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Cys87of simian virus 40 Vp1 are essential in formation of infectious virions
Virus Research 107 (2005) 21–25
Cys254 and Cys49/Cys87of simian virus 40 Vp1 are essential in formation of infectious virions Editte Gharakhanian∗ , Wafa Mana, Manith Norng Department of Biological Sciences, California State University, 1250 Bellflower Blvd., Long Beach, CA 90840-3702, USA Received 30 December 2003; received in revised form 16 June 2004; accepted 16 June 2004 Available online 5 August 2004
Abstract The SV40 capsid is composed of pentameric capsomeres of Vp1. We have previously shown that disulfide linkages at Vp1 Cys9, Cys104, and Cys207 are essential in formation of infectious virions. Here, the role of the remaining four cysteines was explored. Single, double, and quadruple cys → ser mutant genomes at Vp1 Cys49, Cys87, Cys254, and Cys267 codons were generated and transfected into CV-1 cells. The quadruple mutant Vp1 continued to localize to the nucleus and to bind DNA, but resulted in no plaques. SV40Vp1.Cys254 was the only single mutant with complete defect in plaque formation. The double mutant at Vp1.Cys49.Cys87 showed complete defect in plaque formation, while single mutants at the two residues resulted in plaques, suggesting a cumulative effect. All mutants defective in plaque formation continued to localize viral proteins in the nucleus. Taken together, our results suggest that Cys254 and the Cys49/Cys87 combination are essential in late stages of infectious virion formation. Published by Elsevier B.V. Keywords: SV40; Vp1; Cys49/Cys87; Cys254
The capsomeric unit of the polyomavirus simian virus 40 (SV40) is a pentamer of the major structural protein Vp1. Seventy two pentameric capsomeres assemble into the icosahedral capsid around an SV40 mini-chromosome; the minor structural proteins Vp2 and Vp3 form a bridge between the Vp1 capsomeres and the viral genome (Rayment et al., 1982; Baker et al., 1988; Liddington et al., 1991; Stehle et al., 1996). In permissive simian cells, the viral structural proteins Vp1, Vp2, and Vp3 are synthesized in the cytoplasm late during the infection and are transported to the nucleus for assembly (Tooze, 1980). SV40 has been identified as an attractive potential vector for highefficiency gene transfer into various human cells, including hematopoietic cells of the bone marrow (Sandalon et al., 1997; Sandalon and Oppenheim, 1997; Rund et al., 1998; Dalyot-Herman et al., 1999; Goldstein et al., 2002). SV40 assembly and dissociation studies have implicated both ∗
Corresponding author. Tel.: +1 562 985 4803; fax: +1 562 985 8878. E-mail address: [email protected] (E. Gharakhanian).
0168-1702/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.virusres.2004.06.006
permanent intermolecular disulfide bonds between Vp1 molecules and transitory intramolecular and intermolecular disulfide bonds in stable capsid formation (Brady et al., 1977, 1978, 1979; Ng and Bina, 1981; Kosukegawa et al., 1996; Li et al., 2002). Vp1 contains seven cysteines, any or all of which may be involved in disulfide bridges. Mercury-labelling experiments suggested that Vp1 cysteines at positions 9, 104, and 207 may be involved in disulfide linkage (Liddington et al., 1991). In consequent structural studies of assembled virions, interpentamer disulfides between Cys104 residues were detected (Stehle et al., 1996). Studies of SV40 virion-like particles (VLP’s) in insect cells have implicated interpentamer disulfides between Vp1 Cys9 residues (Ishizu et al., 2001). We have reported that Vp1 Cys9, Cys104, and Cys207 are essential for formation of disulfide-linked post-pentameric complexes in vitro (Jao et al., 1999), as well as for formation of infectious virions in vivo (Gharakhanian et al., 2001). In our in vitro studies, mutant Vp1 with cys → ser changes at all the remaining four cysteines—Cys49, Cys87, Cys254, and Cys267—continued
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to form disulfide-linked post-pentameric complexes analogous to wild-type Vp1 (Jao et al., 1999). Here, we report on the individual and cooperative roles of those four cysteines in the formation of infectious virions during the natural course of infection. While the quadruple mutant Vp1 was completely defective in plaque formation, it continued to localize to the nucleus and bind SV40 DNA. Cys254 of Vp1 was the only one of the four cysteines singularly essential for plaque formation. The two double mutants at Vp1 Cys49.Cys87 and Cys254.Cys267 were also defective in plaque formation, with the former showing cumulative effects at work at Cys49 and Cys87. All mutants defective in plaque formation continued to localize T-Ag and mutant Vp1 to the nucleus, but showed no cytopathic effect (CPE). The mammalian expression vector pSV40 contains the wild-type SV40 genome and has been described before (Clever and Kasamatsu, 1993). Vp1 sequences containing site-directed cys → ser codon changes from various pSp6Vp1 mutant plasmids described in Jao et al. (1999) were subcloned into pSV40 to generate a quadruple mutant plasmid at Cys49, Cys87, Cys254, and Cys267 (referred to as pSV40Vp1.4X), two double mutants pSV40Vp1.C49S.C87S and pSV40Vp1.C254S.C267S,
and four single mutants pSV40Vp1.C49S, pSV40Vp1.C87S, pSV40Vp1.C254S, and pSV40Vp1.C267S. Mutant plasmids were confirmed by DNA sequencing (DNA Sequencing Facility, California State U., Northridge). Mutant and wild-type SV40 genomes were excised from pSV40 and were used in transient lipid-mediated DNA transfections (Lipofectamine, Gibco/BRL) of simian CV-1 cells (ATCC). Mock transfections included all reagents and manipulations minus input DNA. Transfected cells were tracked for CPE and plaque formation by light microscopy and plaque assays, respectively (Fig. 1a and b). CPE was detected by day 6 post-transfection (p.t.) in cells transfected with wild-type SV40, SV40Vp1.C49S, SV40Vp1.C87S, and SV40Vp1.C267S; onset and patterns of CPE were identical to wild-type and all resulted in plaques. While the titers of SV40Vp1.C87S and SV40Vp1.C267S were only slightly lower than wild-type, SV40Vp1.C49S consistently resulted in smaller plaques and a titer that was lower than wild-type by one to two orders of magnitude. Cells transfected with SV40Vp1.4x, SV40Vp1.C49S.C87S, SV40Vp1.C254S.C267S, or the SV40Vp1.C254S single mutant showed no CPE when monitored up to 20 days p.t. and resulted in no plaques. Lysates of cells transfected with plaque formation-defective mutants were used for infections and
Fig. 1. (a) Summary of transfection and infection phenotypes for SV40 Vp1 mutants. Data reflect separate experiments. (b) Plaque assays following transfection and infection. CV-1 cells were transfected with linear SV40 DNA (row A) or infected with plaque-purified virions (row B) as indicated and were stained with crystal violet 14 days after transfection or 12 days after infection.
E. Gharakhanian et al. / Virus Research 107 (2005) 21–25
resulted in no plaques (data not shown). Plaques generated following transfections were picked at 14 days p.t. and were subjected to plaque purification. Virions were isolated by a novel small-scale virus mini-preparation we have described elsewhere (Orlando et al., 2000; Gharakhanian et al., 2001) and were used in subsequent infections. In order to further map the block in infectious virion formation seen with SV40Vp1.4X, SV40Vp1C49S.C87S, SV40Vp1.C254S.C267S, and SV40Vp1.C254S, earlier events of the permissive infection were studied. For immunofluorescent microscopy studies, CV-1 cells were seeded onto glass coverslips and transfected with wild-type or mutant SV40 genomes defective in plaque formation, or mock-transfected, as described above. Transfected cells were harvested 48 h p.t. and were subjected to indirect doublelabelled immunofluorescent microscopy studies using MAbs against large T-Ag (CalBiochem) and polyclonal antibodies against GST-Vp1 (kind gift of Ariella Oppenheim, Hebrew University-Hadassah Medical School, Jerusalem, Israel). Each mutant transfection showed wild-type patterns of expression and nuclear localization of T-Ag and Vp1 (Fig. 2). Anti-T-Ag staining was used to assess possible variations in transfection efficiencies and frequencies of nuclear localization of Vp1 among different mutants. Both wild-type and mutant plasmids led to transfection efficiencies of 10–14% as defined by T-Ag staining. In all transfections, 30–40% of positively transfected cells showed nuclear Vp1. Next, the DNA-binding ability of mutant Vp1 was tested. A recent study of deletion and site-directed mutants at the N-terminus of Vp1 has mapped the DNA-binding domain to the first 21 amino acids of the protein and has also suggested the possible involvement of residues at the base of the pentamer in DNA binding (Li et al., 2001). Since Cys49 and Cys254 are located at the base of each monomer (Liddington et al., 1991), DNA binding of the quadruple mutant was tested via an in vitro DNA-binding assay we have described previously (Gharakhanian et al., 2001). Briefly, 35 S-labelled wildtype and 4x mutant Vp1 were expressed in rabbit reticulocyte lysates from Sp6Vp1 or Sp6Vp1.4x plasmids described in Jao et al. (1999), and their binding to immobilized SV40 DNA was measured (Fig. 3). Vp1.4X showed DNA binding indistinguishable from wild-type Vp1. Since Vp1.4X showed no defect, the double or single mutants of Vp1 were not tested. With nuclear localization and DNA-binding functions unaffected in the non-infectious mutants, attempts were made to assess the packaging step of the infection. Packaging of viral DNA was assessed following transfections with wild-type or mutant SV40. Cells were harvested at 72 h p.t. and packaged DNA was isolated as described previously (Orlando et al., 2000; Gharakhanian et al., 2003). Briefly, cellular debris was removed by centrifugation and supernatants were treated with excess DNase to remove all non-packaged DNA, followed by proteinase K, DTT, and organic extraction to remove capsid proteins. Two-fifths of each viral DNA yield was linearized with BamHI, electrophoresed, and subjected to chemiluminescent Southern blotting using SV40 DNA as
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Fig. 2. SV40Vp1.4X, SV40Vp1.C49S.C87S, SV40Vp1.C254S.C267S, and SV40Vp1.C254S lead to expression and nuclear localization of T-Ag and Vp1. CV-1 cells were grown on coverslips and were transfected with linear wild-type or mutant SV40 DNA as indicated. Cells were harvested 48 h post-transfection by methanol fixation and were subjected to doublelabelled indirect immunofluorescent microscopy using mouse anti-Tag (␣TAg) and rabbit anti-GST-Vp1 (␣GST-Vp1) followed by TITC-conjugated rabbit anti-mouse and FITC-conjugated goat anti-rabbit secondary antibodies (SIGMA). Cells were mounted on slides and observed on an Olympus microscope using a 60× objective.
probe (AlkPhos Direct labeling kit, Amersham Pharmacia). The results were inconclusive, since in repeated experiments, extent of packaging was variable in each of the non-infectious mutants (data not presented). In this study, we have generated SV40 single, double, and quadruple mutants with conservative cys → ser codon changes at Vp1 Cys49, Cys87, Cys254, and Cys267 and have studied them for expression and nuclear localization of early and late SV40 gene products, Vp1.4x DNA binding, onset of CPE, and production of infectious virions in permissive CV-1 cells. In our in vitro studies, the quadruple mutant Vp1 continued to form disulfide-linked post-pentameric complexes (Jao et al., 1999). In the current in vivo study, the SV40 genome
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Fig. 3. Vp1.4x does not exhibit DNA-binding defects. [35 S] methioninelabelled products of wild-type SV40- and SV40Vp1.4X-coupled transcription/translation were incubated with SV40 DNA immobilized on PVDF membranes. DNA binding is expressed as the percentage of input cpm retained on the membrane. Values represent mean of two independent experiments.
encoding the same quadruple mutant Vp1 failed to result in CPE or plaques, but continued to correctly localize SV40 proteins to the nucleus and to bind SV40 DNA. Thus, none of Vp1 Cys49, Cys87, Cys254, and Cys267 residues have a singular or cumulative role in nuclear localization of Vp1 or T-Ag, DNA binding, or pentameric and post-pentameric Vp1 multimerization. When each of the four cysteines were studied individually, only Vp1 Cys254 was singularly essential for eliciting CPE and infectious virion formation. Vp1 Cys254 is located on an internal short loop that connects strands G2 and H (G2H loop) and is in close proximity to the calcium-binding sites of Vp1; most importantly, it is the only one of the four cysteines which is conserved across the polyomavirus family (Gharakhanian, 1988; Liddington et al., 1991). A study of single mutants at all seven Vp1 cysteines in a genetically engineered, non-overlapping SV40 genome has shown drastic reduction in infectivity only with mutants at Cys254 (Li et al., 2000). No disulfides have been detected at Cys254 in virion structural studies (Liddington et al., 1991; Stehle et al., 1996), and a mutant Vp1C254S continues to form disulfide-linked post-pentameric complexes when expressed in rabbit reticulocyte lysates (Jao et al., 1999). A cys → ser mutant SV40 Vp1 at Cys254 has been expressed in a baculovirus system and forms protease-resistant VLP’s analogous to wild-type, leading the authors to also conclude that Cys254 is not involved in disulfide-linked viral assembly (Ishizu et al., 2001). Both our SV40 genome studies and the studies of Li et al. (2000) using a genetically engineered SV40 genome suggest a critical role for a cysteine residue at Vp1 aa254 in the life cycle of SV40. While single mutants SV40Vp1.C49S and SV40Vp1. C87S continued to form infectious virions, the double mutant SV40Vp1.C49S.C87S was completely defective in plaque formation or CPE, suggesting a cumulative effect when both cysteines are mutated. While no disulfides at these two residues have been detected in structural studies of assem-
bled virions (Liddington et al., 1991; Stehle et al., 1996), the two could be involved in transient intramolecular disulfides that have been recently reported as early intermediates of assembly (Li et al., 2002). Cys49 and Cys87 lie in sufficiently close proximity to each other to form a disulfide bridge within a single Vp1 monomer (Liddington et al., 1991). The distinct infectivity difference between the two single mutants and the double mutant suggests that each can bond with another unidentified Vp1 cysteine when its original partner is mutated, but not when both are mutated. Our inconsistent DNA packaging results suggest that these mutants are lacking a predictable, consistent packaging pathway, such that random interactions only sometimes lead to packaged DNA. Transient disulfides may be significant in promoting correct folding and facilitating progression of a predictable assembly pathway. We have previously shown that in rabbit reticulocyte lysates, Vp1.4X forms 7S pentameric complexes in 1 h post-translation and proceeds to form 12S multi-pentameric complexes in 3 h post-translation as assessed by sucrose gradient sedimentation profiles; this progression of Vp1 multimerization is indistinguishable from wild type Vp1 (Jao et al., 1999). The fact that even the 4X mutant continues to bind DNA and to form post-pentameric Vp1 complexes suggests that the Cys49/ Cys87 combination may be essential in maintaining a consistent assembly pathway for later stages of DNA packaging. Future biochemical and electron microscopic analyses of SV40Vp1.C49S.C87S and SV40Vp1.C254S and SV40Vp1.4x can further shed light on the state and structure of these non-infectious mutant complexes. Acknowledgements We thank Dr. Ariella Oppenheim for kind gifts of antiGST-Vp1 antibodies. This work was made possible by a grant from the National Science Foundation-Research for Undergraduate Institutions (MCB-9630904) to E.G. W. Mana and M. Norng were partially supported by Howard Hughes Foundation and NSF-RUI, respectively. References Baker, T.S., Drake, J., Bina, M., 1988. Reconstruction of the three dimensional structure of simian virus 40 and visualization of the chromatic core. Proc. Natl. Acad. Sci. USA 85, 422–426. Brady, J.N., Winston, V.D., Consigli, R.A., 1977. Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions. J. Virol. 23, 717–724. Brady, J.N., Winston, V.D., Consigli, R.A., 1978. Characterization of a DNA–protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N -tetraacetic acid and dithiothreitol. J. Virol. 27, 193–204. Brady, J.N., Kendall, J.D., Consigli, R.A., 1979. In vitro reassembly of infectious polyoma virions. J. Virol. 32, 640–647. Clever, J., Kasamatsu, H., 1993. Identification of a DNA-binding domain in simian virus 40 capsid proteins Vp2 and Vp3. J. Biol. Chem. 268, 20877–20883.
E. Gharakhanian et al. / Virus Research 107 (2005) 21–25 Dalyot-Herman, N., Rund, D., Oppenheim, A., 1999. Expression of betaglobulin in primary erythroid progenitors of beta-thalassemia patients using an SV40-based gene delivery system. J. Hematother. Stem Cell Res. 8, 573–574. Gharakhanian, E., 1988. Studies on the intermolecular interactions and nuclear transport of SV40 structural proteins. Doctoral Dissertation, UCLA. Gharakhanian, E., Fasching, C.L., Orlando, S.J., Perez, A.R., 2001. Cys(9), Cys(104) and Cys(207) of simian virus 40 Vp1 are essential for infectious virion formation in CV-1 cells. J. Gen. Virol. 82, 1935–1939. Gharakhanian, E., Munoz, L., Mayorca, L., 2003. The simian virus 40 minor structural protein Vp3, but not Vp2, is essential for infectious virion formation. J. Gen. Virol. 84, 2111–2116. Goldstein, H., Pettoello-Mantovani, M., Anderson, C.M., Cordelier, P., Pomerantz, R.J., Strayer, D.S., 2002. Gene therapy using a simian virus 40-derived vector inhibits the development of in vivo human immunodeficiency virus type 1 infection of severe combined immunodeficiency mice implanted with human fetal thymic and liver tissue. J. Infect. Dis. 185, 1425–1430. Ishizu, K.I., Watanabe, H., Han, S.I., Kanesahi, S.N., Hoque, M., Yajima, H., Kataoka, K., Handa, H., 2001. Roles of disulfide linkage and calcium ion-mediated interactions in assembly and disassembly of virus-like particles composed of simian virus 40 VP1 capsid protein. J. Virol. 75, 61–72. Jao, C.C., Weidman, M.K., Perez, A.R., Gharakhanian, E., 1999. Cys9, Cys104 and Cys207 of simian virus 40 Vp1 are essential for interpentamer disulfide-linkage and stabilization in cell-free lysates. J. Gen. Virol. 80, 2481–2489. Kosukegawa, A., Arisaka, F., Takayama, M., Yajima, H., Kaidow, A., Handa, H., 1996. Purification and characterization of virus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells. Biochim. Biophys. Acta 1290, 37–45. Liddington, R.C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T.L., Harrison, S.C., 1991. Structure of simian virus 40 at 3.8-A resolution. Nature 354, 278–284.
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Li, P.P., Nakanishi, A., Tran, M.A., Salazar, A.M., Liddington, R.C., Kasamatsu, H., 2000. Role of simian virus 40 Vp1 cysteines in virion infectivity. J. Virol. 74, 11388–11393. Li, P.P., Nakanishi, A., Shum, D., Sun, P.C., Salazar, A.M., Fernandez, C.F., Chan, S.W., Kasamatsu, H., 2001. Simian virus 40 Vp1 DNAbinding domain is functionally separable from the overlapping nuclear localization signal and is required for effective virion formation and full viability. J. Virol. 75, 7321–7329. Li, P.P., Nakanishi, A., Clark, S.W., Kasamatsu, H., 2002. Formation of transitory intrachain and interchain disulfide bonds accompanies the folding and oligomerization of simian virus 40 Vp1 in the cytoplasm. Proc. Natl. Acad. Sci. USA 99, 1353–1358. Ng, S.C., Bina, M., 1981. Disulfide bonds protect the encapsidated chromosomes of simian virus 40. FEBS Lett. 130, 47– 49. Orlando, J.S., Nabavi, M., Gharakhanian, E., 2000. Rapid small-scale isolation of SV40 virions and SV40 DNA. J. Virol. Methods 90, 109–114. Rayment, I., Baker, T.S., Caspar, D.L.D., 1982. Polyoma virus capsid structure at 22.5A resolution. Nature 295, 110–115. Rund, D., Dagan, M., Daylot-Herman, N., Kimchi-Sarfaty, C., Schoenlein, P.V., Gottesman, M.N., Oppenheim, A., 1998. Efficient transduction of human hematopoietic cells with the human multidrug resistance gene 1 via SV40 pseudovirions. Hum. Gene Ther. 20, 207– 609. Sandalon, Z., Dalyot-Herman, N., Oppenheim, A.B., Oppenheim, A., 1997. In vitro self assembly of SV40 pseudovirions: vector development for gene therapy. Hum. Gene Ther. 8, 843–849. Sandalon, Z., Oppenheim, A., 1997. Self-assembly and protein–protein interactions between the SV40 capsid proteins produced in insect cells. Virology 237, 414–421. Stehle, T., Gamblin, S.J., Yan, Y., Harrison, S.C., 1996. The structure of simian virus 40 refined at 3.1A resolution. Structure 4, 165– 182. Tooze, J. (Ed.), 1980. DNA Tumor Viruses, second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Cys254 and Cys49/Cys87of simian virus 40 Vp1 are essential in formation of infectious virions Editte Gharakhanian∗ , Wafa Mana, Manith Norng Department of Biological Sciences, California State University, 1250 Bellflower Blvd., Long Beach, CA 90840-3702, USA Received 30 December 2003; received in revised form 16 June 2004; accepted 16 June 2004 Available online 5 August 2004
Abstract The SV40 capsid is composed of pentameric capsomeres of Vp1. We have previously shown that disulfide linkages at Vp1 Cys9, Cys104, and Cys207 are essential in formation of infectious virions. Here, the role of the remaining four cysteines was explored. Single, double, and quadruple cys → ser mutant genomes at Vp1 Cys49, Cys87, Cys254, and Cys267 codons were generated and transfected into CV-1 cells. The quadruple mutant Vp1 continued to localize to the nucleus and to bind DNA, but resulted in no plaques. SV40Vp1.Cys254 was the only single mutant with complete defect in plaque formation. The double mutant at Vp1.Cys49.Cys87 showed complete defect in plaque formation, while single mutants at the two residues resulted in plaques, suggesting a cumulative effect. All mutants defective in plaque formation continued to localize viral proteins in the nucleus. Taken together, our results suggest that Cys254 and the Cys49/Cys87 combination are essential in late stages of infectious virion formation. Published by Elsevier B.V. Keywords: SV40; Vp1; Cys49/Cys87; Cys254
The capsomeric unit of the polyomavirus simian virus 40 (SV40) is a pentamer of the major structural protein Vp1. Seventy two pentameric capsomeres assemble into the icosahedral capsid around an SV40 mini-chromosome; the minor structural proteins Vp2 and Vp3 form a bridge between the Vp1 capsomeres and the viral genome (Rayment et al., 1982; Baker et al., 1988; Liddington et al., 1991; Stehle et al., 1996). In permissive simian cells, the viral structural proteins Vp1, Vp2, and Vp3 are synthesized in the cytoplasm late during the infection and are transported to the nucleus for assembly (Tooze, 1980). SV40 has been identified as an attractive potential vector for highefficiency gene transfer into various human cells, including hematopoietic cells of the bone marrow (Sandalon et al., 1997; Sandalon and Oppenheim, 1997; Rund et al., 1998; Dalyot-Herman et al., 1999; Goldstein et al., 2002). SV40 assembly and dissociation studies have implicated both ∗
Corresponding author. Tel.: +1 562 985 4803; fax: +1 562 985 8878. E-mail address: [email protected] (E. Gharakhanian).
0168-1702/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.virusres.2004.06.006
permanent intermolecular disulfide bonds between Vp1 molecules and transitory intramolecular and intermolecular disulfide bonds in stable capsid formation (Brady et al., 1977, 1978, 1979; Ng and Bina, 1981; Kosukegawa et al., 1996; Li et al., 2002). Vp1 contains seven cysteines, any or all of which may be involved in disulfide bridges. Mercury-labelling experiments suggested that Vp1 cysteines at positions 9, 104, and 207 may be involved in disulfide linkage (Liddington et al., 1991). In consequent structural studies of assembled virions, interpentamer disulfides between Cys104 residues were detected (Stehle et al., 1996). Studies of SV40 virion-like particles (VLP’s) in insect cells have implicated interpentamer disulfides between Vp1 Cys9 residues (Ishizu et al., 2001). We have reported that Vp1 Cys9, Cys104, and Cys207 are essential for formation of disulfide-linked post-pentameric complexes in vitro (Jao et al., 1999), as well as for formation of infectious virions in vivo (Gharakhanian et al., 2001). In our in vitro studies, mutant Vp1 with cys → ser changes at all the remaining four cysteines—Cys49, Cys87, Cys254, and Cys267—continued
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to form disulfide-linked post-pentameric complexes analogous to wild-type Vp1 (Jao et al., 1999). Here, we report on the individual and cooperative roles of those four cysteines in the formation of infectious virions during the natural course of infection. While the quadruple mutant Vp1 was completely defective in plaque formation, it continued to localize to the nucleus and bind SV40 DNA. Cys254 of Vp1 was the only one of the four cysteines singularly essential for plaque formation. The two double mutants at Vp1 Cys49.Cys87 and Cys254.Cys267 were also defective in plaque formation, with the former showing cumulative effects at work at Cys49 and Cys87. All mutants defective in plaque formation continued to localize T-Ag and mutant Vp1 to the nucleus, but showed no cytopathic effect (CPE). The mammalian expression vector pSV40 contains the wild-type SV40 genome and has been described before (Clever and Kasamatsu, 1993). Vp1 sequences containing site-directed cys → ser codon changes from various pSp6Vp1 mutant plasmids described in Jao et al. (1999) were subcloned into pSV40 to generate a quadruple mutant plasmid at Cys49, Cys87, Cys254, and Cys267 (referred to as pSV40Vp1.4X), two double mutants pSV40Vp1.C49S.C87S and pSV40Vp1.C254S.C267S,
and four single mutants pSV40Vp1.C49S, pSV40Vp1.C87S, pSV40Vp1.C254S, and pSV40Vp1.C267S. Mutant plasmids were confirmed by DNA sequencing (DNA Sequencing Facility, California State U., Northridge). Mutant and wild-type SV40 genomes were excised from pSV40 and were used in transient lipid-mediated DNA transfections (Lipofectamine, Gibco/BRL) of simian CV-1 cells (ATCC). Mock transfections included all reagents and manipulations minus input DNA. Transfected cells were tracked for CPE and plaque formation by light microscopy and plaque assays, respectively (Fig. 1a and b). CPE was detected by day 6 post-transfection (p.t.) in cells transfected with wild-type SV40, SV40Vp1.C49S, SV40Vp1.C87S, and SV40Vp1.C267S; onset and patterns of CPE were identical to wild-type and all resulted in plaques. While the titers of SV40Vp1.C87S and SV40Vp1.C267S were only slightly lower than wild-type, SV40Vp1.C49S consistently resulted in smaller plaques and a titer that was lower than wild-type by one to two orders of magnitude. Cells transfected with SV40Vp1.4x, SV40Vp1.C49S.C87S, SV40Vp1.C254S.C267S, or the SV40Vp1.C254S single mutant showed no CPE when monitored up to 20 days p.t. and resulted in no plaques. Lysates of cells transfected with plaque formation-defective mutants were used for infections and
Fig. 1. (a) Summary of transfection and infection phenotypes for SV40 Vp1 mutants. Data reflect separate experiments. (b) Plaque assays following transfection and infection. CV-1 cells were transfected with linear SV40 DNA (row A) or infected with plaque-purified virions (row B) as indicated and were stained with crystal violet 14 days after transfection or 12 days after infection.
E. Gharakhanian et al. / Virus Research 107 (2005) 21–25
resulted in no plaques (data not shown). Plaques generated following transfections were picked at 14 days p.t. and were subjected to plaque purification. Virions were isolated by a novel small-scale virus mini-preparation we have described elsewhere (Orlando et al., 2000; Gharakhanian et al., 2001) and were used in subsequent infections. In order to further map the block in infectious virion formation seen with SV40Vp1.4X, SV40Vp1C49S.C87S, SV40Vp1.C254S.C267S, and SV40Vp1.C254S, earlier events of the permissive infection were studied. For immunofluorescent microscopy studies, CV-1 cells were seeded onto glass coverslips and transfected with wild-type or mutant SV40 genomes defective in plaque formation, or mock-transfected, as described above. Transfected cells were harvested 48 h p.t. and were subjected to indirect doublelabelled immunofluorescent microscopy studies using MAbs against large T-Ag (CalBiochem) and polyclonal antibodies against GST-Vp1 (kind gift of Ariella Oppenheim, Hebrew University-Hadassah Medical School, Jerusalem, Israel). Each mutant transfection showed wild-type patterns of expression and nuclear localization of T-Ag and Vp1 (Fig. 2). Anti-T-Ag staining was used to assess possible variations in transfection efficiencies and frequencies of nuclear localization of Vp1 among different mutants. Both wild-type and mutant plasmids led to transfection efficiencies of 10–14% as defined by T-Ag staining. In all transfections, 30–40% of positively transfected cells showed nuclear Vp1. Next, the DNA-binding ability of mutant Vp1 was tested. A recent study of deletion and site-directed mutants at the N-terminus of Vp1 has mapped the DNA-binding domain to the first 21 amino acids of the protein and has also suggested the possible involvement of residues at the base of the pentamer in DNA binding (Li et al., 2001). Since Cys49 and Cys254 are located at the base of each monomer (Liddington et al., 1991), DNA binding of the quadruple mutant was tested via an in vitro DNA-binding assay we have described previously (Gharakhanian et al., 2001). Briefly, 35 S-labelled wildtype and 4x mutant Vp1 were expressed in rabbit reticulocyte lysates from Sp6Vp1 or Sp6Vp1.4x plasmids described in Jao et al. (1999), and their binding to immobilized SV40 DNA was measured (Fig. 3). Vp1.4X showed DNA binding indistinguishable from wild-type Vp1. Since Vp1.4X showed no defect, the double or single mutants of Vp1 were not tested. With nuclear localization and DNA-binding functions unaffected in the non-infectious mutants, attempts were made to assess the packaging step of the infection. Packaging of viral DNA was assessed following transfections with wild-type or mutant SV40. Cells were harvested at 72 h p.t. and packaged DNA was isolated as described previously (Orlando et al., 2000; Gharakhanian et al., 2003). Briefly, cellular debris was removed by centrifugation and supernatants were treated with excess DNase to remove all non-packaged DNA, followed by proteinase K, DTT, and organic extraction to remove capsid proteins. Two-fifths of each viral DNA yield was linearized with BamHI, electrophoresed, and subjected to chemiluminescent Southern blotting using SV40 DNA as
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Fig. 2. SV40Vp1.4X, SV40Vp1.C49S.C87S, SV40Vp1.C254S.C267S, and SV40Vp1.C254S lead to expression and nuclear localization of T-Ag and Vp1. CV-1 cells were grown on coverslips and were transfected with linear wild-type or mutant SV40 DNA as indicated. Cells were harvested 48 h post-transfection by methanol fixation and were subjected to doublelabelled indirect immunofluorescent microscopy using mouse anti-Tag (␣TAg) and rabbit anti-GST-Vp1 (␣GST-Vp1) followed by TITC-conjugated rabbit anti-mouse and FITC-conjugated goat anti-rabbit secondary antibodies (SIGMA). Cells were mounted on slides and observed on an Olympus microscope using a 60× objective.
probe (AlkPhos Direct labeling kit, Amersham Pharmacia). The results were inconclusive, since in repeated experiments, extent of packaging was variable in each of the non-infectious mutants (data not presented). In this study, we have generated SV40 single, double, and quadruple mutants with conservative cys → ser codon changes at Vp1 Cys49, Cys87, Cys254, and Cys267 and have studied them for expression and nuclear localization of early and late SV40 gene products, Vp1.4x DNA binding, onset of CPE, and production of infectious virions in permissive CV-1 cells. In our in vitro studies, the quadruple mutant Vp1 continued to form disulfide-linked post-pentameric complexes (Jao et al., 1999). In the current in vivo study, the SV40 genome
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Fig. 3. Vp1.4x does not exhibit DNA-binding defects. [35 S] methioninelabelled products of wild-type SV40- and SV40Vp1.4X-coupled transcription/translation were incubated with SV40 DNA immobilized on PVDF membranes. DNA binding is expressed as the percentage of input cpm retained on the membrane. Values represent mean of two independent experiments.
encoding the same quadruple mutant Vp1 failed to result in CPE or plaques, but continued to correctly localize SV40 proteins to the nucleus and to bind SV40 DNA. Thus, none of Vp1 Cys49, Cys87, Cys254, and Cys267 residues have a singular or cumulative role in nuclear localization of Vp1 or T-Ag, DNA binding, or pentameric and post-pentameric Vp1 multimerization. When each of the four cysteines were studied individually, only Vp1 Cys254 was singularly essential for eliciting CPE and infectious virion formation. Vp1 Cys254 is located on an internal short loop that connects strands G2 and H (G2H loop) and is in close proximity to the calcium-binding sites of Vp1; most importantly, it is the only one of the four cysteines which is conserved across the polyomavirus family (Gharakhanian, 1988; Liddington et al., 1991). A study of single mutants at all seven Vp1 cysteines in a genetically engineered, non-overlapping SV40 genome has shown drastic reduction in infectivity only with mutants at Cys254 (Li et al., 2000). No disulfides have been detected at Cys254 in virion structural studies (Liddington et al., 1991; Stehle et al., 1996), and a mutant Vp1C254S continues to form disulfide-linked post-pentameric complexes when expressed in rabbit reticulocyte lysates (Jao et al., 1999). A cys → ser mutant SV40 Vp1 at Cys254 has been expressed in a baculovirus system and forms protease-resistant VLP’s analogous to wild-type, leading the authors to also conclude that Cys254 is not involved in disulfide-linked viral assembly (Ishizu et al., 2001). Both our SV40 genome studies and the studies of Li et al. (2000) using a genetically engineered SV40 genome suggest a critical role for a cysteine residue at Vp1 aa254 in the life cycle of SV40. While single mutants SV40Vp1.C49S and SV40Vp1. C87S continued to form infectious virions, the double mutant SV40Vp1.C49S.C87S was completely defective in plaque formation or CPE, suggesting a cumulative effect when both cysteines are mutated. While no disulfides at these two residues have been detected in structural studies of assem-
bled virions (Liddington et al., 1991; Stehle et al., 1996), the two could be involved in transient intramolecular disulfides that have been recently reported as early intermediates of assembly (Li et al., 2002). Cys49 and Cys87 lie in sufficiently close proximity to each other to form a disulfide bridge within a single Vp1 monomer (Liddington et al., 1991). The distinct infectivity difference between the two single mutants and the double mutant suggests that each can bond with another unidentified Vp1 cysteine when its original partner is mutated, but not when both are mutated. Our inconsistent DNA packaging results suggest that these mutants are lacking a predictable, consistent packaging pathway, such that random interactions only sometimes lead to packaged DNA. Transient disulfides may be significant in promoting correct folding and facilitating progression of a predictable assembly pathway. We have previously shown that in rabbit reticulocyte lysates, Vp1.4X forms 7S pentameric complexes in 1 h post-translation and proceeds to form 12S multi-pentameric complexes in 3 h post-translation as assessed by sucrose gradient sedimentation profiles; this progression of Vp1 multimerization is indistinguishable from wild type Vp1 (Jao et al., 1999). The fact that even the 4X mutant continues to bind DNA and to form post-pentameric Vp1 complexes suggests that the Cys49/ Cys87 combination may be essential in maintaining a consistent assembly pathway for later stages of DNA packaging. Future biochemical and electron microscopic analyses of SV40Vp1.C49S.C87S and SV40Vp1.C254S and SV40Vp1.4x can further shed light on the state and structure of these non-infectious mutant complexes. Acknowledgements We thank Dr. Ariella Oppenheim for kind gifts of antiGST-Vp1 antibodies. This work was made possible by a grant from the National Science Foundation-Research for Undergraduate Institutions (MCB-9630904) to E.G. W. Mana and M. Norng were partially supported by Howard Hughes Foundation and NSF-RUI, respectively. References Baker, T.S., Drake, J., Bina, M., 1988. Reconstruction of the three dimensional structure of simian virus 40 and visualization of the chromatic core. Proc. Natl. Acad. Sci. USA 85, 422–426. Brady, J.N., Winston, V.D., Consigli, R.A., 1977. Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions. J. Virol. 23, 717–724. Brady, J.N., Winston, V.D., Consigli, R.A., 1978. Characterization of a DNA–protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N -tetraacetic acid and dithiothreitol. J. Virol. 27, 193–204. Brady, J.N., Kendall, J.D., Consigli, R.A., 1979. In vitro reassembly of infectious polyoma virions. J. Virol. 32, 640–647. Clever, J., Kasamatsu, H., 1993. Identification of a DNA-binding domain in simian virus 40 capsid proteins Vp2 and Vp3. J. Biol. Chem. 268, 20877–20883.
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