JOURNAL OF VIROLOGY, Mar. 1991, p. 1623-1627 0022-538X/91/031623-05$02.00/0 Copyright C) 1991, American Society for Microbiology
Vol. 65, No. 3
Biogenesis of Type I Cytopathic Vacuoles in Semliki Forest Virus-Infected BHK Cells JOHAN PERANEN* AND LEEVI KAARIAINEN Institute of Biotechnology, University of Helsinki, Valimotie 7, SF-00380 Helsinki, Finland Received 26 July 1990/Accepted 11 December 1990
We investigated the biogenesis of type I cytopathic vacuoles (CPVIs) in Semliki Forest virus (SFV)-infected cells by immunofluorescence and electron microscopy. By using the tsl mutant of SFV at the restrictive temperature to avoid superinfection, we showed that the multiplicity of infection affects the time of appearance but not the number of CPVIs in a cell. Formation of CPVIs did not require incoming virus particles, because they were found in BHK cells transfected with infectious RNA from the SFV prototype strain or tsl mutant. When the SFV gene for nsP3 was expressed alone in BHK cells, the nsP3 protein was localized to numerous vesiclelike structures and large vacuoles. The nsP3 protein may function as an anchoring protein for the RNA replication complex of SFV. Semliki Forest virus (SFV), an alphavirus, is an enveloped positive single-stranded RNA virus which enters cells by the endocytic pathway (13). In this process, the capsid is delivered into the cytoplasm. In an unknown manner, the SFV genome, 42S RNA, is liberated from the capsid and translated into a large polyprotein, P1234, which is processed into four nonstructural proteins (nsPl to nsP4) (11, 24). The nonstructural proteins of SFV are thought to be responsible for SFV-specific RNA replication and transcription (11). Recent studies with Sindbis virus have allowed assignment of potential functions for the individual nonstructural proteins (8, 9, 16, 19). The nsPl protein is probably both an initiation factor in minus-strand RNA synthesis and a methyltransferase, while nsP2 is a proteinase (2) and a regulator of 26S RNA synthesis (9). The nsP3 protein, a phosphoprotein, is essential for RNA synthesis but has an unknown function (9, 18). It is widely believed that nsP4 is the elongation factor in the RNA polymerase complex (8). During alphavirus infection, two unique membranous structures appear. One is type I cytopathic vacuole (CPVI), which appears early in infection (1 to 2 h postinfection [p.i.]) (1, 4, 7). The other is CPVII, which is found late in infection (7). CPVIs (0.6 to 2.0 p.m in diameter) have 50-nm invaginations, spherules, on their surfaces. They have been regarded as sites for alphavirus RNA synthesis (4, 7). Recently it has been shown that CPVIs are modified endosomes and lysosomes with replicase-specific nonstructural proteins (nsP3 and nsP4 of Sindbis virus) located on their surfaces (5). In this study, we investigated the biogenesis of CPVI formation in SFV-infected BHK cells. We used RNA and DNA transfections in combination with indirect immunofluorescence and electron microscopy to find out the role of the virus particle in the formation of CPVIs. In addition, we showed that one of the nonstructural proteins, nsP3, has an affinity for vesicle and vacuolelike structures when expressed alone in BHK cells. By using monospecific anti-nsP3 antibodies in combination with immunofluorescence microscopy, we showed recently that SFV-specific nonstructural protein nsP3 is localized to large cytoplasmic vesicles in virus-infected BHK cells (18). Crude cell fractionation showed that about 80% of *
nsP3, together with nsPl and nsP4, was localized in a mitochondrial pellet fraction (P15) which has virtually all of the virus-specific RNA polymerase activity (18). Thus, nsP3 serves as a useful marker for the sites of RNA replication, as recently shown for Sindbis virus (5). The distribution of nsP3 at different times p.i. was studied by indirect immunofluorescence (14) using tsl-infected BHK cells (Fig. 1). BHK21 cells, the SFV prototype strain, and the tsl mutant of SFV were propagated as described previously (12, 20). We used the tsl mutant because it makes the nonstructural proteins in excess (15). However, the overall staining pattern of tsl-infected cells was otherwise identical to that seen in SFV prototype-infected cells. To avoid asynchronous infection, we used a high multiplicity of infection (MOI) of 200 PFU per cell (Fig. 1B, C, D, G, and J). A further advantage of tsl is elimination of superinfection when cultures are incubated at the restrictive temperature of 39°C (12, 21). At this temperature, no viruses are produced because of a mutation in E3 that arrests the envelope proteins of tsl in the endoplasmic reticulum (23). At 1 h p.i., the anti-nsP3 antibody stained small irregular spots that seemed to be distributed near the plasma membrane (Fig. 1B). Mock-infected cells showed no specific staining (Fig. 1A). Already at 2 h p.i., some cells contained large vesiclelike structures which were randomly distributed throughout the cytoplasm (Fig. 1C). From 3 h on, large vesicles were predominant (Fig. 1D), sometimes very huge, as shown for the SFV prototype (Fig. 1E). Another interesting nsP3-specific staining pattern could be seen starting from 2 h p.i. Large patches, composed of very small dots arranged in rows, were seen at the plasma membrane (PM). In some cells, only this staining was seen (Fig. 1L). It is plausible that these patches represent spherules arranged in large groups at the PM, as has been shown by electron microscopy (5). The identity of nsP3-positive structures, seen by immunofluorescence microscopy, was investigated by acid phosphatase (a lysosomal enzyme) or horseradish peroxidase (HRP; an endosomal marker) staining. Acid phosphatase staining was done as described previously (6), except for an additional 10 min of incubation in 1% sodium sulfate in HN buffer (40 mM HEPES [N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid; pH 7.2], 80 mM NaCl). The reaction was stopped by rinsing three times with HN buffer. BHK cells infected with tsl (MOI, 200 PFU per cell) were stained
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FIG. 1. Indirect immunofluorescence, by anti-nsP3 antibody, of BHK cells infected with the SFV prototype strain or tsl mutant. The MOI was 200 PFU per cell unless otherwise indicated. Panels: A, mock-infected cell; B, 1 h p.i. with tsl; C, 2 h p.i. with tsl; D, 4 h p.i. with tsl; E, 3.5 h p.i. with the prototype strain; F, 4 h p.i. with tsl (MOI, 0.2 PFU per cell); G, 4 h p.i. with tsl; H, same as in panel G but stained for acid phosphatase; I, 6 h p.i. with tsl (MOI, 0.2 PFU per cell); J, cell transfected with 42S RNA from tsl at 7 h posttransfection and stained by antienvelope antibody; K, same as in panel J but stained by anti-nsP3 antibody; L, 4 h p.i. tsl (PM).
at 4 h p.i. for acid phosphatase and immunostained by affinity-purified anti-nsP3 antibody. Many of the nsP3stained structures were also positively stained for acid phosphatase, showing their lysosomal origin (Fig. 1G and H). Some of the nsP3-positive vesicles at 2 h p.i. contained the HRP marker, indicating that the viral protein had asso-
ciated with endosomes formed early during infection (17). These results are in agreement with previously presented electron microscopic observations, according to which CPVIs are modified endosomes and lysosomes (5). When a very low MOI of 0.2 PFU per cell was used, positively stained cells (2 to 10%) were first detected at 4 h
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VOL. 65, 1991
p.i. (Fig. 1F). In these cells, the typical vacuolar and/or PM staining was seen but it was fainter and there were fewer vacuolar structures than in cells infected with a high MOI of 200 PFU per cell. At 6 h p.i., the situation for the positive cells corresponded to that seen with the high MOI at 4 h p.i., with numerous brightly stained vacuolar structures (Fig. 11). These data show that one incoming virion is capable of inducing the formation of numerous CPVIs in the absence of virus production, meaning that the number of CPVIs formed cannot be directly dependent on the virus dose. The delayed appearance of CPVIs when a low MOI was used after infection with a low virus dose suggests strongly that RNA replication and synthesis of virus-specific proteins are required for production of CPVIs. Since virus production can be obtained by protein-free 42S RNA, we wanted to study whether CPVI structures are formed under these conditions. We used the lipofection method (3) to transfect BHK cells with 42S RNA from SFV or tsl virions (23). Monolayers of BHK cells on cover slips or 35-mm-diameter dishes (40 to 80% confluency) were rinsed three times with Opti-MEM I medium (GIBCO) supplemented with penicillin (100 IU/ml) and streptomycin (100 ,ug/liter). The transfection mixture was prepared by mixing 1 to 2 ,ug of 42S RNA in 0.5 ml of Opti-MEM I medium with 10 ,ug of cationic liposomes (1 mg/ml) (Lipofectin kit; Bethesda Research Laboratories, Inc., Gaithersburg, Md.) in 0.5 ml of Opti-MEM I medium. The cells were immediately incubated with the transfection mixture for various times. The appearance of CPVs was monitored by immunofluorescence or electron microscopy. Simultaneous localization of SFV envelope proteins (El and E2) and nsP3 was achieved by using a double-labeling method for immunofluorescence microscopy (17). To exclude the possibility of superinfection, we used 42S RNA from tsl virions for transfection studies. BHK cells transfected with tsl 42S RNA at 39°C were fixed at 7 h posttransfection; 1 to 10% of the cells were transfected. These cells showed a typical endoplasmic reticulum staining pattern with antienvelope antibodies, similar to that found in tsl-infected BHK cells in which transport of envelope proteins is arrested in the endoplasmic reticulum (23) (Fig. 1J). As expected, none of tsl 42S RNA-transfected cells exhibited PM staining for SFV envelope proteins (data not shown). When anti-nsP3 staining of the same cells was investigated, large vacuolar structures were seen (Fig. 1K). These vacuoles were also positive for acid phosphatase, indicating that they were of lysosomal origin (data not shown). We also studied the ultrastructure of CPVs in BHK cells transfected with 42S RNA of the SFV prototype strain. This was possible because of the high transfection efficiency achieved by the lipofection procedure (10 to 40% of the SFV prototype 42S RNA-transfected cells were positively stained by anti-nsP3 at 3 h posttransfection, as judged by immunofluorescence microscopy). The transfected cells were processed for electron microscopy as previously described for SFV-infected cells (21). CPVIs were rarely found at 2 h posttransfection. CPVIs at 3 h had many spherules, which consisted of typical dense, star-shaped structures with thin spokes radiating from the central mass (Fig. 2, inset). At 5 h posttransfection, the CPVIs were generally somewhat larger and had a lysosomal appearance (data not shown). At that time, they were often located close to the rough endoplasmic reticulum (data not shown). There was no difference between CPVIs from transfected cells and those from infected cells. At 3 h posttransfection, no budding viruses were
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FIG. 2. Electron microscopy of BHK cells transfected with 42S RNA from the prototype strain of SFV at 3 h posttransfection. The inset is an enlargement of a spherule. Bar, 0.3 ,um; bar in the inset, 60 nm.
detected, and even at 5 h, budding profiles were rare. The electron microscopy and double-labeling studies of SFV 42S
RNA-transfected BHK cells showed that 42S RNA alone is capable of directing CPVI formation. We have recently constructed an expression vector, pS VNS3, containing the gene for nsP3 under control of the early simian virus 40 promoter (17). This gene for nsP3 encodes an additional methionine at its N terminus, as the result of introduction of a translation start signal to the gene. BHK cells were transfected with pSVNS3 and processed for immunofluorescence microscopy on the following day. When the nsP3-positive cells were monitored, it soon became clear that nsP3 was localized to numerous vesiclelike structures similar to those found in SFV-infected cells. In some cells, these structures seemed to contact each other (Fig. 3A). In cells expressing high levels of nsP3, the vesicles were replaced by very large vacuolelike structures which were easily detected by Nomarski optics (Fig. 3C and D). Thus, it is possible that the large vacuolar structures were formed from fusing vesicles. These structures differed from those found in SFV-infected cells in that they had neither the clear outer boundary nor the empty looking inside space typical of large CPVs in infected BHK cells (Fig. 1). In contrast to infected cells, no nsP3 was seen at the plasma membrane of pSVNS3-transfected cells. BHK cells transfected with control plasmid pBR322 contained no nsP3-
positive staining (data not shown). To clarify the identity of these vesiclelike structures in cells with pSVNS3-transfected BHK cells, we stained theshowed no acid phosphatase. Acid phosphatase staining overlap with nsP3 staining in transfected cells, indicating that they were not lysosomes (Fig. 3E and F). We used HRP
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H FIG. 3. Immunofluorescence of BHK cells transfected with plasmid pSVNS3 containing cDNA encoding full-length nsP3. Panels: A, anti-nsP3 antibody; B, Nomarski image of panel A; C, anti-nsP3 antibody; D, Nomarski image of panel C; E, anti-nsP3 antibody; F, acid phosphatase staining of panel E; G, anti-nsP3 antibody; H, endosomal staining of panel G by HRP-DAB.
as an endosomal marker by incubating transfected cells with HRP (10 mg/ml) for 2 h at 20°C. The cells were then fixed (14) and stained for HRP as previously described (10). No overlap between HRP and nsP3 was detected, suggesting that these vesiclelike structures were not newly formed endosomes (Fig. 3G and H). The identity of the vesiclelike
structures in cells expressing nsP3 remains unknown. Studies intended to elucidate both what region on nsP3 is essential for targeting nsP3 to vacuolar structures and the mode of interaction of nsP3 with these structures are in progress. The data reported here permit us to draw some new
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VOL. 65, 1991
conclusions regarding the biogenesis of CPVIs in alphavirusinfected cells. We showed that the incoming virus particle is not a prerequisite for CPVI formation and that the spherules of CPVIs cannot be remnants of incoming viruses. Thus, it seems likely that the site of replication is determined by a specific targeting mechanism. The exact nature of this mechanism is unclear, but viral, and probably cellular, protein synthesis is required. By using the ts6 mutant of Sindbis virus, Sreevalsan has shown that the input viral RNA attaches to membranes under conditions in which no new viral RNAs are synthesized when primary viral translation is allowed to occur (22). This means that one or some of the alphavirus nonstructural proteins are required for targeting of genome RNA to membranes. We think that nsP3 is involved in this process, because it is found in association with vesicles and vacuolar structures reminiscent of CPVIs when expressed alone. However, other virus-specific proteins are most likely needed in combination with nsP3 for formation of spherules on endosomes and lysosomes in alphavirus-infected cells. We thank Annikki Kallio and Anssi Morttinen for excellent technical assistance and Carl-Henrik von Bonsdorff for helpful
10. 11. 12.
13.
14.
15. 16.
discussions. This work was supported by the Sigrid Juselius Foundation. 1. 2. 3.
4.
5. 6. 7.
8.
9.
REFERENCES Acheson, N. H., and I. Tamm. 1967. Replication of Semliki Forest virus: an electron microscopy study. Virology 32:128143. Ding, M., and M. J. Schlesinger. 1989. Evidence that Sindbis virus nsP2 is an autoprotease which processes the nonstructural polyprotein. Virology 171:280-284. Feigner, P. L., T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen. 1987. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84:7413-7417. Friedman, R. M., J. G. Levin, P. M. Grimley, and I. K. Berezesky. 1972. Membrane-associated replication complex in arbovirus infection. J. Virol. 10:504-515. Froshauer, S., J. Kartenbeck, and A. Helenius. 1988. Alphavirus RNA replication occurs on the cytoplasmic surface of endosomes and lysosomes. J. Cell Biol. 107:2075-2086. Griffiths, G. W. 1979. Transport of glial cell acid phosphatase by endoplasmic reticulum into damaged axons. J. Cell Sci. 36:361389. Grimley, P. M., I. K. Berezesky, and R. Friedman. 1968. Cytoplasmic structures associated with an arbovirus infection: loci of viral ribonucleic acid synthesis. J. Virol. 2:1326-1338. Hahn, Y. S., A. Grakoui, C. M. Rice, E. G. Strauss, and J. H. Strauss. 1989. Mapping of RNA- temperature-sensitive mutants of Sindbis virus: complementation group F mutants have lesions in nsP4. J. Virol. 63:1194-1202. Hahn, Y. S., E. G. Strauss, and J. H. Strauss. 1989. Mapping of RNA- temperature-sensitive mutants of Sindbis virus: assign-
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ment of complementation groups A, B, and G to nonstructural proteins. J. Virol. 63:3142-3150. Heuser, J. 1989. Changes in lysosome shape and distribution correlated with changes in cytoplasmic pH. J. Cell Biol. 108: 855-864. Kiaariainen, L., K. Takkinen, S. Keranen, and H. Soderlund. 1987. Replication of the genome of alphaviruses. J. Cell Sci. Suppl. 7:231-250. Kerainen, S., and L. Kaariainen. 1974. Isolation and basic characterization of temperature-sensitive mutants from Semliki Forest virus. Acta Pathol. Microbiol. Scand. Sect. B 82:810820. Kielian, M. C., and A. Helenius. 1986. Entry of alphaviruses, p. 91-119. In S. Schlesinger and M. J. Schlesinger (ed.), The Togaviridae and Flaviviridae. Plenum Publishing Corp., New York. Kuismanen, E., K. Hedman, J. Saraste, and R. F. Pettersson. 1982. Uukuniemi virus maturation: accumulation of virus particles and viral antigens in the Golgi complex. Mol. Cell. Biol. 2: 1444-1458. Lachmi, B., and L. Kaariainen. 1976. Sequential translation of nonstructural proteins in cells infected with a Semliki Forest virus mutant. Proc. Natl. Acad. Sci. USA 73:1936-1940. Mi, S., R. Durbin, H. V. Huang, C. M. Rice, and V. Stollar. 1989. Association of the Sindbis virus RNA methyltransferase activity with the nonstructural protein nsPl. Virology 170:385391. Peranen, J. Unpublished data. Peranen, J., K. Takkinen, N. Kalkkinen, and L. Kaariainen. 1988. Semliki Forest virus-specific nonstructural protein nsP3 is a phosphoprotein. J. Gen. Virol. 69:2165-2178. Rice, C. M., R. Lewis, J. H. Strauss, and H. V. Huang. 1987. Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J. Virol. 61:3809-3819. Saraste, J., L. Kaariainen, H. Soderlund, and S. Keranen. 1977. RNA synthesis directed by a temperature-sensitive mutant of Semliki Forest virus. J. Gen. Virol. 37:399-406. Saraste, J., C.-H. von Bonsdorff, K. Hashimoto, L. Kaariainen, and S. Keranen. 1980. Semliki Forest virus mutants with temperature-sensitive transport defect of envelope proteins. Virology 100:229-245. Sreevalsan, T. 1970. Association of viral ribonucleic acid with cellular membranes in chick embryo cells infected with Sindbis virus. J. Virol. 6:438-444. Syvaoja, P., J. Peranen, M. Suomalainen, S. Keranen, and L. Kaariainen. 1990. A single amino acid change in E3 of tsl mutant inhibits the intracellular transport of SFV envelope protein complex. Virology 174:658-666. Takkinen, K. 1986. Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus. Nucleic Acids Res. 14:5667-5681. Tuomi, K., L. Kaariainen, and H. Soderlund. 1975. Quantitation of Semliki Forest virus RNAs in infected cells using 32P equilibrium labelling. Nucleic Acids Res. 2:555-565.
Vol. 65, No. 3
Biogenesis of Type I Cytopathic Vacuoles in Semliki Forest Virus-Infected BHK Cells JOHAN PERANEN* AND LEEVI KAARIAINEN Institute of Biotechnology, University of Helsinki, Valimotie 7, SF-00380 Helsinki, Finland Received 26 July 1990/Accepted 11 December 1990
We investigated the biogenesis of type I cytopathic vacuoles (CPVIs) in Semliki Forest virus (SFV)-infected cells by immunofluorescence and electron microscopy. By using the tsl mutant of SFV at the restrictive temperature to avoid superinfection, we showed that the multiplicity of infection affects the time of appearance but not the number of CPVIs in a cell. Formation of CPVIs did not require incoming virus particles, because they were found in BHK cells transfected with infectious RNA from the SFV prototype strain or tsl mutant. When the SFV gene for nsP3 was expressed alone in BHK cells, the nsP3 protein was localized to numerous vesiclelike structures and large vacuoles. The nsP3 protein may function as an anchoring protein for the RNA replication complex of SFV. Semliki Forest virus (SFV), an alphavirus, is an enveloped positive single-stranded RNA virus which enters cells by the endocytic pathway (13). In this process, the capsid is delivered into the cytoplasm. In an unknown manner, the SFV genome, 42S RNA, is liberated from the capsid and translated into a large polyprotein, P1234, which is processed into four nonstructural proteins (nsPl to nsP4) (11, 24). The nonstructural proteins of SFV are thought to be responsible for SFV-specific RNA replication and transcription (11). Recent studies with Sindbis virus have allowed assignment of potential functions for the individual nonstructural proteins (8, 9, 16, 19). The nsPl protein is probably both an initiation factor in minus-strand RNA synthesis and a methyltransferase, while nsP2 is a proteinase (2) and a regulator of 26S RNA synthesis (9). The nsP3 protein, a phosphoprotein, is essential for RNA synthesis but has an unknown function (9, 18). It is widely believed that nsP4 is the elongation factor in the RNA polymerase complex (8). During alphavirus infection, two unique membranous structures appear. One is type I cytopathic vacuole (CPVI), which appears early in infection (1 to 2 h postinfection [p.i.]) (1, 4, 7). The other is CPVII, which is found late in infection (7). CPVIs (0.6 to 2.0 p.m in diameter) have 50-nm invaginations, spherules, on their surfaces. They have been regarded as sites for alphavirus RNA synthesis (4, 7). Recently it has been shown that CPVIs are modified endosomes and lysosomes with replicase-specific nonstructural proteins (nsP3 and nsP4 of Sindbis virus) located on their surfaces (5). In this study, we investigated the biogenesis of CPVI formation in SFV-infected BHK cells. We used RNA and DNA transfections in combination with indirect immunofluorescence and electron microscopy to find out the role of the virus particle in the formation of CPVIs. In addition, we showed that one of the nonstructural proteins, nsP3, has an affinity for vesicle and vacuolelike structures when expressed alone in BHK cells. By using monospecific anti-nsP3 antibodies in combination with immunofluorescence microscopy, we showed recently that SFV-specific nonstructural protein nsP3 is localized to large cytoplasmic vesicles in virus-infected BHK cells (18). Crude cell fractionation showed that about 80% of *
nsP3, together with nsPl and nsP4, was localized in a mitochondrial pellet fraction (P15) which has virtually all of the virus-specific RNA polymerase activity (18). Thus, nsP3 serves as a useful marker for the sites of RNA replication, as recently shown for Sindbis virus (5). The distribution of nsP3 at different times p.i. was studied by indirect immunofluorescence (14) using tsl-infected BHK cells (Fig. 1). BHK21 cells, the SFV prototype strain, and the tsl mutant of SFV were propagated as described previously (12, 20). We used the tsl mutant because it makes the nonstructural proteins in excess (15). However, the overall staining pattern of tsl-infected cells was otherwise identical to that seen in SFV prototype-infected cells. To avoid asynchronous infection, we used a high multiplicity of infection (MOI) of 200 PFU per cell (Fig. 1B, C, D, G, and J). A further advantage of tsl is elimination of superinfection when cultures are incubated at the restrictive temperature of 39°C (12, 21). At this temperature, no viruses are produced because of a mutation in E3 that arrests the envelope proteins of tsl in the endoplasmic reticulum (23). At 1 h p.i., the anti-nsP3 antibody stained small irregular spots that seemed to be distributed near the plasma membrane (Fig. 1B). Mock-infected cells showed no specific staining (Fig. 1A). Already at 2 h p.i., some cells contained large vesiclelike structures which were randomly distributed throughout the cytoplasm (Fig. 1C). From 3 h on, large vesicles were predominant (Fig. 1D), sometimes very huge, as shown for the SFV prototype (Fig. 1E). Another interesting nsP3-specific staining pattern could be seen starting from 2 h p.i. Large patches, composed of very small dots arranged in rows, were seen at the plasma membrane (PM). In some cells, only this staining was seen (Fig. 1L). It is plausible that these patches represent spherules arranged in large groups at the PM, as has been shown by electron microscopy (5). The identity of nsP3-positive structures, seen by immunofluorescence microscopy, was investigated by acid phosphatase (a lysosomal enzyme) or horseradish peroxidase (HRP; an endosomal marker) staining. Acid phosphatase staining was done as described previously (6), except for an additional 10 min of incubation in 1% sodium sulfate in HN buffer (40 mM HEPES [N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid; pH 7.2], 80 mM NaCl). The reaction was stopped by rinsing three times with HN buffer. BHK cells infected with tsl (MOI, 200 PFU per cell) were stained
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FIG. 1. Indirect immunofluorescence, by anti-nsP3 antibody, of BHK cells infected with the SFV prototype strain or tsl mutant. The MOI was 200 PFU per cell unless otherwise indicated. Panels: A, mock-infected cell; B, 1 h p.i. with tsl; C, 2 h p.i. with tsl; D, 4 h p.i. with tsl; E, 3.5 h p.i. with the prototype strain; F, 4 h p.i. with tsl (MOI, 0.2 PFU per cell); G, 4 h p.i. with tsl; H, same as in panel G but stained for acid phosphatase; I, 6 h p.i. with tsl (MOI, 0.2 PFU per cell); J, cell transfected with 42S RNA from tsl at 7 h posttransfection and stained by antienvelope antibody; K, same as in panel J but stained by anti-nsP3 antibody; L, 4 h p.i. tsl (PM).
at 4 h p.i. for acid phosphatase and immunostained by affinity-purified anti-nsP3 antibody. Many of the nsP3stained structures were also positively stained for acid phosphatase, showing their lysosomal origin (Fig. 1G and H). Some of the nsP3-positive vesicles at 2 h p.i. contained the HRP marker, indicating that the viral protein had asso-
ciated with endosomes formed early during infection (17). These results are in agreement with previously presented electron microscopic observations, according to which CPVIs are modified endosomes and lysosomes (5). When a very low MOI of 0.2 PFU per cell was used, positively stained cells (2 to 10%) were first detected at 4 h
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VOL. 65, 1991
p.i. (Fig. 1F). In these cells, the typical vacuolar and/or PM staining was seen but it was fainter and there were fewer vacuolar structures than in cells infected with a high MOI of 200 PFU per cell. At 6 h p.i., the situation for the positive cells corresponded to that seen with the high MOI at 4 h p.i., with numerous brightly stained vacuolar structures (Fig. 11). These data show that one incoming virion is capable of inducing the formation of numerous CPVIs in the absence of virus production, meaning that the number of CPVIs formed cannot be directly dependent on the virus dose. The delayed appearance of CPVIs when a low MOI was used after infection with a low virus dose suggests strongly that RNA replication and synthesis of virus-specific proteins are required for production of CPVIs. Since virus production can be obtained by protein-free 42S RNA, we wanted to study whether CPVI structures are formed under these conditions. We used the lipofection method (3) to transfect BHK cells with 42S RNA from SFV or tsl virions (23). Monolayers of BHK cells on cover slips or 35-mm-diameter dishes (40 to 80% confluency) were rinsed three times with Opti-MEM I medium (GIBCO) supplemented with penicillin (100 IU/ml) and streptomycin (100 ,ug/liter). The transfection mixture was prepared by mixing 1 to 2 ,ug of 42S RNA in 0.5 ml of Opti-MEM I medium with 10 ,ug of cationic liposomes (1 mg/ml) (Lipofectin kit; Bethesda Research Laboratories, Inc., Gaithersburg, Md.) in 0.5 ml of Opti-MEM I medium. The cells were immediately incubated with the transfection mixture for various times. The appearance of CPVs was monitored by immunofluorescence or electron microscopy. Simultaneous localization of SFV envelope proteins (El and E2) and nsP3 was achieved by using a double-labeling method for immunofluorescence microscopy (17). To exclude the possibility of superinfection, we used 42S RNA from tsl virions for transfection studies. BHK cells transfected with tsl 42S RNA at 39°C were fixed at 7 h posttransfection; 1 to 10% of the cells were transfected. These cells showed a typical endoplasmic reticulum staining pattern with antienvelope antibodies, similar to that found in tsl-infected BHK cells in which transport of envelope proteins is arrested in the endoplasmic reticulum (23) (Fig. 1J). As expected, none of tsl 42S RNA-transfected cells exhibited PM staining for SFV envelope proteins (data not shown). When anti-nsP3 staining of the same cells was investigated, large vacuolar structures were seen (Fig. 1K). These vacuoles were also positive for acid phosphatase, indicating that they were of lysosomal origin (data not shown). We also studied the ultrastructure of CPVs in BHK cells transfected with 42S RNA of the SFV prototype strain. This was possible because of the high transfection efficiency achieved by the lipofection procedure (10 to 40% of the SFV prototype 42S RNA-transfected cells were positively stained by anti-nsP3 at 3 h posttransfection, as judged by immunofluorescence microscopy). The transfected cells were processed for electron microscopy as previously described for SFV-infected cells (21). CPVIs were rarely found at 2 h posttransfection. CPVIs at 3 h had many spherules, which consisted of typical dense, star-shaped structures with thin spokes radiating from the central mass (Fig. 2, inset). At 5 h posttransfection, the CPVIs were generally somewhat larger and had a lysosomal appearance (data not shown). At that time, they were often located close to the rough endoplasmic reticulum (data not shown). There was no difference between CPVIs from transfected cells and those from infected cells. At 3 h posttransfection, no budding viruses were
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FIG. 2. Electron microscopy of BHK cells transfected with 42S RNA from the prototype strain of SFV at 3 h posttransfection. The inset is an enlargement of a spherule. Bar, 0.3 ,um; bar in the inset, 60 nm.
detected, and even at 5 h, budding profiles were rare. The electron microscopy and double-labeling studies of SFV 42S
RNA-transfected BHK cells showed that 42S RNA alone is capable of directing CPVI formation. We have recently constructed an expression vector, pS VNS3, containing the gene for nsP3 under control of the early simian virus 40 promoter (17). This gene for nsP3 encodes an additional methionine at its N terminus, as the result of introduction of a translation start signal to the gene. BHK cells were transfected with pSVNS3 and processed for immunofluorescence microscopy on the following day. When the nsP3-positive cells were monitored, it soon became clear that nsP3 was localized to numerous vesiclelike structures similar to those found in SFV-infected cells. In some cells, these structures seemed to contact each other (Fig. 3A). In cells expressing high levels of nsP3, the vesicles were replaced by very large vacuolelike structures which were easily detected by Nomarski optics (Fig. 3C and D). Thus, it is possible that the large vacuolar structures were formed from fusing vesicles. These structures differed from those found in SFV-infected cells in that they had neither the clear outer boundary nor the empty looking inside space typical of large CPVs in infected BHK cells (Fig. 1). In contrast to infected cells, no nsP3 was seen at the plasma membrane of pSVNS3-transfected cells. BHK cells transfected with control plasmid pBR322 contained no nsP3-
positive staining (data not shown). To clarify the identity of these vesiclelike structures in cells with pSVNS3-transfected BHK cells, we stained theshowed no acid phosphatase. Acid phosphatase staining overlap with nsP3 staining in transfected cells, indicating that they were not lysosomes (Fig. 3E and F). We used HRP
1626
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H FIG. 3. Immunofluorescence of BHK cells transfected with plasmid pSVNS3 containing cDNA encoding full-length nsP3. Panels: A, anti-nsP3 antibody; B, Nomarski image of panel A; C, anti-nsP3 antibody; D, Nomarski image of panel C; E, anti-nsP3 antibody; F, acid phosphatase staining of panel E; G, anti-nsP3 antibody; H, endosomal staining of panel G by HRP-DAB.
as an endosomal marker by incubating transfected cells with HRP (10 mg/ml) for 2 h at 20°C. The cells were then fixed (14) and stained for HRP as previously described (10). No overlap between HRP and nsP3 was detected, suggesting that these vesiclelike structures were not newly formed endosomes (Fig. 3G and H). The identity of the vesiclelike
structures in cells expressing nsP3 remains unknown. Studies intended to elucidate both what region on nsP3 is essential for targeting nsP3 to vacuolar structures and the mode of interaction of nsP3 with these structures are in progress. The data reported here permit us to draw some new
NOTES
VOL. 65, 1991
conclusions regarding the biogenesis of CPVIs in alphavirusinfected cells. We showed that the incoming virus particle is not a prerequisite for CPVI formation and that the spherules of CPVIs cannot be remnants of incoming viruses. Thus, it seems likely that the site of replication is determined by a specific targeting mechanism. The exact nature of this mechanism is unclear, but viral, and probably cellular, protein synthesis is required. By using the ts6 mutant of Sindbis virus, Sreevalsan has shown that the input viral RNA attaches to membranes under conditions in which no new viral RNAs are synthesized when primary viral translation is allowed to occur (22). This means that one or some of the alphavirus nonstructural proteins are required for targeting of genome RNA to membranes. We think that nsP3 is involved in this process, because it is found in association with vesicles and vacuolar structures reminiscent of CPVIs when expressed alone. However, other virus-specific proteins are most likely needed in combination with nsP3 for formation of spherules on endosomes and lysosomes in alphavirus-infected cells. We thank Annikki Kallio and Anssi Morttinen for excellent technical assistance and Carl-Henrik von Bonsdorff for helpful
10. 11. 12.
13.
14.
15. 16.
discussions. This work was supported by the Sigrid Juselius Foundation. 1. 2. 3.
4.
5. 6. 7.
8.
9.
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