Role of the membrane anchoring and cytoplasmic domains in intracellular transport and localization of viral glycoproteins.

The role of the transmembrane and the cytoplasmic regions of viral glycoproteins namely, the envelope glycoprotein gD of herpes simplex virus and the integral membrane glycoprotein E3-11.6 K of the nonenveloped adenovirus that are localized in the nuclear envelope has been studied. Chimeras of the cell surface glycoprotein G of vesicular stomatitis virus containing the transmembrane and (or) the cytoplasmic-tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K protein were examined for their intracellular transport and localization. The results show that hybrids containing the membrane anchoring and (or) the cytoplasmic tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K glycoprotein were localized in the nuclear envelope as well as in the endoplasmic reticulum and the Golgi complex. These results suggest that the membrane anchoring and the cytoplasmic domains of herpes simplex virus glycoproteins gD, as well as the adenovirus integral membrane protein E3-11.6 K, were necessary for localization in the nuclear envelope and could influence retention in the endoplasmic reticulum and the Golgi complex.     

Cell surface mucin-type glycoproteins and mucin-like domains.

Cell surface mucins and mucin-like domains comprise a diverse and heterogeneous group of cell surface glycoproteins. The heterogeneity results from both genetic variations in the polypeptides and carbohydrate differences. Mucins form extended rods from the cell surface. The mucin domains apparently serve a protective function, protecting the glycoproteins from cell surface proteolysis and protecting the cells from attack by other cells. Biosynthesis of mucin oligosaccharides is initiated near the transit of the proteins from the endoplasmic reticulum to the Golgi and proceeds rapidly during passage to the cell surface. In some carcinomas a second O-glycosylation pathway adds new oligosaccharides to the glycoproteins after they have reached the cell surface, presumably during recycling.     

Role of the cytoplasmic tail domains of Bunyamwera orthobunyavirus glycoproteins Gn and Gc in virus assembly and morphogenesis

The M RNA genome segment of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, encodes a precursor polyprotein that is proteolytically cleaved to yield two structural proteins, Gn and Gc, and a nonstructural protein called NSm. Gn and Gc are type I integral transmembrane glycoproteins. The Gn protein contains a predicted cytoplasmic tail (CT) of 78 residues, and Gc has a shorter CT of 25 residues. Little is known about the role of the Gn and Gc CT domains in the virus replication cycle. We generated a series of mutant glycoprotein precursor constructs containing either deletions or alanine substitutions in the CT domains of Gn and Gc. We examined the effects of these mutations on glycoprotein maturation, cell surface expression, and low pH-induced syncytium formation. In addition, the effects of these mutations were also assessed using a reverse genetics-based virus assembly assay and a virus rescue system. Our results show that the CT domains of both Gn and Gc play crucial roles in BUNV-mediated membrane fusion, virus assembly, and morphogenesis.     

Localization of cell surface glycoproteins in membrane domains associated with the underlying filament network

To visualize the localization of cell surface constituents in relation to the plasma membrane-associated filament network, we developed a method based on a combination of immunogold labeling and dry-cleaving. For labeling we used trinitrophenyl-derivatized ligand, anti-TNP antibodies, and protein A-coated colloidal gold. Dry-cleaving (Mesland, D. A. M., H. Spiele, and E. Roos, 1981, Exp. Cell Res., 132: 169-184) involves cleavage of lightly fixed critical point-dried cells by means of adhesive tape. Since cells cleave close to the cell surface, the remaining layer is thin enough to be examined in transmission electron microscopy. Using this method, we studied concanavalin A-binding constituents on the medium-facing surface of H35 hepatoma cells. The distribution of the gold particles, which was partly dispersed and partly patchy, coincided strikingly with membrane-associated filaments, and label was virtually absent from areas overlying openings in the filament network. In stereo pairs we observed the label to be localized to areas of somewhat enhanced electron density at the plane of the membrane. These areas were interconnected in a pattern congruent with the filament network. Preliminary observations on wheat germ agglutinin receptors on the hepatoma cells as well as concanavalin A receptors on isolated hepatocytes yielded comparable results. It thus appears that surface glycoproteins, although seemingly randomly distributed as observed in thin sections, may actually be localized to particular membrane domains associated with underlying filaments.     

A tyrosine-based motif in the cytoplasmic tail of pseudorabies virus glycoprotein B is important for both antibody-induced internalization of viral glycoproteins and efficient cell-to-cell spread

Pseudorabies virus (PRV), a swine alphaherpesvirus, is capable of causing viremia in vaccinated animals. Two mechanisms that may help PRV avoid recognition by the host immune system during this viremia are direct cell-to-cell spread in tissue and antibody-induced internalization of viral cell surface glycoproteins in PRV-infected blood monocytes, the carrier cells of the virus in the blood. PRV glycoprotein B (gB) is crucial during both processes. Here we show that mutating a tyrosine residue located in a YXXPhi motif in the gB cytoplasmic tail results in decreased efficiency of cell-to-cell spread and a strong reduction in antibody-induced internalization of viral cell surface glycoproteins. Mutating the dileucine motif in the gB tail led to an increased cell-to-cell spread of the virus and the formation of large syncytia.     

Interaction of Sendai virions with resealed human erythrocyte ghosts. Lateral mobility of the viral glycoproteins in the cell membrane following fusion

Two independent methods demonstrated that resealed human erythrocyte ghosts undergo Sendai virus-mediated cell-cell fusion to a much lower degree (about 4%) than intact erythrocytes, in spite of similar levels of viral envelope-cell fusion in the two preparations. Fluorescence photobleaching recovery (FPR) showed similar lateral mobilities of the viral glycoproteins following fusion with either ghosts or whole erythrocytes. It is suggested that although viral glycoprotein mobilization in the cell membrane is essential for cell-cell fusion, the target cell properties are also important; in the absence of the required cellular parameters, the mobilization may not be a sufficient condition.     

Cell surface transport, oligomerization, and endocytosis of chimeric type II glycoproteins: role of cytoplasmic and anchor domains.

We investigated the role of cytoplasmic and anchor domains of type II glycoproteins in intracellular transport, oligomerization, and endocytosis by expressing the wild-type and chimeric genes in mammalian cells. Chimeric genes were constructed by exchanging the DNA segments that encode the cytoplasmic and anchor domains between the human influenza virus (A/WSN/33) neuraminidase (NA) and transferrin receptor (TR). The chimeric proteins in which domains were exchanged precisely were productively targeted to the cell surface. However, the proteins appeared to assemble differently in the intracellular compartment. For example, while TR existed predominantly as a dimer, NATR delta 90, containing the cytoplasmic and signal-anchor domains of NA and the ectodomain of TR, was present as a tetramer, a dimer, and a monomer. Similarly, the influenza virus NA existed predominantly as a tetramer but TRNA delta 35, in which the cytoplasmic and signal-anchor domains of TR were joined to the ectodomain of NA, existed predominantly as a dimer, suggesting that the cytoplasmic and anchor domains of type II glycoproteins affect the subunit assembly of heterologous ectodomains. In addition, we analyzed the role of the cytoplasmic domain in endocytosis. NA and NATR delta 90 did not undergo endocytosis, whereas both TR and TRNA delta 35 were internalized efficiently, demonstrating that the NH2 cytoplasmic domain of TR was capable of internalizing a heterologous ectodomain (NA) from the cell surface.     

Role of the transmembrane and cytoplasmic domains in the assembly and surface exposure of the platelet integrin GPIIb/IIIa.

Integrins are alpha beta heterodimers that play a major role in cell-cell contacts and in interactions between cells and extracellular matrices. Identification of structural domains that are critical for the expression of such receptors at the cell surface in a functional conformation is one of the major issues that has not yet been resolved. In the present study, the role of the cytoplasmic and transmembrane domains of each of the subunits has been examined using platelet GPIIb/IIIa as a prototypic integrin. GPIIb/IIIa (alpha IIb/beta 3) is a member of the integrin family and functions as a receptor for fibrinogen, fibronectin, von Willebrand factor, and vitronectin at the surface of activated platelets. Human megakaryocyte GPIIb and GPIIIa cDNAs were used to create a GPIIb mutant coding for the extracellular GPIIb heavy chain alone (GPIIb delta 1) and a GPIIIa mutant lacking the transmembrane and cytoplasmic domains (GPIIIa delta m). Full length and mutant cDNAs were subcloned into the expression vector pECE and used to transfect COS cells. The formation of heterodimers and their cellular localization was analyzed by immunoprecipitation and immunofluorescence labeling using anti-platelet GPIIb/IIIa antibodies. We show here that the extracellular domains of alpha and beta subunits are able to form a heterodimer, although with a lower efficiency, in the absence of the transmembrane and cytoplasmic domains. The presence of the cytoplasmic and transmembrane domains in the alpha subunit is, however, necessary for expression at the surface of the cell whereas the corresponding domains of the beta subunit are not required.     

Autonomous roles for the cytoplasmic domains of the CD2 and CD4 T cell surface antigens.

CD2 and CD4 are single chain transmembrane T cell surface molecules that are involved in signal transduction. Chimaeric constructs from rat CD2 and CD4 antigens were expressed in the Jurkat human T cell line to examine the role of extracellular, transmembrane and cytoplasmic domains in mediating functions controlled by CD2 and CD4. The results show that the large rise in concentration of cytoplasmic free Ca2+ mediated via CD2 crosslinking is controlled by the cytoplasmic domain and does not require the CD2 transmembrane and extracellular domains. Similarly the CD4 cytoplasmic domain alone was shown to encode the specificity for binding to the p56lck tyrosine kinase and to control down-modulation of CD4 after treatment with phorbol ester. Evidence was obtained that down-modulation of CD4 occurs when p56lck dissociates from the cytoplasmic domain due to phosphorylation of Ser 405.     

Deletion of hydrophobic domains of viral glycoproteins increases the level of their production in Escherichia coli.

Overexpression of glycoprotein-encoding genes in Escherichia coli sometimes results in toxicity to the host and low protein yields. One possible explanation for this phenomenon is the presence of hydrophobic amino acid (aa) domains approx. 15-20 aa in length in the overproduced protein. As an initial test of this hypothesis, regions of hydrophobicity located within the envelope glycoproteins of HIV-1 and HTLV-1 were identified by computer analysis, and subsequently deleted by site-directed mutagenesis. The parent and modified envelope genes were expressed in bacteria using both lambda pL and T7 inducible expression systems. Removal of the hydrophobic domains reduced the apparent toxicity and significantly increased the accumulation of recombinant protein from undetectable levels to approx. 10-15% of total cellular protein.     

Studies on the function of cell surface glycoproteins. II. Possible role of surface glycoproteins in the control of cytoskeletal organization and surface morphology

Immunoglobulin from goat antiserum directed against purified surface membranes from transformed BHK21/C13 cells (anti-M) has been shown to cause both control and transformed hamster cells to round and detach from the substrate (see accompanying paper). This paper documents the effects of the antiserum on the cytoskeletal organization and cell surface morphology of control BHK21/C13 cells examined by scanning and transmission electron microscopy. As a result of antiserum-induced rounding, the normally smooth cell surface becomes covered with filopodia and blebs, and the organization of all three components of the filamentous cytoskeleton is altered. In terms of cell surface morphology and cytoskeletal organization, the cells resemble rounded, postmitotic or trypsinized BHK cells rather than cells treated with either anticytoskeletal drugs or lectins. Immunocytochemical and radioimmune assay experiments support the suggestion that the rounding reaction induced by anti-M serum results from the specific interaction of antibodies with molecules on the cell surface. It is suggested that anti-M serum induces alterations in cytoskeletal organization via a transmembrane signal and that cytoskeletal reorganization is a fundamental part of the rounding and detachment process.     

Structure-function relationships in the mast cell high affinity receptor for IgE. Role of the cytoplasmic domains and of the beta subunit.

To define functionally critical regions of the high affinity receptor for IgE (Fc epsilon RI), we stably transfected P815 cells with mutated cDNAs coding for subunits with truncated cytoplasmic domains (CD). In addition, to examine further the role of the beta subunit, stable transfectants expressing chimeric Fc epsilon RI without beta subunits were generated. Transfectants were tested for receptor-mediated changes in intracellular Ca2+, for stimulated hydrolysis of phosphoinositides, and for protein tyrosine phosphorylation. In all cases these biochemical signals were affected coordinately, suggesting that they are coupled, possibly in a single pathway. Truncation of the alpha subunit or of the NH2-terminal CD of the beta subunit had no effect, but Fc epsilon RIs with beta subunits missing the COOH-terminal CD were inactive. Interestingly, receptors in cells transfected only with human Fc epsilon RI(alpha) (which utilize the gamma chains endogenously synthesized by the P815 cells but which contain no beta subunits) responded normally. Therefore, the beta subunit influences the functions studied but is not essential. Although structural analysis excluded a straightforward mechanism, truncation of the CD of the gamma chain led to loss of signaling.     

Role of HIV-1 Gag domains in viral assembly

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed.     

Role of HIV-1 Gag domains in viral assembly

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed.     

Role of signal recognition particle in the membrane assembly of Sindbis viral glycoproteins

We have investigated the role of signal recognition particle (SRP) in the biosynthesis of Sindbis glycoproteins by translating the viral 26S mRNA in a wheat-germ cell-free system. SRP was shown to have no effect on the synthesis or proteolytic processing of the cytoplasmic C protein. In contrast, the membrane integration and the proteolytic processing of the viral glycoproteins PE2 and E1 were demonstrated to be SRP-dependent. In the absence of microsomal membranes, SRP caused an arrest of the synthesis of the viral glycoproteins. This arrest could be released by the addition of salt-extracted microsomal membranes. Synchronization experiments indicated that the uncleaved signal sequence of PE2 was recognized by SRP after at most 130 amino acids of PE2 had been polymerized. No apparent interaction of SRP with a putative signal sequence of E1 and/or a 6-kDa peptide could be detected.     

An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface

A Tyr to Cys mutation at amino acid position 723 in the cytoplasmic domain of the simian immunodeficiency virus (SIV) transmembrane (TM) molecule has been shown to increase expression of envelope glycoproteins on the surface of infected cells. Here we show that Tyr- 723 contributes to a sorting signal that directs the rapid endocytosis of viral glycoproteins from the plasma membrane via coated pits. On cells infected by SIVs with a Tyr at position 723, envelope glycoproteins were transiently expressed on the cell surface and then rapidly endocytosed. Similar findings were noted for envelope molecules expressed in the absence of other viral proteins. Immunoelectron microscopy demonstrated that these molecules were localized in patches on the cell surface and were frequently associated with coated pits. In contrast, envelope glycoproteins containing a Y723C mutation were diffusely distributed over the entire plasma membrane. To determine if an internalization signal was present in the SIV TM, chimeric molecules were constructed that contained the CD4 external and membrane spanning domains and a SIV TM cytoplasmic tail with a Tyr or other amino acids at SIV position 723. In Hela cells stably expressing these molecules, chimeras with a Tyr-723 were rapidly endocytosed, while chimeras containing other amino acids at position 723, including a Phe, were internalized at rates only slightly faster than a CD4 molecule that lacked a cytoplasmic domain. In addition, the biological effects of the internalization signal were evaluated in infectious viruses. A mutation that disrupted the signal and as a result, increased the level of viral envelope glycoprotein on infected cells, was associated with accelerated infection kinetics and increased cell fusion during viral replication. These results demonstrate that a Tyr-dependent motif in the SIV TM cytoplasmic domain can function as an internalization signal that can modulate expression of the viral envelope molecules on the cell surface and affect the biological properties of infectious viruses. The conservation of an analogous Tyr in all human and simian immunodeficiency viruses suggests that this signal may be present in other primate lentiviruses and could be important in the pathogenesis of these viruses in vivo.