Retrovirus Molecular Biology Group, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK1
Oxford BioMedica (UK) Ltd, Oxford Science Park, Oxford OX4 4GA, UK2
Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester M60 1QD, UK3
Author for correspondence: Kyriacos Mitrophanous. Fax +44 1865 783001. e-mail k.mitrophanous{at}oxfordbiomedica.co.uk
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Abstract |
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We therefore used a myc epitope tag, as it is very small and therefore should not influence the localization (Wilson et al., 1996 ; Elefanty et al., 1996
). A mycHis tag was placed at the C terminus of S2 by inserting the S2 sequence into pCDNA3.1MycHisA (Invitrogen). A myc tag was placed at the N terminus of S2 by inserting an appropriate synthetic oligonucleotide into pCI-S2. As shown in Fig. 2(a)
, both of the S2 proteins tagged with the myc epitope showed cytoplasmic localization in 293T and COS7 cells. Interestingly, the staining was punctate compared with the control lacZmycHis and the localization appeared to be biased towards the perinuclear region. This suggests an association of S2 with subcellular structures. The immunostaining was repeated with cells that were fixed without permeabilization, and no staining for S2 was observed (data not shown). This suggests that S2 is confined to the internal structures of the cytoplasm. The fact that localization was strikingly cytoplasmic irrespective of the fusion at the N or C terminus suggests that the previous data with the GFP fusion at the C terminus were artifactual.
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Given that S2 could diffuse to the nucleus, its restriction to the cytoplasm must be active rather than passive. This could be achieved either by a rapid nuclear export mechanism that simply prevented detection of nuclear-localized S2 or by the binding of S2 to cytoplasmic structures. The punctate staining in the cytoplasm was suggestive of an interaction with cellular structures. Primary sequence analysis of S2 revealed a nucleoporin motif (Fig. 1a). Nucleoporin motifs mediate binding to the cytoplasmic face of the nuclear envelope. The cell culture-adapted avirulent strain pSPEIAV19 has an amino acid change in the potential nucleoporin motif and this could serve to disrupt the classical consensus nucleoporin motif (Cook et al., 1998
), thereby influencing the localization of OptS219. We therefore made OptS2PV, based on the S2 sequence of EIAV strain EIAVPV, which has a potential nucleoporin motif. When the localization of this protein was studied in COS7 cells, the staining was still found in the cytoplasm and was still punctate (Fig. 2d
). These data suggest that a potential nucleoporin motif in S2 does not dictate the staining pattern and imply that the attenuation of pSPEIAV19 is not due to differences in S2 sequences. This may not be surprising, as it is thought that more than one nucleoporin motif is required for localization to the nuclear membrane (Nigg, 1997
).
It is possible that overexpression of S2 could influence its subcellular localization. The mycHis tag was therefore fused to OptS2PV and its localization was compared with the wild-type S2 sequence fused to mycHis. As shown in Fig. 2(f), Western blot analysis using anti-myc MAbs revealed a higher level of expression of OptS2PVmycHis compared with wild-type S2. Immunofluorescence analysis of OptS2PVmycHis revealed the same localization and punctate quality of staining of S2mycHis (Fig. 2e
). With S2mycHis, only 3% of the cells were stained, whereas with OptS2PVmycHis it was over 80% of the cells. We also examined whether there was any difference in the localization of S2 when it was co-expressed with other EIAV proteins. We could not detect any differences in the pattern of immunostaining, which indicates that localization of S2 may not be affected by other viral proteins (data not shown). Taken together, the data indicate strongly that S2 is naturally localized to the cytoplasm. During localization studies, it was found that the anti-S2N antibody against the region Ala12Pro28 could not detect any mammalian cell-expressed S2 proteins, while detecting E. coli-expressed TrxS2. This indicates that S2 may be modified post-translationally, at least in the region of the epitope to which the anti-S2N antibody binds.
We next purified the S2 protein fused with Trx to investigate S2-interacting proteins. Equivalent amounts of Trx or TrxS2 purified from E. coli were dialysed against coupling buffer (MOPSNaOH pH 7·9, 0·3 M NaCl, 0·5% glycerol) and coupled with affigel-10 (Bio-Rad) according to the manufacturers instructions. TrxS2-bound resin was incubated with lysates from 293T cells transfected with an EIAV gag/pol-expressing construct, pONY3.1, which expresses all EIAV proteins except envelope (Mitrophanous et al., 1999 ). Trx-bound resin incubated with cell lysates was used as a negative control. As shown in Fig. 3
(a
, b
), the Gag precursor (p55) bound TrxS2 and not Trx, which indicates that S2 interacts directly with the Gag precursor. In order to examine whether any cellular (293T) proteins bind TrxS2 non-specifically, the TrxS2-bound resin was incubated with a cell lysate from pCI-neo-transfected 293T cells, but no band was detected (Fig. 3a
, lane 6). In order to confirm the S2Gag interaction in vivo, co-immunoprecipitation was carried out with the codon-optimized S2 plasmid pOptS2PVMycHis and the EIAV gag/pol-expression plasmid pONY3.1
S2. The results showed that S2 interacts with the EIAV Gag precursor in vivo (Fig. 3c
). The possible interaction of S2 with the EIAV envelope was also investigated, but it was found that the EIAV envelope did not co-precipitate with S2 (data not shown).
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In conclusion, our study shows that S2 is a cytoplasmic protein that can interact with Gag but is not incorporated preferentially into particles. This indicates that S2 may not function in the early phase of the life-cycle. The possible association of S2 with other intracellular organelles indicated by the punctate pattern of staining and the S2Gag interaction suggests that S2 may be involved in virus particle assembly.
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Acknowledgments |
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Received 16 March 2000;
accepted 8 June 2000.
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