Intracellular Localization of Vaccinia Virus ... - Journal of Virology

JOURNAL OF VIROLOGY, Nov. 2000, p. 10535–10550 0022-538X/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 22

Intracellular Localization of Vaccinia Virus Extracellular Enveloped Virus Envelope Proteins Individually Expressed Using a Semliki Forest Virus Replicon† MARIÁ M. LORENZO,1 INMACULADA GALINDO,1 GARETH GRIFFITHS,2 1 AND RAFAEL BLASCO * Departamento de Mejora Gene´tica y Biotecnologıá—I.N.I.A., E-28040 Madrid, Spain,1 and European Molecular Biology Laboratory, Cell Biology Programme, 69117 Heidelberg, Germany2 Received 30 May 2000/Accepted 15 August 2000

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. Viruses belonging to the family Poxviridae are large, DNAcontaining viruses whose replication cycle takes place in the cytoplasm of infected cells (27). Vaccinia virus, a representative of the genus Orthopoxvirus and the best-studied member of the family, is the model system of choice to study the morphogenesis and transmission of the poxvirus particles. Late steps in the replication cycle of the virus involve the assembly of infectious intracellular mature virions (IMV) that remain in the cytosol and can be released mechanically by breaking the cells. Transport of virions to the cell surface involves the wrapping of IMV particles by intracellular vesicles derived from the transGolgi network (TGN) (36) to form double-membrane-bound viruses called intracellular enveloped virions (IEV) that are transported to the cell periphery, where the outer membrane fuses with the plasma membrane. Intracellular transport of virus, which can occur by the induction of actin polymerization or by another mechanism(s), is dependent on the acquisition of the envelope (4, 5). The enveloped form of the virus found in the extracellular space has one more membrane than IMV and may remain cell associated (cell-associated enveloped virus [CEV]) or may be released from the cell (extracellular enveloped virus [EEV]). Much research has been directed to the biochemical and functional characterization of the EEV envelope, since the envelope plays a crucial role in virus dissemi-

nation (29) as well as in the establishment of immunological protection (1, 2, 8, 9, 29, 42). In addition, the EEV envelope may participate in virus evasion of the immune response (41, 43). To date, six vaccinia virus proteins have been reported to be present in the EEV envelope. Four of these proteins (A33R, A34R, A56R, and B5R) are glycoproteins, with most of the protein being extracellular (10, 11, 18, 24, 31, 39). Protein A36R is a type Ib transmembrane protein with a large cytosolic domain (33), and protein F13L is a peripheral membrane protein which associates with the membrane by a palmitic acid moiety (15, 16, 37). All of the proteins except A56R (the virus hemagglutinin) have roles in virus wrapping or in the induction of actin tails (4, 5, 12, 32–34, 46, 47). Besides their function in contributing to IMV wrapping, IEV transport, and CEV or EEV infectivity, we hypothesized that at least some of the EEV envelope proteins have to interact with cellular structures to determine the cellular compartment for wrapping and to perform functions related to the transport and egress of enveloped virions. In an attempt to unravel the relevant cell biological features of these proteins, we have carried out their individual expression in cells and studied their intracellular fates in the absence of vaccinia virus infection.

* Corresponding author. Mailing address: Dpt. Mejora Gene´tica y Biotecnologıá, I.N.I.A., Ctra. La Corun ã km 7.5, E-28040 Madrid, Spain. Phone: 34-91-347 39 13. Fax: 34-91-357 22 93. E-mail: blasco @inia.es. † Dedicated to the memory of Spanish virologist Eladio Vin ˜uela.

Materials. BHK-21 (ATCC CCL10) cells were grown in BHK medium GMEM containing 5% fetal bovine serum, 3 g of tryptose phosphate broth/ml, and 0.01 M HEPES in a 5% CO2 atmosphere at 37°C. Anti-A33R and anti-A34R rabbit polyclonal antisera were provided by M. Way (European Molecular Biology Laboratory). Anti-A36R mouse monoclonal antibody was made available

MATERIALS AND METHODS

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by G. L. Smith (Oxford University). Monoclonal antibody B2D10, anti-A56R, was kindly provided by Y. Ichihashi. Rat monoclonal antibodies 19C2 (anti-B5R) and 15B6 (anti-F13L) were kindly made available by G. Hiller. Vaccinia viruses W⌬B5R, W-B5R⌬SCR1-4, and W-B5R⌬c have been described previously (17, 23) and were kindly provided by S. Isaacs (University of Pennsylvania). The plasmids for the Semliki Forest virus (SFV) expression system were provided by H. Garoff (Karolinska Institute). Plasmid construction. The coding sequence of the A33R, A36R, and A56R genes were amplified by PCR using vaccinia virus genomic DNA as the template and were inserted into plasmid pSFV1 (20). The A33R gene was amplified using the oligonucleotide primers A335⬘Bam2 (5⬘-GACATAAATAGGATCCATTA CCATGATG-3⬘) (the BamHI site is underlined) and A33R3⬘Sma (5⬘-CATTT ATTAATGTACCCGGGTAAATATTAG-3⬘) (the SmaI site is underlined). The PCR product was cut with BamHI and SmaI and inserted into plasmid pSFV-1 to generate pSVF-A33R. The A36R gene was similarly amplified using the oligonucleotide primers A36R5⬘Bam (5⬘-CGTATATTGAGGATCCAGAA ATGATGC-3⬘) (the BamHI site is underlined) and A36R3⬘Sma (5⬘-CTTCAA TTTTATAACCCGGGAACTAATC-3⬘) (the SmaI site is underlined). The PCR product was cut with BamHI and SmaI and inserted into plasmid pSFV-1 to generate pSVF-A36R. The A56R gene was amplified using the oligonucleotide primers HA5⬘Bam (5⬘-AAATCACTTTGGATCCTAATATGACACG-3⬘) (the BamHI site is underlined) and HA3⬘Sma (5⬘-TTTTACTATCCCGGGATTTA TGTAAG-3⬘) (the SmaI site is underlined). The PCR product was cut with BamHI and SmaI and inserted into plasmid pSFV-1 to generate pSVF-A56R. The A34R gene was amplified using the oligonucleotide primers A34R5⬘ (5⬘-T TGTAGGATCCTCAATGAAATCGCT-3⬘) and A34R3⬘ (5⬘-CGTACGGATC CGACTTATTATT-3⬘) (the BamHI sites are underlined). The PCR product was cut with BamHI, inserted into plasmid pGAT to generate pGAT-A34R, and subsequently subcloned into the BamHI site of pSFV1 to generate pSVF-A34R. The B5R gene was amplified using the oligonucleotide primers B5R5⬘ (5⬘-CTA TTTCTAGACCCGGGAATAAAAA-3⬘) (the XbaI and SmaI sites are underlined) and B5R3⬘ (5⬘-TTAATTATGGTACCGGATTTAT-3⬘) (the KpnI site is underlined). The PCR product was cut with XbaI and KpnI, inserted into plasmid pGEM7 to generate pGEM-B5R, and subsequently subcloned into the SmaI site of pSFV1 to generate pSVF-B5R. The F13L gene was excised from pUC19F13L (7) and subcloned into the BamHI site of pSFV1(NruI) to generate pSVFF13L. The gene encoding GFP-S65T was obtained by PCR from VV GFP-S65T genomic DNA (22) using the oligonucleotide primers GFP5⬘EcoRI (5⬘-GGGT ACCGGTAGAATTCATGAGTAAAGG-3⬘) (the EcoRI site is underlined) and GFP3⬘HindIII (5⬘-TTCTACGAATGAAGCTTGTATAGTTCATCC-3⬘) (the HindIII site is underlined). The PCR product was cut with EcoRI and HindIII and inserted into plasmid pRB21-VP1 (35) to generate pRB-GFPc. Oligonucleotide GFP3⬘HindIII was designed to eliminate the stop codon of green fluorescent protein (GFP) and introduces a HindIII site downstream, changing the C terminus of the GFP-S65T protein by introducing two extra amino acids. A fusion of GFP with the C-terminal portion of protein B5R was achieved by recloning this version of the GFP gene in plasmid pRB-VP1mB5R (A. SanzParra and R. Blasco, unpublished data), which contains the 3⬘ end of the B5R gene, generated by PCR with the oligonucleotides 5⬘-CCCGGTACCAATGCC ATCGTTAAATACCT-3⬘ (the HindIII site is underlined) and 5⬘-GGGCCATG GTTACGGTAGCAATTTATGGA-3⬘ (the NcoI site is underlined). The resulting plasmid was termed pRB-GFPmB5R. The vaccinia virus recombinants v-GFP.c and v-GFP.mB5R were obtained by recombination of plasmids pRBGFP.c and pRB-GFP.mB5R into virus vRB12 (6). The modified versions GFPc and GFP.mB5R were also expressed using the SFV system. For this purpose, the corresponding genes were amplified from plasmids pRB-GFP.c and pRBGFP.mB5R using the oligonucleotides 5⬘-TTTTTTTTTGGATCCTAAATAAA TAAGGAATT-3⬘ (the BamHI site is underlined) and 5⬘-AAAATTATTTACC CGGGCCTCCATGG-3⬘ (the SmaI site is underlined) and subcloned into pSFV1 between BamHI and SmaI restriction sites to generate pSVF-GFPc and pSFV-GFP.mB5R. SFV expression. For each construct, 3 ␮g of linearized recombinant pSFV plasmid was used as a template for in vitro transcription with SP6 RNA polymerase (Pharmacia Biotech). For in vivo packaging of recombinant RNA into SFV particles, in vitro-transcribed RNA was electroporated into BHK-21 cells together with SFV helper RNA (3, 21). After 24 h, SFV particles in the culture medium were collected and frozen rapidly to be stored as a virus stock. The titers of stocks were determined by infecting cells in coverslips with serial dilutions of the stocks followed by indirect immunofluorescence assay for the expressed proteins. Western blotting. To obtain cell extracts, monolayers were washed with phosphate-buffered saline (PBS) and incubated for 20 min on ice with lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1 ␮g of leupeptin/ml, 1 mM phenylmethylsulfonyl fluoride). The cells were scraped and recovered. Proteins were resolved by electrophoresis in sodium dodecyl sulfate– 12% polyacrylamide gels and transferred onto nitrocellulose membranes by electroblotting (17 V for 1 h and 30 min at room temperature). After transfer, the membranes were blocked in PBS buffer containing 5% nonfat dry milk and then incubated overnight at 4°C with primary antibody diluted in PBS containing 1% nonfat dry milk, 0.05% Tween 20 (1:500 for anti-A33R, 1:100 for anti-A34R, 1:100 for anti-A36R, 1:100 for anti-A56R, 1:25 for anti-B5R, and 1:50 for anti-

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FIG. 1. Distribution of vaccinia virus EEV envelope proteins. Immunofluorescence staining was performed on nonpermeabilized cells (NP) and Triton X-100-permeabilized cells (P). Note the bright central staining in permeabilized cells, which corresponds to the Golgi complex area.

F13L). After being washed extensively with PBS–0.05% Tween 20, the membranes were incubated for 1 h at room temperature with horseradish peroxidaseconjugated secondary antibody diluted 1:3,000 in PBS–0.05% Tween 20 (sheep anti-rat immunoglobulin [Ig], sheep anti-mouse Ig, or sheep anti-rabbit Ig [Amersham]). After being washed, the membranes were incubated for 1 min with a 1:1 mix of solution A (2.5 mM luminol [Sigma]–0.4 mM p-coumaric acid [Sigma]–100 mM Tris HCl [pH 8.5]) and solution B (0.018% H2O2–100 mM Tris HCl [pH 8.5]) and exposed to an autoradiographic film. Immunofluorescence microscopy. Cells grown on round coverslips were infected with vaccinia virus at a multiplicity of infection of 10 PFU per cell or with 200 ␮l of supernatants containing SFV particles. After 6.5 h of infection, the cells were incubated with 10 ␮g of cycloheximide/ml for 30 min. The cells were washed twice with PBS, fixed for 15 min at room temperature with cold 4% paraformaldehyde, and permeabilized by incubation for 15 min with PBS–0.1% Triton X-100. After incubation with PBS–glycine (0.1 M), the coverslips were incubated with primary antibody diluted in PBS–20% horse serum (1:100 for anti-A33R, 1:50 for anti-A34R, 1:100 for anti-A36R, 1:100 for anti-A56R, 1:100 for antiB5R, and 1:50 for anti-F13L), washed, and incubated with secondary antibody diluted in PBS–20% horse serum (1:200 rabbit anti-mouse Ig-tetramethyl rhodamine isocyanate [TRITC] or 1:200 swine anti-rabbit Ig-TRITC [Dako, Glostrup, Denmark]). Some preparations were also incubated with 0.02 mg of TRITC-conjugated Triticum vulgaris lectin/ml together with the secondary anti-

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body (Sigma). Both incubations were carried out at room temperature for 30 min. Finally, the coverslips were washed, mounted with FluorSave reagent (Calbiochem), and observed by fluorescence microscopy. Electron microscopy. For electron microscopy, electroporation of RNA was used in order to obtain a high percentage of transfected cells (20). BHK-21 cells grown in 83-cm2 flasks to 80% confluence were transfected by electroporation with RNA transcribed in vitro from the pSFV constructs. Parallel transfections with pSFV-GFP were carried out to monitor the transfection efficiency, which was close to 100%. After 8 h, the cells were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS for 15 min at room temperature. After being washed twice with PBS, the cells were maintained in 4% paraformaldehyde until they were sectioned. For this, the cell pellets were immersed in 2 M sucrose for 30 min and then cryosectioned by the Tokuyasu procedure. The sections were incubated with the primary antibodies followed by protein A-gold and then dried using a mixture of methyl cellulose and uranyl acetate (see reference 14 for details).

RESULTS Localization of EEV envelope proteins during vaccinia virus infection. Models for EEV formation and release imply that EEV envelope proteins must be present in the wrapping membranes, derived from the TGN. We first compared the distribution of the six known EEV envelope proteins in vaccinia virus-infected cells at late infection times (Fig. 1). The presence of the proteins at the cell surface was revealed in nonpermeabilized cells, which were compared with parallel immunofluorescences on Triton X-100-permeabilized cells. In permeabilized cells, proteins A36R, B5R, and F13L produced the typical EEV envelope pattern, with a strong juxtanuclear signal and peripheral staining of virions. However, proteins A33R, A34R, and A56R significantly deviated from this pattern. Proteins A33R and A34R were concentrated in a central area that was clearly more extended than the Golgi area and also in numerous small membrane structures. These structures were smaller and more abundant in the case of anti-A33R staining. Protein A56R was present both in the juxtanuclear area and in the plasma membrane but was not apparent in viruslike structures. Therefore, the EEV envelope proteins A36R, B5R, and F13L have the typical EEV envelope pattern, whereas proteins A33R, A34R, and A56R have a different distribution, which is partially coincident with the pattern of the former proteins. Surface labeling also revealed differences among the different proteins. Such labeling for proteins B5R and A34R revealed a punctate pattern that presumably represents CEV. In contrast, protein A56R efficiently reached the cell surface but did not produce a punctate pattern reminiscent of virus particles. Anti-A33R surface labeling produced more numerous and smaller surface dots than A34R or B5R labeling. Proteins A36R and F13L are reportedly located on the cytosolic face of the EEV envelope. A36R was not detectable in nonpermeabilized cells, consistent with most of the protein being cytosolic. In contrast, F13L was detected in some surface virions, indicating that the outer envelope of some CEV is permeabilized under these conditions. These results indicate that, although there is a coincidence of these proteins in the wrapping membranes, their distributions in infected cells overlap only partially. In addition, from these results it is not clear that proteins A36R and A56R

FIG. 2. Western blots of proteins expressed using SFV replicons. For each protein, Western blotting was carried out on cell extracts from mock-infected cells (lanes A), cells infected with vaccinia virus WR (lanes B and F) or vaccinia

virus deficient in the corresponding protein (lanes C and G), control SFV expressing GFP (lanes D and H) or SFV expressing the corresponding protein (lanes E and I). Lanes A to E correspond to extracts prepared at 7 h postinfection, and lanes F to I correspond to extracts prepared at 24 h postinfection. Protein molecular weight markers are shown at the left of each panel. The arrow at the right of each panel indicates the position of the full-size protein.

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FIG. 3. Localization of A33R. Immunofluorescence staining was carried out on BHK-21 cells fixed at 7 h postinfection. Both nonpermeabilized (NP) and Triton X-100-permeabilized (P) cells are shown. The cells were either mock infected (M), infected with vaccinia virus (V), or infected with SFV-A33R particles (S). Note the fine punctate surface staining in cells infected with SFV-A33R.

represent bona fide EEV envelope proteins. On one hand, protein A56R is present in the Golgi area but does not seem to accumulate in virionlike structures. In addition, protein A36R, which is distributed intracellularly like other EEV envelope proteins, is not detected in permeabilized-cell surface virions. These observations point to A36R being an IEV, rather than an EEV, component. Expression of EEV envelope proteins in the SFV system. We wished to study the cell biological features of the six EEV envelope proteins, in particular with reference to the intracellular targeting signals that could direct these proteins to the TGN. Thus, the genes for the six known EEV envelope proteins were cloned and expressed by using an SFV replicon. Figure 2 shows the detection of the protein products by immunoblotting with specific antibodies. In all cases, SFV-expressed proteins were similar in size to the native proteins. In the case of protein A36R, two smaller proteins were detected

which were absent in vaccinia virus-infected cells. These probably represent proteolytic products of the full-size protein. A33R protein. As noted above, the distribution of A33R in vaccinia virus-infected cells was different from that of the other EEV envelope proteins. In permeabilized cells, a strong juxtanuclear staining, significantly larger than the Golgi area (as defined by the remaining EEV proteins or by wheat germ agglutinin [WGA] staining) was prominent (Fig. 3). In addition, a fine punctate pattern was visible in nonpermeabilized cells. When expressed in the absence of vaccinia virus infection (using the SFV-A33R construct), the protein produced surface staining similar to that seen in vaccinia virus-infected cells. In contrast, the level of intracellular labeling was relatively low and clearly distinguishable from that of vaccinia virus-infected cells. We consider it likely that the A33R surface labeling during vaccinia virus infection did not reveal enveloped virions alone,

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FIG. 4. Localization of A33R expressed from SFV-A33R by immunogold staining. Note the high concentration of label on the plasma membrane (arrowheads). Details of membrane labeling of a microvilluslike projection are shown in the inset. Bars, 100 nm.

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FIG. 5. Localization of A34R. (A) Immunofluorescence staining was carried out on BHK-21 cells fixed at 7 h postinfection. Both nonpermeabilized (NP) and Triton X-100-permeabilized (P) cells are shown. The cells were either mock infected (M), infected with vaccinia virus (V), or infected with SFV-A34R particles (S). (B) Costaining with rhodamine-labeled WGA. Cells infected with vaccinia virus (V) or with SFV-A34R (S) were fixed and permeabilized and subjected to WGA and anti-A34R labeling. The same cells are shown on the left and right. Note the localization of A34R in the Golgi region in vaccinia virus-infected cells and the more extended localization in SFV-A34R-infected cells.

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FIG. 6. Localization of A36R protein. (A) Immunofluorescence staining was carried out on BHK-21 cells fixed at 7 h postinfection. Both nonpermeabilized (NP) and Triton X-100-permeabilized (P) cells are shown. The cells were either mock infected (M), infected with vaccinia virus (V), or infected with SFV-A36R particles (S). (B) Costaining with rhodamine-labeled phalloidin to reveal actin fibers. Cells infected with SFV-A36R were fixed and permeabilized and subjected to phalloidin and anti-A36R labeling. The same cells are shown on the left and right. Note the localization of A36R in stress fibers and the strong labeling at the tips of cell projections.

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FIG. 7. Immunoelectron microscopy of cells expressing A36R protein. (A) Uniform high labeling of the plasma membrane. There is also some label on the membrane of a putative endosome (E), whereas the mitochondrion (M) is unlabeled. (B) Details of plasma membrane labeling (small arrowheads), including a prominent surface projection (large arrowhead). (C) Heavy labeling for A36R in membrane structures that are in continuity (arrowhead) with the nuclear envelope (N, nucleus). Note the label in vesicles close to the nucleus and in the nuclear envelope (arrows). (D) Significant labeling (arrows) on one side of the Golgi stacks (G). The arrowheads indicate putative clathrin-coated vesicles, whose presence is an indication that the labeled aspect of the Golgi is the TGN. Bars, 100 nm.

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FIG. 7—Continued.

FIG. 8. Localization of A56R. Immunofluorescence staining was carried out on BHK-21 cells fixed at 7 h postinfection. Both nonpermeabilized (NP) and Triton X-100-permeabilized (P) cells are shown. The cells were either mock infected (M), infected with vaccinia virus (V), or infected with SFV-A56R particles (S). Note the strong labeling of the plasma membrane in both vaccinia virus- and SFV-A56R-infected cells.

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FIG. 9. Localization of B5R protein. (A) Immunofluorescence staining was carried out on BHK-21 cells fixed at 7 h postinfection. Both nonpermeabilized (NP) and Triton X-100-permeabilized (P) cells are shown. The cells were either mock infected (M), infected with vaccinia virus (V), or infected with SFV-B5R particles (S). (B) Costaining with rhodamine-labeled WGA. Cells infected with vaccinia virus (V) or SFV-B5R (S) were fixed and permeabilized and subjected to WGA and anti-B5R labeling. The same cells are shown on the right and left. Note that localization of B5R is coincident with the Golgi region in both vaccinia virus-infected cells and SFV-B5R-infected cells.

since the structures labeled were more numerous than surface virions as revealed by anti-B5R antibody. The fine punctate staining at the cell surface was also seen in A33R-expressing cells, indicating that, in the absence of vaccinia virions, the protein similarly accumulated in small, abundant cell surface locations. In an attempt to identify the A33R-containing structures at the cell surface, we carried out immunoelectron microscopy (Fig. 4). While the level of intracellular labeling was low, strong plasma membrane labeling was apparent and was concentrated in cell surface microvillus-like projections, as was clearly seen in areas where these were concentrated (Fig. 4). A34R protein. In vaccinia virus-infected cells, labeling with anti-A34R antibody revealed the characteristic pattern in which Golgi area staining, as well as a peripheral punctate staining corresponding to virions and smaller structures, was apparent (Fig. 5A). Expression of the protein in the absence of vaccinia virus infection produced a completely different immunofluorescence pattern. The protein was retained intracellularly, was enriched in the perinuclear area, and was not transported in

significant amounts to the cell surface. To ascertain whether the perinuclear staining corresponded to the Golgi area, double labeling with anti-A34R antibody and WGA was carried out (Fig. 5B). The immunofluorescence pattern showed that, while in vaccinia virus-infected cells the strong juxtanuclear staining overlapped well with the position of the Golgi complex, the perinuclear labeling pattern seen in SFV-A34R-infected cells did not coincide with the position of the Golgi complex, although they were in roughly the same area (Fig. 5B). A36R protein. The A36R protein is a type Ib transmembrane protein, with most of the protein facing the cytosol. Since the epitope recognized by the antibody is on the cytoplasmic domain, immunofluorescence labeling with anti-A36R antibody was seen only after permeabilization of the cells (Fig. 6A). In vaccinia virus-infected cells, A36R staining was similar to that of other EEV envelope proteins. In contrast, immunofluorescence in cells infected with SFV-A36R showed a complex staining pattern. Staining was noted in an area in proximity to

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FIG. 9—Continued.

the nucleus. Also, plasma membrane staining was evident and was more pronounced at the tips of cell projections. In addition, fibrillar structures, typically running from side to side in the cell, were labeled. These structures were actin filaments, as demonstrated by double labeling with fluorescently labeled phalloidin (Fig. 6B). We carried out immunoelectron microscopy on transfected cells expressing A36R (Fig. 7). In agreement with the immunofluorescence results, the protein showed plasma membrane localization, with particularly strong labeling at cell projections (Fig. 7A and B). The staining also revealed strong labeling of cytoplasmic vesicles in areas close to the nuclear membrane (Fig. 7C) and on one side of the Golgi stacks that could correspond to the TGN (Fig. 7D). A56R protein. Protein A56R, the viral hemagglutinin, is a type I glycoprotein which is heavily glycosylated. In vaccinia virus-infected cells it was present in the Golgi region as well as at the cell surface. We did not detect A56R enrichment in any defined structures that could represent virions, although the protein has been described as a component of the EEV envelope. When expressed separately, the protein was efficiently transported to the cell surface, producing immunofluorescence images similar to those of vaccinia virus-infected cells (Fig. 8). B5R protein. The B5R protein is a type I transmembrane glycoprotein with homology to complement control proteins. As mentioned above, immunofluorescence on vaccinia virusinfected cells showed B5R in a juxtanuclear area, as well as in enveloped virions. When expressed from the SFV construct, the protein was efficiently retained intracellularly in a region close to the nucleus (Fig. 9A). The area labeled with anti-B5R antibody corresponded to the Golgi region, as suggested by double-labeling experiments with WGA (Fig. 9B). We also carried out immunoelectron microscopy of cells expressing B5R (Fig. 10). In agreement with the immunofluorescence results, the protein was associated with vesicles, which were

often found in the proximity of the nucleus. Thus, except for the absence of enveloped virions, the distribution of the protein expressed by itself was indistinguishable from that seen in vaccinia virus-infected cells. F13L protein. The F13L protein is the most abundant protein in the EEV envelope. It is palmitylated and associates with the cytoplasmic side of the membrane. In permeabilized vaccinia virus-infected cells, F13L showed a pattern similar to that of the B5R protein, except for some diffuse labeling. Expression from the SFV construct resulted in a different localization pattern (Fig. 11). The protein was not visible in nonpermeabilized cells, as would be expected from the protein topology. In permeabilized cells, the protein was distributed throughout the cytoplasm, producing a diffuse staining pattern. B5R chimeric constructs. The localization of B5R in the absence of vaccinia virus infection indicates that the B5R protein contains Golgi localization signals. In order to determine the portion of the protein responsible for Golgi targeting, we expressed fusion proteins in which the GFP was fused to the 42-amino-acid C-terminal portion of B5R, encompassing the transmembrane and cytosolic portions of the protein. These chimeric genes were introduced for expression in vaccinia virus, as well as in SFV constructs. Expression in the context of vaccinia virus infection resulted in a pattern that was similar to that of normal B5R (Fig. 12), indicating that the fusion protein was normally targeted to the Golgi complex and was incorporated efficiently into wrapped virions. Interestingly, when GFPB5R was expressed independently (Fig. 12) of the SFV construct, the fusion protein also accumulated in the juxtanuclear region, demonstrating that the C-terminal 42 amino acids are sufficient for retention of the protein. Immunofluorescence in B5R-deficient virus. The abovementioned results point to B5R as a candidate to determine the Golgi complex as the retention site for the remaining EEV envelope proteins. Therefore, we tested whether the wrapping

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FIG. 10. Immunoelectron microscopy of cells expressing B5R protein. (A) Overview of labeling, with prominently labeled intracellular vesicles (arrows). There is little labeling on the plasma membrane (P), and one gold particle is seen over the nuclear envelope (N, nucleus). (B and C) Details of the vesicle labeling. Bars, 100 nm.

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FIG. 11. Localization of F13L protein. Immunofluorescence staining was carried out on BHK-21 cells fixed at 7 h postinfection. Both nonpermeabilized (NP) and Triton X-100-permeabilized (P) cells are shown. The cells were either mock infected (M), infected with vaccinia virus (V), or infected with SFV-F13L particles (S).

process was altered when B5R was absent or modified. We used F13L as a marker for the wrapping compartment. Figure 13 shows the distribution of F13L in cells infected with mutant viruses lacking either the complete B5R gene (W-B5R⫺), the cytoplasmic tail (W-B5R⌬c), or most of the luminal domain (W-B5R⌬SCR1-4). A decrease in the number of individual virions was visible in cells infected with W-B5R⫺, consistent with the role of B5R in virus envelopment (12, 46). In these cells, the F13L protein was efficiently localized to the Golgi area. Interestingly, deletion of the B5R cytoplasmic tail, where Golgi localization signals have been described, had no effect on F13L distribution (Fig. 13, W-B5R⌬c). This observation is consistent with the normal plaque phenotype of this mutant virus (23). These results did not reveal any major change in the distribution of F13L as a result of B5R mutation, suggesting that the absence of B5R protein by itself does not affect the determination of the wrapping compartment.

DISCUSSION At the beginning of this study, we anticipated that at least some of the proteins of the EEV envelope contained signals responsible for their retention in the Golgi complex. If at least one was retained, other EEV-specific proteins could be indirectly retained in the wrapping vesicles by protein-protein interactions. Indeed, such interactions between some of these proteins have been postulated (33). From this point of view, the protein(s) responsible for TGN targeting should in principle be identifiable by individual expression of the different EEV envelope proteins. The results presented here show a more complex scenario. On one hand, B5R is the only EEV envelope protein that, when expressed by itself, showed a clear-cut Golgi complex localization similar to that seen in vaccinia virus-infected cells, indicating that it contains Golgi localization signals. This conclusion is also supported by other recent studies. Thus, a number of lines of evidence point to

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FIG. 12. Localization of B5R protein chimeras. Cells infected with the indicated vaccinia viruses (WR-GFP or WR-GFPmB5R) or with the corresponding SFV replicons (SFV-GFP or SFV-GFPmB5R) are shown.

protein B5R as the major determinant of the Golgi complex as the wrapping compartment. However, the subcellular compartment for the wrapping process does not seem to be altered in B5R-deficient virus, suggesting that other proteins which show a partial Golgi localization, such as A33R, A34R (this report), and F13L (7), may also contribute to this process. In agreement with this idea, a recent report showed that retention of a significant fraction of the B5R protein in the endoplasmic reticulum does not affect the wrapping process but rather leads to a reduction in the amount of B5R incorporated into EEV (26). Several of the EEV-specific proteins have been expressed previously in transfected cells (7, 19, 38, 40). In general, our results using the SFV expression system are in good agreement with these studies. It is to be noted that protein F13L showed a somewhat different pattern when expressed transiently (19) in a stable cell line (7) or by SFV-directed expression (this report). Since membrane association of F13L depends on palmitylation, it is likely that differences in the amount and/or the period of expression may account for these different patterns. One important observation is that different EEV envelope proteins showed different distributions in vaccinia virus-infected cells (Fig. 1). From the immunofluorescence patterns on vaccinia virus-infected cells, proteins B5R, F13L, and A36R appear to be the most specific markers for the EEV envelope. In contrast, proteins A33R, A34R, and A56R showed significant deviation from the typical pattern, suggesting that they are targeted to a number of additional membrane locations in the

cell. Thus, it is likely that multiple protein-protein interactions of different efficiencies, as well as the relative concentrations of the various proteins, determine both the intracellular distributions of individual proteins and their degree of incorporation into the wrapping membranes. A56R, the viral hemagglutinin, is a heavily glycosylated protein that has been shown to be present in purified EEV preparations (28, 30). However, our results and previous reports (38, 40) show that it is efficiently transported to the plasma membrane. It is not clear whether A56R is enriched in the EEV envelope, since viruslike structures are not visible in infected cells by anti-A56R staining. Therefore, as an alternative to specific incorporation in the wrapping membranes, it is possible that, in the absence of specific retention and due to its transit through the Golgi complex, some of the protein may be passively incorporated in the EEV envelope. In fact, nonspecific incorporation of many membrane proteins, including host proteins, in the EEV envelope has recently been demonstrated (43), reinforcing the idea that enrichment, rather than the presence of a particular protein in the EEV envelope, is required to demonstrate specific retention of that protein and selective incorporation into the TGN. The A36R protein also requires special consideration. On one hand, the localization of this protein during vaccinia virus infection indicates that it is specifically targeted to the Golgi complex and incorporated in the wrapping membranes. Despite this, and in contrast to other EEV envelope proteins, we consider it likely that A36R is an IEV but not an EEV protein. We have consistently observed that a proportion of paraform-

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FIG. 13. Localization of EEV envelope proteins in vaccinia virus B5R mutants. Cells infected with the indicated vaccinia viruses were fixed and stained with antibody to F13L protein. Wild-type virus (WR) or mutant viruses lacking most of the B5R gene (W-B5R⫺), most of the extracellular domain (W-B5R⌬SCR1-4), or the cytoplasmic tail (W-B5R⌬c) were used. Note the juxtanuclear localization of F13L in all cases. Differences in the number of enveloped virions reflect differences caused by B5R mutation.

aldehyde-fixed CEVs have envelopes that have been disrupted, as demonstrated by labeling with anti-F13L antibodies (Fig. 11) or antibodies to IMV surface proteins that are inaccessible to intact CEV or EEV (44). In contrast, anti-A36R antibody did not label CEV under the same conditions, and labeling of intracellular A36R required permeabilization of the cells with detergent. These observations may suggest that A36R is exclusively in the outer membrane of IEV and is excluded from EEV. SFV expression of A36R also shows localization of the protein at cell projections and association with actin fibers. The latter observation suggests specific interactions of B5R with cytoskeletal elements, whose significance goes beyond the scope of this report. Interestingly, A36R has been identified as a key protein in the induction of actin polymerization by IEV (13, 33). Our results also complement studies of the characterization of functional domains within B5R protein. The extracellular domain, which shows similarity to complement control proteins, has been shown to be involved in retention of viruses at the plasma membrane (17, 25). Other reports indicate that the transmembrane domain is important for the function of the protein in the wrapping process (17, 23, 25). The complete B5R can be targeted to the Golgi complex in the absence of other viral proteins (reference 19 and this report). We have extended these studies to show that the C-terminal 42 amino acids of the B5R protein, spanning the transmembrane and cytosolic domains, are sufficient to confer Golgi complex targeting on a GFP fusion protein. When the chimeric GFP fusion protein was expressed from a vaccinia virus recombinant,

it was also incorporated into EEV (data not shown). Significantly, the C terminus of B5R has also been shown to mediate incorporation of chimeric human immunodeficiency virus EnvB5R proteins into the virus particle (19). A recent report has revealed a contribution of certain residues within the cytoplasmic tail to the process of intracellular transport of the protein (45). However, we present evidence here that deletion of the cytoplasmic tail of B5R does not significantly affect either the virus wrapping process or the Golgi complex targeting of other EEV envelope proteins. Taken together, these results imply that multiple proteins in the EEV envelope contribute to determine the wrapping compartment and that several of these proteins interact specifically with different cellular membrane structures. ACKNOWLEDGMENTS This work was supported by contracts CT94-0496 and CT98-0225 from the European Commission and grant PB98-0046 from Direccio ń General de Investigacio ń Cientı´fica y Tećnica, Spain. Maria Lorenzo was the recipient of a fellowship from Instituto Nacional de Investigaciones Agrarias, Spain. We thank Yasuo Ichihachi, Stuart Isaacs, Geoffrey Smith, and Michael Way for gifts of antibodies and virus recombinants and Henrik Garoff for assistance with the SFV expression system. REFERENCES 1. Appleyard, G., and C. Andrews. 1974. Neutralizing activities of antisera to poxvirus soluble antigens. J. Gen. Virol. 23:197–200. 2. Appleyard, G., A. J. Hapel, and E. A. Boulter. 1971. An antigenic difference between intracellular and extracellular rabbitpox virus. J. Gen. Virol. 13:9– 17.

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3. Berglund, P., M. Sjoberg, H. Garoff, G. J. Atkins, B. J. Sheahan, and P. Liljestrom. 1993. Semliki Forest virus expression system: production of conditionally infectious recombinant particles. Bio/Technology 11:916–920. 4. Blasco, R., and B. Moss. 1991. 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. J. Virol. 65:5910–5920. 5. Blasco, R., and B. Moss. 1992. Role of cell-associated enveloped vaccinia virus in cell-to-cell virus spread. J. Virol. 66:4170–4179. 6. Blasco, R., and B. Moss. 1995. Selection of recombinant vaccinia viruses on the basis of plaque formation. Gene 158:157–162. 7. Borrego, B., M. M. Lorenzo, and R. Blasco. 1999. Complementation of P37 (F13L gene) knock-out in vaccinia virus by a cell line expressing the gene constitutively. J. Gen. Virol. 80:425–432. 8. Boulter, E. A., and G. Appleyard. 1973. Differences between extracellular and intracellular forms of poxvirus and their implications. Prog. Med. Virol. 16:86–108. 9. Boulter, E. A., H. T. Zwartouw, D. H. J. Titmuss, and H. B. Maber. 1971. The nature of the immune state produced by inactivated vaccinia virus in rabbits. Am. J. Epidemiol. 94:612–620. 10. Duncan, S. A., and G. L. Smith. 1992. Identification and characterization of an extracellular envelope glycoprotein affecting vaccinia virus egress. J. Virol. 66:1610–1621. 11. Engelstad, M., S. T. Howard, and G. L. Smith. 1992. A constitutively expressed vaccinia gene encodes a 42-kDa glycoprotein related to complement control factors that forms part of the extracellular virus envelope. Virology 188:801–810. 12. Engelstad, M., and G. L. Smith. 1993. The vaccinia virus 42-kDa envelope protein is required for the envelopment and egress of extracellular virus and for virus virulence. Virology 194:627–637. 13. Frischknecht, F., V. Moreau, S. Rottger, S. Gonfloni, I. Reckmann, G. Superti-Furga, and M. Way. 1999. Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling. Nature 401:926–929. 14. Griffiths, G. 1993. Fine structure immunocytochemistry. Springer-Verlag, Heidelberg, Germany. 15. Grosenbach, D. W., and D. E. Hruby. 1998. Analysis of a vaccinia virus mutant expressing a nonpalmitylated form of p37, a mediator of virion envelopment. J. Virol. 72:5108–5120. 16. Grosenbach, D. W., D. O. Ulaeto, and D. E. Hruby. 1997. Palmitylation of the vaccinia virus 37-kDa major envelope antigen. Identification of a conserved acceptor motif and biological relevance. J. Biol. Chem. 272:1956–1964. 17. Herrera, E., M. del Mar Lorenzo, R. Blasco, and S. N. Isaacs. 1998. Functional analysis of vaccinia virus B5R protein: essential role in virus envelopment is independent of a large portion of the extracellular domain. J. Virol. 72:294–302. 18. Isaacs, S. N., E. J. Wolffe, L. G. Payne, and B. Moss. 1992. Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope. J. Virol. 66:7217–7224. 19. Katz, E., E. J. Wolffe, and B. Moss. 1997. The cytoplasmic and transmembrane domains of the vaccinia virus B5R protein target a chimeric human immunodeficiency virus type 1 glycoprotein to the outer envelope of nascent vaccinia virions. J. Virol. 71:3178–3187. 20. Liljestrom, P., and H. Garoff. 1991. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Bio/Technology 9:1356–1361. 21. Liljestro ¨m, P., and H. Garoff. 1994. Expression of proteins using Semliki Forest Virus vectors, p. 16.20.1–16.20.16. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman, and K. Struhl (ed.), Current protocols in molecular biology, suppl. 25. Wiley Interscience, New York, N.Y. 22. Lorenzo, M. M., and R. Blasco. 1998. PCR-based method for the introduction of mutations in genes cloned and expressed in vaccinia virus. BioTechniques. 24:308–313. 23. Lorenzo, M. M., E. Herrera, R. Blasco, and S. N. Isaacs. 1998. Functional analysis of vaccinia virus B5R protein: role of the cytoplasmic tail. Virology 252:450–457. 24. Martinez Pomares, L., R. J. Stern, and R. W. Moyer. 1993. The ps/hr gene (B5R open reading frame homolog) of rabbitpox virus controls pock color, is a component of extracellular enveloped virus, and is secreted into the medium. J. Virol. 67:5450–5462. 25. Mathew, E., C. M. Sanderson, M. Hollinshead, and G. L. Smith. 1998. The

J. VIROL.

26.

27. 28. 29. 30. 31. 32.

33. 34.

35.

36. 37. 38. 39. 40. 41. 42. 43.

44.

45. 46.

47.

extracellular domain of vaccinia virus protein B5R affects plaque phenotype, extracellular enveloped virus release, and intracellular actin tail formation. J. Virol. 72:2429–2438. Mathew, E. C., C. M. Sanderson, R. Hollinshead, M. Hollinshead, R. Grimley, and G. L. Smith. 1999. The effects of targeting the vaccinia virus B5R protein to the endoplasmic reticulum on virus morphogenesis and dissemination. Virology 265:131–146. Moss, B. 1990. Poxviridae and their replication, p. 2079–2111. In B. N. Fields and D. M. Knipe (ed.), Virology, 2nd ed. Raven Press, New York, N.Y. Payne, L. G. 1979. Identification of the vaccinia hemagglutinin polypeptide from a cell system yielding large amounts of extracellular enveloped virus. J. Virol. 31:147–155. Payne, L. G. 1980. Significance of extracellular enveloped virus in the in vitro and in vivo dissemination of vaccinia. J. Gen. Virol. 50:89–100. Payne, L. G., and E. Norrby. 1976. Presence of haemagglutinin in the envelope of extracellular vaccinia virus particles. J. Gen. Virol. 32:63–72. Roper, R. L., L. G. Payne, and B. Moss. 1996. Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene. J. Virol. 70:3753–3762. Roper, R. L., E. J. Wolffe, A. Weisberg, and B. Moss. 1998. The envelope protein encoded by the A33R gene is required for formation of actincontaining microvilli and efficient cell-to-cell spread of vaccinia virus. J. Virol. 72:4192–4204. Rottger, S., F. Frischknecht, I. Reckmann, G. L. Smith, and M. Way. 1999. Interactions between vaccinia virus IEV membrane proteins and their roles in IEV assembly and actin tail formation. J. Virol. 73:2863–2875. Sanderson, C. M., F. Frischknecht, M. Way, M. Hollinshead, and G. L. Smith. 1998. Roles of vaccinia virus EEV-specific proteins in intracellular actin tail formation and low pH-induced cell-cell fusion. J. Gen. Virol. 79:1415–1425. Sanz-Parra, A., R. Blasco, F. Sobrino, and V. Ley. 1998. Analysis of the B and T cell response in guinea pigs induced with recombinant vaccinia expressing foot-and-mouth disease virus structural proteins. Arch. Virol. 143: 389–398. Schmelz, M., B. Sodeik, M. Ericsson, E. J. Wolffe, H. Shida, G. Hiller, and G. Griffiths. 1994. Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network. J. Virol. 68:130–147. Schmutz, C., L. Rindisbacher, M. C. Galmiche, and R. Wittek. 1995. Biochemical analysis of the major vaccinia virus envelope antigen. Virology 213:19–27. Shida, H. 1986. Nucleotide sequence of the vaccinia virus hemagglutinin gene. Virology 150:451–462. Shida, H., and S. Dales. 1981. Biogenesis of vaccinia: carbohydrate of the hemagglutinin molecules. Virology 111:56–72. Shida, H., and S. Matsumoto. 1983. Analysis of the hemagglutinin glycoprotein from mutants of vaccinia virus that accumulates on the nuclear envelope. Cell 33:423–434. Smith, G. L. 1999. Vaccinia virus immune evasion. Immunol. Lett. 65:55–62. Turner, G. S., and E. J. Squires. 1971. Inactivated smallpox vaccine: immunogenicity of inactivated intracellular and extracellular vaccinia virus. J. Gen. Virol. 13:19–25. Vanderplasschen, A., E. Mathew, M. Hollinshead, R. B. Sim, and G. L. Smith. 1998. Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope. Proc. Natl. Acad. Sci. USA 95:7544–7549. Vanderplasschen, A., and G. L. Smith. 1997. A novel virus binding assay using confocal microscopy: demonstration that the intracellular and extracellular vaccinia virions bind to different cellular receptors. J. Virol. 71:4032– 4041. Ward, B. M., and B. Moss. 2000. Golgi network targeting and plasma membrane internalization signals in vaccinia virus B5R envelope protein. J. Virol. 74:3771–3780. Wolffe, E. J., S. N. Isaacs, and B. Moss. 1993. Deletion of the vaccinia virus B5R gene encoding a 42-kilodalton membrane glycoprotein inhibits extracellular virus envelope formation and dissemination. J. Virol. 67:4732–4741. (Erratum, 67:5709–5711.) Wolffe, E. J., E. Katz, A. Weisberg, and B. Moss. 1997. The A34R glycoprotein gene is required for induction of specialized actin-containing microvilli and efficient cell-to-cell transmission of vaccinia virus. J. Virol. 71: 3904–3915.