Abstract
The sodium-dependent neutral amino acid transporter type 2 (ASCT2) was recently identified as a cell surface receptor for endogenously inherited retroviruses of cats, baboons, and humans as well as for horizontally transmitted type-D simian retroviruses. By functional cloning, we obtained 10 full-length 2.9-kilobase pair (kbp) cDNAs and two smaller identical 2.1-kbp cDNAs that conferred susceptibility to these viruses. Compared with the 2.9-kbp cDNA, the 2.1-kbp cDNA contains exonic deletions in its 3' noncoding region and a 627-bp 5' truncation that eliminates sequences encoding the amino-terminal portion of the full-length ASCT2 protein. Although expression of the truncated mRNA caused enhanced amino acid transport and viral receptor activities, the AUG codon nearest to its 5' end is flanked by nucleotides that are incompatible with translational initiation and the next in-frame AUG codon is far downstream toward the end of the protein coding sequence. Interestingly, the 5' region of the truncated ASCT2 mRNA contains a closely linked series of CUG(Leu) and GUG(Val) codons in optimal consensus contexts for translational initiation. By deletion and site-directed mutagenesis, cell-free translation, and analyses of epitope-tagged ASCT2 proteins synthesized intracellularly, we determined that the truncated mRNA encodes multiple ASCT2 isoforms with distinct amino termini that are translationally initiated by a leaky scanning mechanism at these CUG and GUG codons. Although the full-length ASCT2 mRNA contains a 5'-situated AUG initiation codon, a significant degree of leaky scanning also occurred in its translation. ASCT2 isoforms with relatively short truncations were active in both amino acid transport and viral reception, whereas an isoform with a 79-amino acid truncation that lacked the first transmembrane sequence was active only in viral reception. We conclude that ASCT2 isoforms with truncated amino termini are synthesized in mammalian cells by a leaky scanning mechanism that employs multiple alternative CUG and GUG initiation codons.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF334818
The other two cDNA clones that we isolated, which we term ⌬human ASCT2 (hASCT2), had sizes of 2.1 kbp and identical sequences that will be further described below. These larger and smaller cDNAs are compatible with the sizes of major and minor mRNA components that were detected by Northern blot analyses of RNAs from different human tissues [3, 7]
Stable expression of the 2.1-kbp ⌬hASCT2 cDNA in Chinese hamster ovary (CHO) cells caused susceptibility to infections by lacZ pseudotypes of RD114, baboon endogenous virus (BaEV), and type-D simian retroviruses (SRV-1 and SRV-2). These results strongly suggest that the 2.1-kbp ⌬hASCT2 cDNA encodes a functional receptor for RD114, BaEV, and SRVs
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF334818. Non-AUG codons such as CUG(Leu), ACG(Thr), and GUG(Val) can serve as low efficiency initiation sites if they occur within the Kozak consensus context (10, 18 –27) In these cases, the initial amino acid that is incorporated appears to be methionine, apparently because these non-AUG codons can still form weak base pairs with the initiator Met-tRNAMet [26, 28]. A clear illustration of these issues occurs with the Gag protein of murine leukemia viruses, which is inefficiently initiated at a CUG codon and more efficiently by the leaked-through ribosomes at a downstream AUG codon [29]. This produces a larger protein with an amino-terminal signal sequence that is.
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