suggesting that trimming of the N-linked sugar side chain is inhibited.     

Relationship of Reticuloendotheliosis Virus to the Avian Tumor Viruses: Nucleic Acid and Polypeptide Composition

The RNA content and polypeptide composition of reticuloendotheliosis virus (REV) was compared to that of C-type RNA tumor viruses. Two RNA species with approximate sedimentation values of 64S and 4S were observed after sucrose gradient centrifugation of RNA extracted from purified REV. The high-molecular-weight RNA species of REV sedimented slightly faster than that of the Bryan strain of Rous sarcoma virus (RSV). Although these characteristics were consistent with those of other C-type RNA tumor viruses, significant differences were observed when the polypeptide composition of REV was compared with that of RSV possessing envelope determinants of Rous-associated virus RAV-2 and RAV-3. Five polypeptides of which two were glycosylated were resolved by polyacrylamide gel electrophoresis. The major nonglycosylated polypeptide of REV did not comigrate with that of RSV (RAV-2)-RSV(RAV-3). The majority of the group-specific antigen reactivity resides in this major nonglycosylated polypeptide of avian tumor viruses and comigrates when proteins of several avian tumor viruses are subjected to coelectrophoresis. This difference in the migration of the major polypeptide of REV and RSV(RAV-2)-RSV(RAV-3) may explain the absence of avian tumor virus group-specific antigen in REV.     

Glycosylation increases the thermostability of human aquaporin 10 protein

Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.     

P2X4 receptor is a glycosylated cardiac receptor mediating a positive inotropic response to ATP

Although P2X receptors are suggested to play a role in synaptic neurotransmission, the specific physiological role of each P2X receptor subtype remains largely unknown. We used cultured chick embryo ventricular myocytes as a model to study a potential physiological role of the P2X(4) receptor in mediating the positive inotropic effect of ATP. The chick P2X4 receptor (cP2X(4)R) mRNA was expressed in the heart and the pharmacological features of the ATP-induced positive inotropic response were similar to those of the cP2X(4)R in terms of insensitivity to blockade by known P2 receptor antagonists and the ineffectiveness of adenosine 5'-(alpha,beta-methylene)triphosphate as an agonist. Treatment of myocytes with antisense oligonucleotides specific to the 5' region of cP2X(4)R abrogated the P2 agonist-stimulated (45)Ca influx. Similarly, antisense oligonucleotide treatment also blocked the 2-methylthio-ATP-stimulated increase in contractile amplitude. The data suggest that the native P2X(4) receptor is involved in mediating the P2 agonist-stimulated response in the heart. In characterizing the biochemical property of the P2X(4) receptor, antibody against cP2X(4)R detected a 44-kDa and a 58-kDa protein in the immunoblot. Inhibition of N-linked glycosylation by tunicamycin converted the 58-kDa protein to the 44-kDa protein, suggesting that the 58-kDa protein was a glycosylated P2X(4) receptor. The nonglycosylated 44-kDa P2X(4) receptor was resistant to various detergent/aqueous extraction, consistent with a role of glycosylation in maintaining its detergent solubility and hydrophilicity. Cross-linking the cell surface proteins with N-hydroxysuccinimide-SS-biotin followed by affinity precipitation with streptavidin-conjugated agarose and subsequent immunoblotting with anti-cP2X(4)R showed that only the glycosylated 58-kDa P2X(4) receptor was expressed on the cell surface, indicating an important role of glycosylation for the receptor's localization on the plasma membrane. These data revealed a novel physiologic function of the P2X(4) receptor and suggested the importance of N-linked glycosylation in its cell surface expression and detergent solubility.     

Biogenesis of vacuolar membrane glycoproteins of yeast Saccharomyces cerevisiae

To investigate the biogenesis of the yeast vacuole, we have sought novel marker proteins localized to the vacuolar membrane. Glycoproteins were prepared from vacuolar membrane vesicles by concanavalin A-Sepharose column chromatography and used to raise monoclonal antibodies. The antibodies obtained recognize several vacuolar proteins that have N-linked oligosaccharide chains. A set of the antibodies reacts with a vacuolar glycoprotein with a major molecular species of 72 kDa (vgp72), which appears to associate peripherally with the vacuolar membrane. The biosynthesis of vgp72 has been examined in detail by pulse-chase experiments and by analyses using various secretory mutants (sec18, sec7, and sec1) and a vacuolar protease mutant (pep4). vgp72 first appears in the endoplasmic reticulum as a 74-kDa species and is quickly modified in the Golgi apparatus to two distinct species: a 79-kDa form, and a heterogeneously glycosylated form (90-150 kDa). Subsequently, both species are proteolytically processed in the vacuole giving rise to a 72-kDa species as well as heavily glycosylated form. Thus, the biogenesis of vgp72 utilizes the early part of the secretory pathway as is the case of vacuolar soluble enzymes. A unique feature is that two species that are different in the extent of glycosylation appear to follow the same destination to the vacuolar membrane.     

Protein glycosylation regulates the externalization of two distinct classes of glucocorticoid-induced glycoproteins in rat hepatoma cells

The relationship of protein glycosylation to the externalization of glucocorticoid inducible alpha1-acid glycoprotein and mouse mammary tumor virus glycoproteins was examined in M1.54, a clonal population of mouse mammary tumor virus-infected rat hepatoma cells. Multiple freeze-thaw of isolated microsomes revealed that while alpha 1-acid glycoprotein is carried through the cell as a soluble component of vesicles, extracellular viral glycoproteins are initially membrane-associated. At concentrations of tunicamycin that specifically inhibited N-linked protein glycosylation, alpha 1-acid glycoprotein fractionated between the cellular and extracellular compartments. Thus, approximately one half of the newly synthesized, nonglycosylated (22,000 Mr) alpha 1-acid glycoprotein was rapidly secreted with kinetics similar to its glycosylated counterpart (release half-time of 60 min), while the remaining species first localized in an undefined intracellular compartment prior to its slow secretion (release half-time of 24 h). The same distribution of nonglycosylated alpha 1-acid glycoprotein was observed at various absolute levels of polypeptide, suggesting that this was not due simply to the saturation of an efficient secretory pathway at high polypeptide levels. In contrast to alpha 1-acid glycoprotein, no labeled viral antigens were released by tunicamycin-treated M1.54, while a nonglycosylated viral precursor glycopolyprotein was expressed intracellularly. Taken together, these results suggest that carbohydrate attachment strongly regulates the externalization of both alpha 1-acid glycoprotein and mouse mammary tumor virus species, which represent two distinct classes of extracellular glycoproteins.