Department of Biological Sciences, California State University at Long Beach, 1250 Bellflower Blvd, Long Beach, CA 90840-3702, USA1
Author for correspondence: Editte Gharakhanian. Fax +1 562 985 8878. e-mail eghara{at}csulb.edu
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Abstract |
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Simian CV-1 cells were transfected with pSV40 plasmid encoding the entire SV40 genome (Clever et al., 1993 ). Transfected plasmids included wild-type Vp1 codons or single, double and triple mutations at Vp1 Cys9, Cys104 and Cys207 codons, introduced via site-directed mutagenesis (Transformer kit, Clontech). Transfected cells were harvested 40 h post-transfection (p.t.) and were subjected to indirect double-labelled immunofluorescence studies using MAbs against large T-Ag (CalBiochem) and polyclonal antibodies against Vp1 (anti-GSTVp1, a kind gift of A. Oppenheim, Hebrew UniversityHadassah Medical School, Jerusalem, Israel). Each mutant sample showed wild-type patterns of expression and nuclear localization of Vp1 (Fig. 1
). Anti-T-Ag staining was used to allow better assessment of 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 1015% and resulted in nuclear staining of Vp1 in over 60% of transfected cells (data not shown).
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Cells transfected with SV40Vp1 . C9S . C104S, SV40Vp1 . C9S . C207S and SV40Vp1 . 3X showed no sign of CPE by 14 days p.t. Several samples were monitored for extended periods of up to 24 days p.t. Three late-onset CPE clusters were detected in three separate transfections. Late-onset plaques were picked and virions were isolated. Both input DNA and DNA from isolated virions were subjected to PCR amplification and sequencing. All three revertants contained back-mutations to the Cys9 codon; isolated revertant virions showed wild-type infection kinetics (data not shown).
Both SV40 structural studies by others (Liddington et al., 1991 ; Stehle et al., 1996
) and in vitro SV40 Vp1 assembly studies by us (Jao et al., 1999
) have implicated Cys9, Cys104 and Cys207 of Vp1 in disulfide linkage of post-pentameric complexes. Here, by generating a series of single, double and triple mutations at Cys9, Cys104 and/or Cys207 codons of Vp1 and their introduction into permissive cells in the context of an otherwise wild-type SV40 genome, we have assayed the role of the three cysteines in the course of permissive infection in vivo. We have presented evidence for the involvement of SV40 Vp1 Cys9, Cys104 and Cys207 residues in the onset of CPE and in the production of infectious virions in permissive CV-1 cells. The present study indicates that the same three cysteines implicated in stabilization of disulfide-linked Vp1 post-pentameric complexes in vitro are also essential for infectious virion formation in vivo.
Our results also show that none of the three cysteines, individually or in double or triple combinations, is involved in the correct nuclear localization or DNA-binding of Vp1.
All Vp1 mutants defective in plaque formation localized successfully to the nucleus of the host cell and bound DNA, but showed no CPE. This suggests that the Vp1 mutants are defective in proteinprotein interactions involved in stable formation of virions, rather than in their ability to enter the nucleus and bind DNA or in the ability of assembled virions to exit the infected cell or infect a new cell successfully. In fact, we have been unsuccessful in isolating virus-like particles from any of the mutants defective in plaque formation (data not shown). A defect in the early steps of assembly is consistent with our in vitro results, where mutants defective at the same three residues continued to form pentamers but failed to form disulfide-linked post-pentameric complexes in cell-free lysates (Jao et al., 1999 ). Most recently, Cys9 and Cys104 of SV40 Vp1 have been implicated in disulfide-bonded formation of virus-like particles in insect cells (Ishizu et al., 2001
).
Due to the overlapping nature of the Vp2/Vp3 and Vp1 genes, Cys9Ser mutagenesis also resulted in the conservative Leu206
Phe codon change in Vp2/Vp3. Leu206 is the last residue in the proposed nuclear localization signal of Vp3, but is not involved in DNA binding or in Vp1Vp3 interactions (Gharakhanian et al., 1988
; Clever & Kasamatsu, 1991
). The nuclear localization of Vp3 was studied in pSV40Vp1 . C9S transfections using anti-GSTVp3 antibodies (kind gift of A. Oppenheim) and was indistinguishable from that of wild-type Vp3 (S. J. Orlando and E. Gharakhanian, unpublished results).
Several virus dissociation and assembly studies have implicated disulfide bonds in the stabilization of the viral capsid (Brady et al., 1977 , 1978
, 1980
; Ng & Bina, 1981
; Kosukegawa et al., 1996
). The conservative nature of the Cys
Ser mutations, along with the fact that isolated revertants invariably involved back-mutations of Ser
Cys codons, suggests strongly that Cys9, Cys104 and Cys207 exert their role in formation of infectious virions via disulfide bond formation. Both the current in vivo results and our recent in vitro results (Jao et al., 1999
) support a cumulative effect in the role of the cysteines such that all single mutants continue to form infectious virions. However, while all double mutants continued to form at least some post-pentameric complexes in vitro, only the double-mutant Vp1 . C104S . C207S continued to lead to CPE and plaque formation in vivo. This suggests that assembly in cells may require further stabilization and may involve more stringent requirements than that observed in cell-free lysates. Furthermore, since only mutants containing Cys9
Ser mutations in our studies showed defects in plaque formation and since all isolated revertants contained a back-mutation at that site, Cys9 may be the most crucial of the three cysteines for disulfide-linked stabilization, with Cys104 or Cys207 having secondary roles. Li et al. (2000)
have recently suggested the significance of Cys254 of Vp1 in efficient calcium binding and in formation of infectious SV40 virions.
While our results suggest disulfide bonding at Cys9 to be crucial to infectious virion formation, the only disulfide linkage detected in structural studies of SV40 has been between two Cys104 residues (Stehle et al., 1996 ). In all structural studies of SV40, however, the N-terminal 13 amino acids of Vp1 have been disordered, such that structural information was not available for that region (Liddington et al., 1991
; Stehle et al., 1996
; Yan et al., 1996
). Thus, the specific positioning of Cys9 and its interactions have not been established. In the current structural model, the C terminus of Vp1 is the main mechanism of contact between pentamers. C-terminal arms extend from every Vp1 in the pentamer and insert into binding sites on adjacent pentamers such that the acceptor monomer forms a clamp to fix the position of the invading arm. Based on available structural information on Vp1 and our results, the most likely role of Cys9 may be in the stabilization of the N-terminal clamp and its interactions with the C terminus of the invading Vp1.
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Acknowledgments |
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Footnotes |
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c Present address: Department of Biology, University of California, San Diego, CA 92093, USA.
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References |
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Received 11 December 2000;
accepted 21 March 2001.