Department of Biological Sciences, California State University at Long Beach, 1250 Bellflower Blvd, Long Beach, CA 90840-3702, USA
Correspondence
Editte Gharakhanian
eghara{at}csulb.edu
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ABSTRACT |
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Present address: Department of Neurobiology & Anatomy, University of Utah, USA.
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MAIN TEXT |
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SV40 Vp2, SV40
Vp3 and SV40
Vp2/Vp3 genomes were generated by site-directed mutagenesis of the initiation codons of Vp2, Vp3, or both Vp2 and Vp3, respectively, in the parent plasmid pSV40 (Transformer Kit, Clontech). The mammalian expression vector pSV40 contains the wild-type SV40 genome and has been described before (Clever & Kasamatsu, 1993
). To minimize revertants, all three bases of each initiation codon were changed (ATG
GCC); mutations were confirmed by sequencing (DNA Sequencing Facility, California State Univ., 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. Western blots of transfected cell lysates using anti-Vp3 antibodies confirmed lack of Vp2 or Vp3, or both Vp2 and Vp3 expression in cells transfected with SV40
Vp2, SV40
Vp3 or SV40
Vp2/Vp3, respectively (Fig. 1
a). Transfected cells were tracked for cytopathic effect (CPE) and plaque formation by light microscopy and plaque assays (Fig. 1
). CPE was detected by day 711 post-transfection (p.t.) in cells transfected with wild-type SV40 or SV40
Vp2 (Fig. 1b, c
); in repeated experiments, onset of CPE for wild-type- and SV40
Vp2-transfected cells was identical and showed a range relative to passage age of cells. Microscopic monitoring of SV40
Vp3-transfected cells uncovered distinct differences in onset and progression of CPE relative to wild-type (Fig. 1b, c
). A delayed, limited and transient CPE was detected in cells transfected with SV40
Vp3. CPE onset was consistently delayed by 2 days relative to wild-type, cell vesicularization was limited, and CPE was no longer detectable after 4 days post-onset. Cells transfected with SV40
Vp2/Vp3 or mock-transfected showed no CPE when monitored up to 20 days p.t. (Fig. 1b, c
). Repeated plaque assays consistently yielded plaques only in SV40
Vp2- and SV40-transfected plates. Plaques generated following transfections with SV40
Vp2 DNA were picked at 14 days p.t. and were subjected to three rounds of plaque purifications; titres were determined following infections. SV40
Vp2 consistently yielded titres and plaque sizes indistinguishable from wild-type SV40 (Fig. 1c, d
). SV40
Vp2 virions were isolated by a novel small-scale virus mini-preparation that we have described elsewhere (Orlando et al., 2000
). DNA from plaque-purified mutant SV40 was subjected to PCR amplification of the mutated AUG codon region and the amplified fragment was sequenced. Sequencing confirmed ATG
GCC mutation at the Vp2 initiation codon of SV40
Vp2 infectious virions. SV40
Vp2/Vp3- and SV40
Vp3-transfected plates were also picked at 14 and 20 days p.t. but did not yield productive infections (Fig. 1c
) nor amplified SV40 DNA (see Fig. 3b
).
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The individual roles of full-length SV40 Vp2 and Vp3 in the course of a permissive infection have not been adequately explored to date. A recent study on mouse polyoma virus has shown that in the absence of either polyoma Vp2 or Vp3, virus particles formed but showed reduced infectivity (Mannova et al., 2002). In this study, we have generated SV40 mutants with defective initiation codons for Vp2, Vp3, or both, and have studied them for expression and nuclear localization of early and late SV40 gene products, SV40 DNA packaging, onset of CPE and production of infectious virions in permissive CV-1 cells. Our results show that full-length Vp3 is essential for formation of infectious virions, whereas full-length Vp2 is not. All three mutants continued to express and correctly localize to the nucleus both the early gene product, T-Antigen, and the late gene product, major structural protein Vp1. All three continued to package SV40 DNA into DNase-resistant structures. These results suggest that one major role of full-length Vp3 may be at post-packaging stages of the permissive infection. These may include interaction of assembled virion particles with the infected cell for progression of infection, entry into cells for secondary waves of infection, or uncoating in the ER following entry. In our study, SV40
Vp3 led to transient, delayed CPE in CV-1 cells which best supports the first alternative. Interestingly, Gordon-Shaag et al. (2003)
have most recently shown that poly(ADP-ribose) polymerase (PARP) is involved in the magnitude of CPE in SV40-infected CV-1 cells, and that Vp3 stimulates PARP.
Since our mutants contain only mutagenized initiating AUG codons, translation initiation from internal AUGs could theoretically continue. There are no internal AUG codons in the Vp2-specific 118 amino acid coding region; however, there are two internal AUGs in the Vp2/Vp3 common region, which would render polypeptides of length150 and 50 amino acids, corresponding to the Vp2/Vp3 C terminus. In cells transfected with SV40Vp2/Vp3 DNA, immunofluorescent microscopy studies using anti-Vp3 polyclonal antibodies show a weak but positive nuclear staining, and Western blots show a weak Vp3-related 150 amino acid band (data not shown). Thus, an N-terminally truncated Vp3 may be expressed with continued nuclear localization and DNA-binding functions, attributing the observed post-packaging role for Vp3 to the N-terminal domains of the protein.
Our results indicate that infectious SV40 virions can form in the absence of full-length Vp2. SV40VP2 infection is indistinguishable from wild-type SV40 infection as assessed by onset of CPE, plaque size and viral titres. SV40
Vp3 elicits a limited CPE in transfected cells, whereas SV40
Vp2/Vp3 does not, suggesting that Vp2 may have a secondary role in pre-CPE stages of the productive infection. A less likely alternative may be that the Met-118 of Vp2, which is changed to an alanine codon in SV40
Vp3, is essential for infectious virion formation. The only study pertaining to a specific role of SV40 Vp2 in infection dates back to 1977, prior to full knowledge of the SV40 restriction or genomic map (Cole et al., 1977
). In that study, SV40 deletion mutants generated by restriction and S1 nuclease digests were assessed for plaque formation; mutations presumed to map to the N-terminal unique region of Vp2 continued to form plaques and infectious particles but plaque onset was delayed and plaque size was smaller relative to wild-type infections. Our study indicates that a full-length Vp2 is not essential in a productive SV40 infection; it also suggests that the Vp2-unique coding region may be dispensable as long as the Vp3 reading frame and initiating AUG codon are maintained. Dispensability of viral coding regions in formation of infectious virions, is a current area of study in vector development for gene therapy. SV40 has been identified as an attractive vector for high-efficiency gene transfer into various human cells (Sandalon et al., 1997
; Sandalon & Oppenheim, 1997
; Rund et al., 1998
; Dalyot-Herman et al., 1999
; Goldstein et al., 2002
). Chang & Wilson (1986)
have suggested the packaging limit for SV40 to be between 284 and 460 extra base pairs. Removing the Vp2-unique coding sequences along with the 5'-untranslated region would significantly increase the packaging limit of SV40 in gene replacement studies.
Future biochemical and electron microscopic analyses of these mutants can further shed light on the state and structure of the packaged complexes of SV40Vp3 and SV40
Vp2/Vp3, as well as the SV40
Vp2 infectious virions.
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ACKNOWLEDGEMENTS |
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Received 13 December 2002;
accepted 14 April 2003.
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