Intracellular localization of vaccinia virus extracellular enveloped virus envelope proteins individually expressed using a Semliki Forest virus replicon

The extracellular enveloped virus (EEV) form of vaccinia virus is bound by an envelope which is acquired by wrapping of intracellular virus particles with cytoplasmic vesicles containing trans-Golgi network markers. Six virus-encoded proteins have been reported as components of the EEV envelope. Of these, four proteins (A33R, A34R, A56R, and B5R) are glycoproteins, one (A36R) is a nonglycosylated transmembrane protein, and one (F13L) is a palmitylated peripheral membrane protein. During infection, these proteins localize to the Golgi complex, where they are incorporated into infectious virus that is then transported and released into the extracellular medium. We have investigated the fates of these proteins after expressing them individually in the absence of vaccinia infection, using a Semliki Forest virus expression system. Significant amounts of proteins A33R and A56R efficiently reached the cell surface, suggesting that they do not contain retention signals for intracellular compartments. In contrast, proteins A34R and F13L were retained intracellularly but showed distributions different from that of the normal infection. Protein A36R was partially retained intracellularly, decorating both the Golgi complex and structures associated with actin fibers. A36R was also transported to the plasma membrane, where it accumulated at the tips of cell projections. Protein B5R was efficiently targeted to the Golgi region. A green fluorescent protein fusion with the last 42 C-terminal amino acids of B5R was sufficient to target the chimeric protein to the Golgi region. However, B5R-deficient vaccinia virus showed a normal localization pattern for other EEV envelope proteins. These results point to the transmembrane or cytosolic domain of B5R protein as one, but not the only, determinant of the retention of EEV proteins in the wrapping compartment.     

Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks

Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.     

Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein.

There are two types of infectious vaccinia virus particles: intracellular naked virions and extracellular enveloped virions (EEV). To determine the biological role of the enveloped form of vaccinia virus, we produced and characterized a mutant that is defective in EEV formation. The strategy involved replacement by homologous recombination of the gene F13L, encoding a 37,000-Da protein (VP37) that is specific for the outer envelope of EEV, with a selectable antibiotic resistance marker, the Escherichia coli gpt gene. Initial experiments, however, suggested that such a mutation was lethal or prevented plaque formation. By employing a protocol consisting of high-multiplicity passages of intracellular virus from the transfected cells and then limiting dilution cloning, we succeeded in isolating the desired mutant, which was defective in production of plaques and extracellular virus but made normal amounts of intracellular naked virions. Electron microscopic examination indicated that the mutant virus particles, unlike wild type, were neither wrapped with Golgi-derived membranes nor associated with the cell surface. The absence of VP37 did not prevent the transport of the viral hemagglutinin to the plasma membrane but nevertheless abrogated both low-pH- and antibody-mediated cell fusion. These results indicate that VP37 is required for EEV formation and also plays a critical role in the local cell-to-cell transmission of vaccinia virus, perhaps via enveloped virions attached to or released from the cell membrane. By contrast, a mutated virus with a deletion of the K4L open reading frame, which is a homolog of the VP37 gene, was not defective in formation of plaques or EEV.     

Vaccinia Virus Protein F12 Associates with Intracellular Enveloped Virions through an Interaction with A36

Vaccinia virus is the prototypical member of the family Poxviridae. Three morphologically distinct forms are produced during infection: intracellular mature virions (IMV), intracellular enveloped virions (IEV), and extracellular enveloped virions (EEV). Two viral proteins, F12 and A36, are found exclusively on IEV but not on IMV and EEV. Analysis of membranes from infected cells showed that F12 was only associated with membranes and is not an integral membrane protein. A yeast two-hybrid assay revealed an interaction between amino acids 351 to 458 of F12 and amino acids 91 to 111 of A36. We generated a recombinant vaccinia virus that expresses an F12, which lacks residues 351 to 458. Characterization of this recombinant revealed a small-plaque phenotype and a subsequent defect in virus release similar to a recombinant virus that had F12L deleted. In addition, F12 lacking residues 351 to 458 was unable to associate with membranes in infected cells. These results suggest that F12 associates with IEV through an interaction with A36 and that this interaction is critical for the function of F12 during viral egress.     

Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

The wrapping of intracellular mature vaccinia virions by modified trans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.     

Role of cell-associated enveloped vaccinia virus in cell-to-cell spread.

The roles of intracellular naked (INV), cell-associated enveloped (CEV), and extracellular enveloped (EEV) forms of vaccinia virus in cell-to-cell and longer-range spread were investigated by using two closely related strains of vaccinia virus, WR and IHD-J. We confirmed previous results that WR and IHD-J produced similar amounts of INV and formed similar-size primary plaques but that IHD-J produced 10 to 40 times more EEV and spread to distant cells much more efficiently than did WR. Nevertheless, cells infected with WR and IHD-J had similar amounts of CEV, indicating that wrapping and transport of WR virions were unimpaired. A WR mutant with a deletion in VP37, the major outer envelope protein, formed normal amounts of INV; however, the generation of CEV was blocked and plaque formation was inhibited. These results suggested that CEV is the form of virus that mediates cell-to-cell spread. Marker rescue experiments indicated that the differences in EEV production by WR and IHD-J were not due to sequence differences in VP37. The low amount of WR EEV could be attributed to retention of CEV on the cell membrane. In support of this hypothesis, mild treatment with trypsin released as much or more infectious virus from cells infected with WR as it did with cells infected with IHD-J. Most of the virus released by trypsin sedimented with the buoyant density of EEV. Also, addition of trypsin to cells following inoculation with WR led to a comet-shaped distribution of secondary plaques characteristic of IHD-J. These results demonstrated that the release of CEV from the cell surface was limiting for extracellular virus formation and affirmed the role of EEV in long-range spread.     

The Envelope Protein Encoded by the A33R Gene Is Required for Formation of Actin-Containing Microvilli and Efficient Cell-to-Cell Spread of Vaccinia Virus

The vaccinia virus (VV) A33R gene encodes a highly conserved 23- to 28-kDa glycoprotein that is specifically incorporated into the viral outer envelope. The protein is expressed early and late after infection, consistent with putative early and late promoter sequences. To determine the role of the protein, two inducible A33R mutants were constructed, one with the late promoter and one with the early and late A33R promoter elements. Decreased A33R expression was associated with small plaques that formed comets in liquid medium. Using both an antibiotic resistance gene and a color marker, an A33R deletion mutant, vA33Δ, was isolated, indicating that the A33R gene is not essential for VV replication. The plaques formed by vA33Δ, however, were tiny, indicating that the A33R protein is necessary for efficient cell-to-cell spread. Rescue of the large-plaque phenotype was achieved by inserting a new copy of the A33R gene into the thymidine kinase locus, confirming the specific genetic basis of the phenotype. Although there was a reduction in intracellular virus formed in cells infected with vA33Δ, the amount of infectious virus in the medium was increased. The virus particles in the medium had the buoyant density of extracellular enveloped viruses (EEV). Additionally, amounts of vA33Δ cell-associated extracellular enveloped viruses (CEV) were found to be normal. Immunogold electron microscopy of cells infected with vA33Δ demonstrated the presence of the expected F13L and B5R proteins in wrapping membranes and EEV; however, fully wrapped vA33Δ intracellular enveloped viruses (IEV) were rare compared to partially wrapped particles. Specialized actin tails that propel IEV particles to the periphery and virus-tipped microvilli (both common in wild-type-infected cells) were absent in cells infected with vA33Δ. This is the first deletion mutant in a VV envelope gene that produces at least normal amounts of fully infectious EEV and CEV and yet has a small-plaque phenotype. These data support a new model for VV spread, emphasizing the importance of virus-tipped actin tails.     

Role of receptor-mediated endocytosis in the formation of vaccinia virus extracellular enveloped particles

Infectious intracellular mature vaccinia virus particles are wrapped by cisternae, which may arise from trans-Golgi or early endosomal membranes, and are transported along microtubules to the plasma membrane where exocytosis occurs. We used EH21, a dominant-negative form of Eps15 that is an essential component of clathrin-coated pits, to investigate the extent and importance of endocytosis of viral envelope proteins from the cell surface. Several recombinant vaccinia viruses that inducibly or constitutively express an enhanced green fluorescent protein (GFP)-EH21 fusion protein were constructed. Expression of GFP-EH21 blocked uptake of transferrin, a marker for clathrin-mediated endocytosis, as well as association of adaptor protein-2 with clathrin-coated pits. When GFP-EH21 was expressed, there were increased amounts of viral envelope proteins, including A33, A36, B5, and F13, in the plasma membrane, and their internalization was inhibited. Wrapping of virions appeared to be qualitatively unaffected as judged by electron microscopy, a finding consistent with a primary trans-Golgi origin of the cisternae. However, GFP-EH21 expression caused a 50% reduction in released enveloped virions, decreased formation of satellite plaques, and delayed virus spread, indicating an important role for receptor-mediated endocytosis. Due to dynamic interconnection between endocytic and exocytic pathways, viral proteins recovered from the plasma membrane could be used by trans-Golgi or endosomal cisternae to form new viral envelopes. Adherence of enveloped virions to unrecycled viral proteins on the cell surface may also contribute to decreased virus release in the presence of GFP-EH21. In addition to a salvage function, the retrieval of viral proteins from the cell surface may reduce immune recognition.     

An E2–F12 complex is required for intracellular enveloped virus morphogenesis during vaccinia infection

The vaccinia virus protein, F12, has been suggested to play an important role in microtubule-based transport of intracellular enveloped virus (IEV). We found that GFP-F12 is recruited to IEV moving on microtubules but is released from virus particles when they switch to actin-based motility. In the absence of F12, although the majority of IEV remain close to their peri-nuclear site of assembly, a small number of IEV still move with linear trajectories at speeds of 0.85 μm s⁻¹, consistent with microtubule transport. Using a recombinant virus expressing GST-F12, we found that the viral protein E2 interacts directly with F12. In infected cells, GFP-E2 is observed on moving IEV as well as in the Golgi region, but is not associated with actin tails. In the absence of E2L, IEV accumulate in the peri-nuclear region and F12 is not recruited. Conversely, GFP-E2 is not observed on IEV in the absence of F12. Ultra-structural analysis of ΔE2L- and ΔF12L-infected cells reveals that loss of either protein results in defects in membrane wrapping during IEV formation. We suggest that E2 and F12 function as a complex that is necessary for IEV morphogenesis prior to their microtubule-based transport towards the plasma membrane.     

The vaccinia virus A33R protein provides a chaperone function for viral membrane localization and tyrosine phosphorylation of the A36R protein

The products of the A33R and A36R genes of vaccinia virus are incorporated into the membranes of intracellular enveloped virions (IEV). When extracts of cells that had been infected with vaccinia virus and labeled with H(3)(32)PO(4) were immunoprecipitated with antibodies against the A33R protein, two prominent bands were resolved. The moderately and more intensely labeled bands were identified as phosphorylated A33R and A36R proteins, respectively. The immunoprecipitated complex contained disulfide-bonded dimers of A33R protein that were noncovalently linked to A36R protein. Biochemical analysis indicated that the two proteins were phosphorylated predominantly on serine residues, with lesser amounts on threonines. The A36R protein was also phosphorylated on tyrosine, as determined by specific binding to an anti-phosphotyrosine antibody. Serine phosphorylation and A33R-A36R protein complex formation occurred even when virus assembly was blocked at an early stage with the drug rifampin. Tyrosine phosphorylation was selectively reduced in cells infected with F13L or A34R gene deletion mutants that were impaired in the membrane-wrapping step of IEV formation. In addition, tyrosine phosphorylation was specifically inhibited in cells infected with an A33R deletion mutant that still formed IEV. Immunofluorescence and immunoelectron microscopy indicated that in the absence of the A33R protein, the A36R protein was localized in Golgi membranes but not in IEV. In the absence of the A36R protein, however, the A33R protein still localized to IEV membranes. These studies together with others suggest that the A33R protein guides the A36R protein to the IEV membrane, where it subsequently becomes tyrosine phosphorylated as a signal for actin tail formation.     

Electron tomography of RabA4b- and PI-4Kβ1-labeled trans Golgi network compartments in Arabidopsis

The trans Golgi network (TGN) of plant cells sorts and packages Golgi products into secretory (SV) and clathrin-coated (CCV) vesicles. We have analyzed of TGN cisternae in Arabidopsis root meristem cells by cell fractionation and electron microscopy/tomography to establish reliable criteria for identifying TGN cisternae in plant cells, and to define their functional attributes. Transformation of a trans Golgi cisterna into a Golgi-associated TGN cisterna begins with cisternal peeling, the formation of SV buds outside the plane of the cisterna and a 30-35% reduction in cisternal membrane area. Free TGN compartments are defined as cisternae that have detached from the Golgi to become independent organelles. Golgi-associated and free TGN compartments, but not trans Golgi cisternae, bind anti-RabA4b and anti-phosphatidylinositol-4 kinase (PI-4K) antibodies. RabA4b and PI-4Kβ1 localize to budding SVs in the TGN and to SVs en route to the cell surface. SV and CCV release occurs simultaneously via cisternal fragmentation, which typically yields ～30 vesicles and one to four residual cisternal fragments. Early endosomal markers, VHA-a1-green fluorescent protein (GFP) and SYP61-cyan fluorescent protein (CFP), colocalized with RabA4b in TGN cisternae, suggesting that the secretory and endocytic pathways converge at the TGN. pi4k1/pi4k2 knockout mutant plants produce SVs with highly variable sizes indicating that PI-4Kβ1/2 regulates SV size.     

The trans-Golgi network can be dissected structurally and functionally from the cisternae of the Golgi complex by brefeldin A.

TGN38, a transmembrane glycoprotein predominantly localized to the trans-Golgi network, is utilized to study both the structure and function of the trans-Golgi network (TGN). The effects of brefeldin A (BFA) on the TGN were studied in comparison to its documented effects on the Golgi cisternae. During the first 30 min of BFA treatment, the TGN loses its cisternal structure and extends as tubules throughout the cytoplasm. By 60 min, it condenses into a stable structure surrounding the microtubule-organizing center. By electron microscopy, this structure appears as a population of large vesicles, and by immunolabeling, most of these vesicles contain TGN38. TGN38 cycles to the plasma membrane and back, which is shown by addition of TGN38 luminal domain antibodies directly to cell culture media. This results in rapid uptake of antibodies which label the TGN within 30 min, both in its native and BFA-induced conformation. A number of transmembrane proteins have been shown to take this cycling pathway, but TGN38 is unique in that it is the only one predominantly localized to the TGN. To investigate the cycling of TGN38, the endocytic pathway was labeled by internalization of Lucifer Yellow, and in the presence of BFA there was partial colocalization with TGN38. Further studies were carried out in which microtubules were depolymerized, resulting in dispersal of Golgi elements and inhibition of transport from endosomes to lysosomes. TGN38 cycling continues in the absence of microtubules. Taken together, these studies indicate that TGN38 returns from the plasma membrane via the endocytic pathway. We conclude that the TGN is structurally and functionally distinct from the Golgi cisternae, indicating that different molecules control membrane traffic from the Golgi cisternae and from the TGN.     

Entry of the two infectious forms of vaccinia virus at the plasma membane is signaling-dependent for the IMV but not the EEV

The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane. We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation. These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores. The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements. Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.     

Mechanism of Vaccinia Virus Release and Its Specific Inhibition by N1-Isonicotinoyl-N2-3-Methyl-4- Chlorobenzoylhydrazine

The release of vaccinia virus from RK-13 cells and its specific inhibition by N(1)-isonicotinoyl-N(2)-3-methyl-4- chlorobenzoylhydrazine (IMCBH) was studied. Intracellular naked vaccinia virus (INV) was wrapped by intracytoplasmic membranes, forming an intracellular double-membraned virion. Wrapped virions migrated to the cell surface, where the outer virion membrane presumably fused with the plasma membrane, releasing virus surrounded by the inner membrane, referred to as extracellular enveloped vaccinia virus (EEV). At no time was there any evidence that vaccinia virus acquired an envelope by budding of naked virus from the cytoplasmic membrane. Naked virus and double-membraned virus each constituted about one-third of intracellular virus at 8 and 12 h postinfection (p.i.). Beginning at 16 h p.i., the proportion of intracellular virus occurring as double-membraned virus steadily decreased to 1% at 24 h while the proportion of naked virus rose to 87%. IMCBH inhibited the formation of the double-membraned virion and the appearance of EEV while not affecting the production of INV. IMCBH had no effect on INV infectivity or polypeptide composition, on vaccinia virus-specified membrane-associated proteins or glycoproteins, or on hemadsorption. The presence of IMCBH until 4 h p.i. did not decrease the amount of EEV at 48 h p.i., whereas less than 10% of the normal 48-h EEV yield was obtained if the drug was present during the first 16 h p.i. Cell cultures infected at very low multiplicities showed a rapid virus dissemination in the absence of the drug, whereas the presence of IMCBH very effectively inhibited this spread. We conclude that vaccinia virus is liberated via a double-membraned intermediate as an enveloped virion and that it is this extracellular enveloped virus that is responsible for dissemination of infection.     

Redistribution of cellular and herpes simplex virus proteins from the trans-golgi network to cell junctions without enveloped capsids

Herpes simplex virus (HSV) and other alphaherpesviruses assemble enveloped virions in the trans-Golgi network (TGN) or endosomes. Enveloped particles are formed when capsids bud into TGN/endosomes and virus particles are subsequently ferried to the plasma membrane in TGN-derived vesicles. Little is known about the last stages of virus egress from the TGN/endosomes to cell surfaces except that the HSV directs transport of nascent virions to specific cell surface domains, i.e., epithelial cell junctions. Previously, we showed that HSV glycoprotein gE/gI accumulates extensively in the TGN at early times after infection and also when expressed without other viral proteins. At late times of infection, gE/gI and a cellular membrane protein, TGN46, were redistributed from the TGN to epithelial cell junctions. We show here that gE/gI and a second glycoprotein, gB, TGN46, and another cellular protein, carboxypeptidase D, all moved to cell junctions after infection with an HSV mutant unable to produce cytoplasmic capsids. This redistribution did not involve L particles. In contrast to TGN membrane proteins, several cellular proteins that normally adhere to the cytoplasmic face of TGN, Golgi, and endosomal membranes remained primarily dispersed throughout the cytoplasm. Therefore, cellular and viral membrane TGN proteins move to cell junctions at late times of HSV infection when the production of enveloped particles is blocked. This is consistent with the hypothesis that there are late HSV proteins that reorganize or redistribute TGN/endosomal compartments to promote virus egress and cell-to-cell spread.     

Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope.

Using a reverse genetic approach, we have demonstrated that the product of the B5R open reading frame (ORF), which has homology with members of the family of complement control proteins, is a membrane glycoprotein present in the extracellular enveloped (EEV) form of vaccinia virus but absent from the intracellular naked (INV) form. An antibody (C'-B5R) raised to a 15-amino-acid peptide from the translated B5R ORF reacted with a 42-kDa protein (gp42) found in vaccinia virus-infected cells and cesium chloride-banded EEV but not INV. Under nonreducing conditions, an 85-kDa component, possibly representing a hetero- or homodimeric form of gp42, was detected by both immunoprecipitation and Western immunoblot analysis. Metabolic labeling with [3H]glucosamine and [3H]palmitate revealed that the B5R product is glycosylated and acylated. The C-terminal transmembrane domain of the protein was identified by constructing a recombinant vaccinia virus that overexpressed a truncated, secreted form of the B5R ORF product. By N-terminal sequence analysis of this secreted protein, the site of signal peptide cleavage of gp42 was determined. A previously described monoclonal antibody (MAb 20) raised to EEV, which immunoprecipitated a protein with biochemical characteristics similar to those of wild-type gp42, reacted with the recombinant, secreted product of the B5R ORF. Immunofluorescence of wild-type vaccinia virus-infected cells by using either MAb 20 or C'-B5R revealed that the protein is expressed on the cell surface and within the cytoplasm. Immunogold labeling of EEV and INV with MAb 20 demonstrated that the protein was found exclusively on the EEV membrane.     

Identification of the vaccinia hemagglutinin polypeptide from a cell system yielding large amounts of extracellular enveloped virus.

HeLa, SIRC, and RK-13 cells were compared as to their production of intracellular naked vaccinia virus (INV) and extracellular enveloped vaccinia virus (EEV) after infection with vaccinia strains WR and IHD-J. IHD-J produced more EEV from all three cell lines than did WR, although both strains produced approximately the same quantity of INV. The most efficient EEV release was from RK-13 cells infected with IHD-J, which was 200 times more than from WR-infected SIRC cells. This permitted for the first time the purification of milligram quantities of EEV that contained much fewer cell protein contaminants than could be obtained from HeLa or SIRC cells. The INV surface proteins 200K, 95K, 65K, and 13K were present in both HeLa and RK-13 cell-derived INV but were absent in SIRC cell INV. These proteins were absent in EEV from all three cell lines. Four glycoproteins of molecular weights 210 x 10(3) (210K), 110K, 89K, and 42K and five glycoproteins in the 23K to 20K range plus a nonglycosylated protein of 37K were detected in EEV from the hemagglutinin-positive IHD-J vaccinia strain. The 89K glycoprotein was not present in EEV or membranes from cells infected with the hemagglutinin-negative vaccinia strain IHD-W. Antisera to IHD-W lacking hemagglutinin-inhibiting antibodies did not precipitate the 89K glycoprotein of IHD-J. The only glycoprotein that specifically attached to rooster erythrocytes was the 89K glycoprotein. This evidence indicates that the 89K glycoprotein is the vaccinia hemagglutinin.     

Isolation of a fraction enriched in the trans-Golgi network from baby hamster kidney cells

Incubation of cultured cells at 20 degrees C blocks the transport of newly synthesized plasma membrane proteins, and the proteins accumulate intracellularly in a terminally glycosylated form. When baby hamster kidney cells are infected with the ts O45 mutant of vesicular stomatitis virus, and incubated at 20 degrees C, the terminally glycosylated spike glycoprotein G of the virus accumulates in the membranes of a tubular network localized on the trans side of the Golgi cisternae, the trans-Golgi network (TGN). We have used the G protein of ts O45 as a marker for the TGN and isolated a TGN fraction using a combination of conventional cell fractionation techniques and immunoisolation. The TGN was separated from the bulk of the endoplasmic reticulum, mitochondria, lysosomes, plasma membrane, and endosomes, while the activity of trans-Golgi marker galactosyltransferase copurified with the G protein. Using G protein as the TGN marker we have determined that the TGN was enriched 25-fold in the final fraction relative to the total homogenate. Several polypeptides (Mr 75,000, 87,000, 92,000, and 120,000) copurified with the G protein in the isolated TGN fraction and most likely represent resident markers of the compartment.     

Golgi apparatus and TGN during endocytosis

Wheat germ agglutinin labelled with horseradish peroxidase (WGA) was used for analyses of endosomal compartments and Golgi apparatus in HepG(2) hepatoma cells during early and late periods of endocytosis. WGA was rapidly transferred into the Golgi region. Transport of internalised WGA into the Golgi apparatus could be classified in three stages. A short stage I, characterised by predominance of vesicular endosomes, was followed by stage II showing new formations of extended endocytic trans Golgi networks (TGNs); the endocytic TGNs comprised reticular and globular parts, showed intimate associations with segments of the endoplasmic reticulum and budding of multiple coated vesicles. Parts of the endocytic TGNs associated with trans Golgi cisternae and became integrated into Golgi stacks. During stage III, concomitantly with integration into the stacks, the endocytic TGNs decreased in size and stacked Golgi cisternae became prominent endocytic compartments. Our results show that endocytosis of WGA is connected with extensive membrane dynamics at the trans Golgi side: an endocytic TGN is newly formed, increases in size and is consumed again. The findings suggest that incorporation of TGN elements into Golgi stacks provides a mechanism for uptake of internalised WGA into the Golgi apparatus.     

Direct Pathway from Early/Recycling Endosomes to the Golgi Apparatus Revealed through the Study of Shiga Toxin B-fragment Transport

Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37°C, ultrastructural studies on cryosections failed to detect B-fragment–specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor–containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.