1 Institute for Biomedical Research, Georg-Speyer Haus, Paul-Ehrlich-Str. 42-44, D-60596 Frankfurt/Main, Germany
2 Paul Ehrlich Institute, Medical Biotechnology, Paul-Ehrlich-Str. 51-59, D-63225 Langen, Germany
Correspondence
Barbara Schnierle
schnierle{at}em.uni-frankfurt.de
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ABSTRACT |
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MAIN TEXT |
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We generated a replication-competent virus expressing the chimeric GFP-Env1 protein. For this, GFP sequences were inserted into the PRR of pE-MO, a plasmid encoding a replication-competent MoMLV with 53 amino acids of the epidermal growth factor (EGF) fused to the N terminus of Env to target virus binding to the EGF receptor (Buchholz et al., 1998). This resulted in GFP-EMO1 (Fig. 2
A). When NIH 3T3 cells were transfected, the cells released infectious virus that could be passaged further. We also generated GFP-EMO2, a construct carrying, in addition, the translocation domain (TLD) of the bacterial toxin exotoxin A between the EGF and Env sequences (Fig. 2A
). The TLD enables the toxin, after endocytosis by the cell, to escape degradation in the endosomes and to translocate into the cytoplasm (Allured et al., 1986
; Hwang et al., 1987
). We had found recently that the presence of the TLD sequences in Env allowed the generation of a functionally active Env protein but severely reduced transduction titres (Erlwein et al., 2002
). We wanted to investigate whether the replication behaviour of GFP-EMO1 or GFP-EMO2 was affected in comparison to E-MO.
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NIH 3T3 cells transfected with GFP-EMO1 (NIH 3T3/GFP-EMO1 cells) or GFP-EMO2 (NIH 3T3/GFP-EMO2 cells) were cultured for another 19 days until, in each case, the virus had spread through the whole cell culture, as judged by the expression of green fluorescence (Fig. 2B). Virus growth kinetics were monitored by FACS (Fig. 2B
) and titres released from NIH 3T3/GFP-EMO1 remained at 3x103 IU ml-1, while the titre for NIH 3T3/GFP-EMO2 increased and also reached about 103 IU ml-1 after 16 days (Fig. 2B
). As shown by Western blot analysis, viruses released from NIH 3T3/GFP-EMO2 cells contained an Env protein that was slightly smaller than that observed for NIH 3T3/GFP-EMO1 cells (Fig. 3
A, lane 4). To investigate whether this was due to rearrangements in the Env protein of GFP-EMO2, we isolated cellular DNA from both chronically infected cell lines and performed PCR analysis. The primers MLV5'ENV (5'-TAACCCGCGAGGCCCCCTAATCC-3') and BS5 (5'-TCTGAGTCGGATCCCAAATGTAAG-3') bind from position 6663 to 6685 and from position 7578 to 7555 of pE-MO, respectively.
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The PRR of Env has been described to modulate its conformation and fusogenicity (Lavillette et al., 1998; Wu et al., 1998
). Therefore, we wanted to investigate whether the insertion of the GFP sequences altered the usage of the natural receptor, the cationic amino acid transporter mCAT-1 (Albritton et al., 1989
). We carried out a receptor interference assay, exploiting the fact that cells chronically infected with MoMLV cannot be superinfected by another virus utilizing the same receptor (Hunter, 1997
). The packaging cell line TELCeB6 produces the structural proteins Gag and Pol, together with a packagable vector encoding
-galactosidase (
-Gal). TELCeB6 cells were transfected with the plasmids pwt(HX) or pGFP-Env1 and the vector containing supernatant was added to recipient cells. After 2 days, those cells were stained for
-Gal expression (Schnierle et al., 1997
). On NIH 3T3 cells, the titre for wt(HX) and GFP-Env1 was found to be 5x102 UI ml-1. In contrast, no
-Gal expression could be detected for wt(HX) or GFP-Env1 in chronically infected NIH 3T3/GFP-EMO1 cells, demonstrating that superinfection was reduced at least 500-fold and that the presence of foreign sequences in the PRR does not affect the specificity of the Env interaction.
Recently, the insertion of GFP sequences between amino acids 6 and 7 of MoMLV Env has been described (Kizhatil et al., 2001). Despite the successful packaging of the recombinant protein into virions, transduction failed due to a fusion defect until an additional double mutation (Q227
R, D243
Y) was included. In our experiments, GFP-Env2, which has the GFP sequences at the same location (aa 67), was also not able to transduce a reporter gene despite good Env protein production in the cell. However, there seemed to be a packaging defect of GFP-Env2 into the virion (Fig. 1B
). The reason for the discrepancy to the results described by Kizhatil et al. (2001)
is not clear. One explanation could be that GFP-Env2 carries the original GFP (S65
T) sequences rather than enhanced GFP, which contains an additional point mutation in the chromophore and preferred human codons that enhance the translational efficiency of the mRNA. Inefficient translation of GFP-Env2 may interfere with proper folding of the protein and hinder incorporation of the protein into the virion.
In contrast, GFP-Env1, having the sequences of enhanced GFP in the PRR, was fully functional without the need of the additional mutations described (Kizhatil et al., 2001). The fact that a replication-competent virus can be generated on the basis of this construct without loss of titre demonstrates that this recombinant protein is not toxic to the cell and that it can be stably expressed. Furthermore, we demonstrated that labelling Env in the PRR does not change the specificity for its receptor, leaving other regions like the N terminus available for further insertions of ligands to target specific receptors. Recently, a retrovirus library based on feline leukaemia virus subtype A has been described that carries random modifications in the Env molecule to target new receptors (Bupp & Roth, 2002
). The GFP-bearing virus described by us may prove helpful in screening similar libraries based on MoMLV to visualize the virus entry process and to easily monitor the tissue distribution of MoMLV.
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ACKNOWLEDGEMENTS |
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Received 7 August 2002;
accepted 23 October 2002.