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US3 of human cytomegalovirus is an endoplasmic reticulum resident transmembrane glycoprotein that binds to major histocompatibility complex class I molecules and prevents their departure. The endoplasmic reticulum retention signal of the US3 protein is contained in the luminal domain of the protein. To define the endoplasmic reticulum retention sequence in more detail, we have generated a series of deletion and point mutants of the US3 protein. By analyzing the rate of intracellular transport and immunolocalization of the mutants, we have identified Ser58, Glu63, and Lys64 as crucial for retention, suggesting that the retention signal of the US3 protein has a complex spatial arrangement and does not comprise a contiguous sequence of amino acids. We also show that a modified US3 protein with a mutation in any of these amino acids maintains its ability to bind class I molecules; however, such mutated proteins are no longer retained in the endoplasmic reticulum and are not able to block the cell surface expression of class I molecules. These findings indicate that the properties that allow the US3 glycoprotein to be localized in the endoplasmic reticulum and bind major histocompatibility complex class I molecules are located in different parts of the molecule and that the ability of US3 to block antigen presentation is due solely to its ability to retain class I molecules in the endoplasmic reticulum.

Four well-characterized monoclonal antibodies (MAbs) directed against rabies virus glycoprotein (G) were used to study G folding in vivo. Two of the MAbs were able to immunoprecipitate incompletely oxidized folding intermediates. The two others recognized G only after folding was completed. By using these MAbs, the ability of G to undergo low-pH-induced conformational changes during folding was also investigated. It appeared that some domains acquire this ability before folding is completed. In addition, interactions between unfolded G and some of the molecular chaperones were analyzed. Unfolded G was associated with BiP and calnexin. Association with BiP was maximal immediately after the pulse, whereas association with calnexin was maximal after 5 to 10 min of chase. The effects of tunicamycin and castanospermine on chaperone binding and folding were also studied. In the presence of both drugs, calnexin binding was reduced, consistent with the view that calnexin specifically recognizes monoglucosylated oligosaccharides, but some residual binding was still observed, indicating that calnexin also recognizes the polypeptide chain. In the presence of both drugs, association with BiP was increased and prolonged and folding was impaired. However, the global effects of the drugs were different, since folding was much more efficient in the presence of castanospermine than in the presence of tunicamycin. Taken together, these results provide the basis to draw a schematic view of rabies virus glycoprotein folding.

Syncytial mutants of herpes simplex virus (HSV) cause extensive fusion of cultured cells, whereas wild-type HSV primarily causes cell rounding and aggregation. A large fraction of syncytial viruses contain mutations in the UL53 gene, which encodes glycoprotein K (gK). Previously, we demonstrated that wild-type and syncytial forms of gK are expressed at similar levels and possess identical electrophoretic mobilities. Using immunofluorescence, we show that gK is not transported to the surfaces of cells infected with either wild-type or syncytial HSV. Instead, gK accumulates in the perinuclear and nuclear membranes of cells. This finding is in contrast to the behavior of all other HSV glycoproteins described to date, which reach the cell surface. When gK was expressed in the absence of other HSV proteins, using a recombinant adenovirus vector, a similar perinuclear and nuclear pattern was observed. In addition, gK remained sensitive to endoglycosidase H, consistent with the hypothesis that gK does not reach the Golgi apparatus and is retained in the endoplasmic reticulum and nuclear envelope. Therefore, although gK mutations promote fusion between the surface membranes of HSV-infected cells, the glycoprotein does not reach the plasma membrane and, thus, must influence fusion indirectly.

Rabbit polyclonal antibodies (PAbs) directed towards the amino-terminal cysteine-rich 7S domain (PAb anti-7S), the major internal collagenous domain (PAb anti-type IV), and the C-terminal noncollagenous region (PAb anti-NC1) of the type IV collagen molecule were probed by indirect immunofluorescence against Candida albicans blastoconidia and germinated blastoconidia. Most nongerminating cells and mother blastoconidia from which germ tubes originated showed strong fluorescence when PAb anti-7S was used, whereas with PAb anti-type IV, fluorescence was found almost exclusively on the surface of filamentous forms. A patched fluorescent pattern rather than a homogenous confluent fluorescence was observed in all cases. No fluorescent cells were observed with PAb anti-NC1. By Western immunoblotting, PAb anti-type IV cross-reacted primarily with a polypeptide of 37 kDa present in wall extracts obtained from intact cells of both growth forms by treatment with beta-mercaptoethanol, whereas PAb anti-7S recognized a major 58-kDa antigen also present in both extracts, along with some other high-molecular-mass (> 106-kDa) polydisperse species present only in the material released from blastoconidia with beta-mercaptoethanol. No reactive bands were observed when PAb anti-NC1 was used as a probe in Western immunoblotting experiments. The sensitivities or resistances to collagenase digestion of the different polypeptides that cross-reacted with PAbs anti-type IV and anti-7S suggest the existence of cell wall components in C. albicans that contain epitopes that mimic the collagenous domains of the type IV collagen molecule.

Vesicular stomatitis virus (VSV) RNA synthesis requires the template nucleocapsid, the polymerase (L) protein, and the cofactor phosphorylated (P/NS) protein. To determine whether the degree of phosphorylation regulated VSV RNA synthesis, infected Chinese hamster ovary cells were treated with okadaic acid (OKA), a serine/threonine phosphatase inhibitor. OKA reduced viral penetration and uncoating but had little or no effect on primary transcription or viral protein synthesis. However, approximately 80% of total viral RNA synthesis was inhibited when 2 microM or more OKA was added to infected cells after viral uncoating had taken place. Analysis of proteins and RNA species in infected cells labeled with 32P showed that OKA led to hyperphosphorylation of two viral phosphoproteins, the P/NS protein and matrix protein (M), resulting in inhibition of full-length RNA synthesis and subsequent secondary transcription. Pulse-chase experiments demonstrated that the hyperphosphorylated P/NS species was converted rapidly from the less phosphorylated form. Hyperphosphorylated P/NS as well as the less phosphorylated form, but not M, were found to be associated with nucleocapsids isolated from cytoplasmic extracts. These results suggest that phosphorylation played an important role in the regulation between viral transcription and viral RNA replication as well as the turning off of RNA replication. Thus, phosphatase inhibitors promise to be a valuable tool for dissecting the regulatory mechanisms involving phosphorylated viral proteins.

Viruses which cause persistence in the naturally infected host are predicted to have evolved immune evasion mechanisms. Human cytomegalovirus (HCMV) causes significant morbidity and mortality in immunocompromised patients yet persists without clinical manifestations in seropositive individuals who have normal immune function. We report that HCMV infection in vitro impairs major histocompatibility complex class I (MHC-I) assembly accompanied by resistance to killing by cytotoxic CD8+ T lymphocytes. Pulse-chase metabolic labelling experiments show that MHC-I complexes continue to be assembled by both uninfected and HCMV-infected cells. However, MHC-I molecules are unstable in HCMV-infected cells and are rapidly broken down. Endoglycosidase H treatment of immunoprecipitates indicates that the breakdown of MHC-I complexes in HCMV-infected cells occurs primarily in a pre-Golgi compartment. Interference with normal MHC-I assembly and expression, if relevant in vivo, may have implications for the restriction of the diversity of the CD8+ cytotoxic T lymphocyte repertoire directed against HCMV antigens and may be an important mechanism of viral persistence.

The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes.

The influenza A/fowl plague virus/Rostock/34 hemagglutinin (HA), which is cleaved intracellularly and has a high pH threshold (pH 5.9) for undergoing its conformational change to the low-pH form, was expressed from cDNA in CV-1 and HeLa T4 cells in the absence of other influenza virus proteins. It was found, by biochemical assays, that the majority of the HA molecules were in a form indistinguishable from the low-pH form of HA. The acidotropic agent, ammonium chloride, stabilized the accumulation of HA in its native form. Coexpression of HA and the homotypic influenza virus M2 protein, which has ion channel activity, stabilized the accumulation of HA in its pH neutral (native) form, and the M2 protein ion channel blocker, amantadine, prevented the rescue of HA in its native form. These data provide direct evidence that the influenza virus M2 protein ion channel activity can affect the status of the conformational form of cleaved HA during intracellular transport.

Previous studies have shown that trimers of the vesicular stomatitis virus glycoprotein (VSV G protein) are in rapid equilibrium with monomeric subunits after folding and assembly in the endoplasmic reticulum (ER). To determine whether G protein trimers were in equilibrium with monomers in other cellular compartments, we studied heterotrimer formation between VSV G protein and a mutant G protein (G mu protein) containing a 3-amino-acid cytoplasmic domain replacing the normal 29-amino-acid domain. The G mu protein is transported from the ER much more slowly than G protein, although both G and G mu proteins form trimers rapidly in the ER. In coexpression experiments, we observed that VSV G protein molecules exited the ER about sixfold faster than G mu protein molecules, and we observed no heterotrimer formation in the ER, probably because of rapid reassortment of the mutant and wild-type trimers. However, heterotrimer formation between the two proteins was observed after long chase periods that allowed time for trimers of the mutant protein to reach the plasma membrane and reassort with the G protein subunits. Additional studies showed that heterotrimers of the two proteins could form in the Golgi or in the ER if exit of the G protein from either compartment was blocked.

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus (HIV) receptor for infection of human host cells. We have recently demonstrated that Vpu, an HIV type 1 (HIV-1) encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. In this report, we describe an in vitro model system that allowed us to define important parameters for Vpu-dependent CD4 degradation. The rate of CD4 decay in rabbit reticulocyte lysate was approximately one-third of that observed previously in tissue culture experiments in the presence of Vpu (40 versus 12 min) and required no other HIV-1 encoded proteins. Degradation was contingent on the presence of microsomal membranes in the assay and the coexpression of Vpu and CD4 in the same membrane compartment. By using the in vitro degradation assay, the effects of specific mutations in CD4, including C-terminal truncations and glycosylation mutants, were analyzed. The results of these experiments indicate that Vpu has the capacity to induce degradation of glycosylated as well as nonglycosylated membrane-associated CD4. Truncation of 13 C-terminal amino acids of CD4 did not affect the ability of Vpu to induce its degradation. However, the removal of 32 amino acids from the C-terminus of CD4 completely abolished sensitivity to Vpu. This suggests that Vpu targets specific sequences in the cytoplasmic domain of CD4 to induce its degradation. We also analyzed the effects of mutations in Vpu on its biological activity in the in vitro CD4 degradation assay. The results of these experiments suggest that sequences critical for this function of Vpu are located in its hydrophilic C-terminal domain.

Glycoprotein gIV is an envelope component of bovine herpesvirus type 1 and appears to be involved in attachment, penetration, and cell fusion. Four antigenic domains which include both continuous and discontinuous epitopes have been previously defined by competition binding assays using gIV-specific monoclonal antibodies (MAbs). Here we describe the construction of C-terminal truncations and internal deletions in the gIV-encoding gene and analyses of the effects of these mutations on the synthesis, processing, transport, and antigenicity of glycoprotein gIV as expressed by recombinant vaccinia viruses. Wild-type gIV expressed by recombinant vaccinia virus STgIV was indistinguishable from authentic gIV produced in bovine herpesvirus 1-infected cells with respect to molecular weight, processing, transport, and antigenicity. Analysis of the mutant proteins showed that the binding sites for MAbs 9D6 and 3D9S, which recognize linear epitopes, lie between amino acids 164 and 216 and amino acids 320 and 355, respectively. Discontinuous epitopes recognized by MAbs 3E7, 4C1, 2C8, and 3C1 were located between amino acids 19 and 320, whereas amino acids 320 to 355 were critical for binding of MAb 136. All mutant proteins containing amino acids 245 to 320 were processed, possess endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides, and were transported to the cell surface or secreted into the medium. In contrast, mutant proteins missing amino acids 245 to 320 were retained in the rough endoplasmic reticulum. These findings suggest that residues 245 to 320 are important for proper processing and transport of gIV to the cell surface.

Varicella-zoster virus (VZV) specifies the synthesis of at least four families of glycoproteins, which have been designated gpI, gpII, gpIII, and gpIV. In this report we describe the assembly and processing of VZV gpII, a structural protein of an apparent Mr of 140,000, which is the homolog of gB of herpes simplex virus. For these studies, we used two anti-gpII monoclonal antibodies which exhibited both complement-independent neutralization activity and inhibition of virus-induced cell-to-cell fusion. Pulse-chase labeling experiments identified a 124,000-Mr intermediate which was chased to the mature 140,000-Mr product when analyzed in nonreducing gels; in the presence of a reducing agent, the native gp140 was cleaved into two closely migrating species (gp66 and gp68). The biosynthesis of VZV gpII was further analyzed in the presence of the following inhibitors of glycoprotein processing: tunicamycin, monensin, castanospermine, swainsonine, and deoxymannojirimycin. All intermediate and mature forms were digested with endoglycosidases H and F, neuraminidase, and O-glycanase to further define high-mannose, complex, and O-linked glycans. Finally, the addition of sulfate residues was investigated. This characterization of VZV gpII revealed the following results. (i) gp128 and gp124 were early high-mannose forms, (ii) gp126 was an intermediate form with complex N-linked oligosaccharides, (iii) gp130 was a later intermediate with both N-linked and O-linked glycans, and (iv) the mature product gp140 contained a mixture of N-linked and O-linked glycans which were both sialated and sulfated. Further investigations indicated that gpII sulfation was inhibited by tunicamycin and castanospermine but not by deoxymannojirimycin or swainsonine. We also concluded that VZV gpII displayed many biological and biochemical properties similar to those of its herpes simplex virus homolog gB.

To study protein processing in an insect Spodoptera frugiperda (fall armyworm; Sf9) cell line, a 26S cDNA encoding the sequence of Sindbis virus structural proteins (capsid protein, of 30 kilodaltons [kDa]; p62 [the precursor of E3 and E2], of 62 kDa; a 6-kDa peptide; and the E1 protein, of 56 kDa) was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) adjacent to the polyhedrin promoter. By immunoblot analysis with antisera directed against whole Sindbis virus and the individual structural proteins (capsid, E2, and E1), we have shown that polypeptides similar in size and antigenicity to those synthesized in Sindbis virus-infected BHK cells are expressed in Sf9 cells infected with the recombinant baculovirus Ac373-SV26. By pulse-chase labeling in the presence or absence of tunicamycin, by endo-beta-N-acetylglucosaminidase H (endo-H) treatment of the recombinant glycoproteins, and by N-terminal sequence analysis of the E1 envelope glycoprotein, we have further shown that the 26S transcription translation unit of Sindbis virus, although normally encoded by nonnuclear RNA, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared with BHK cells when a baculovirus expression vector is used.

The endoglycosidase endo-beta-N-acetylglucominidase H (endo H) was used to examine the nature of the oligosaccharides associated with the herpes simplex virus type 1 glycoproteins gA, gB, and gC. Immunoprecipitates from detergent extracts of infected cells, using monospecific antisera to gAB and gC, were treated with endo H. The low-molecular-weight precursor to gC, pgC(105), was found to be sensitive to endo H. Removal of the endo H-sensitive oligosaccharide chains from pgC(105) resulted in a protein with an apparent molecular weight of 75,000. In contrast, the fully glycosylated gC was not sensitive to endo H treatment. These results suggested that the oligosaccharide chains of pgC(105) were primarily of the simple high-mannose type. Both gA and gB were sensitive to endo H treatment; however, gB appeared to be only partially susceptible, whereas [3H]mannose-labeled gA was not detectable after endo H treatment. These results that gB contained both complex- and simple-type oligosaccharides, and gA contained only simple-type oligosaccharides. An accumulation of the high-mannose glycoproteins pgC(105) and gA was observed in monensin-treated infected cells with a concomitant inhibition of gB and gC. Glycoproteins gA and pgC(105) synthesized in the presence of monensin were also sensitive to endo H treatment.