The RD114/simian type D retrovirus receptor is a neutral amino acid transporter

Abstract

The RD114/simian type D retroviruses, which include the feline endogenous retrovirus RD114, all strains of simian immunosuppressive type D retroviruses, the avian reticuloendotheliosis group including spleen necrosis virus, and baboon endogenous virus, use a common cell-surface receptor for cell entry. We have used a retroviral cDNA library approach, involving transfer and expression of cDNAs from highly infectable HeLa cells to nonpermissive NIH 3T3 mouse cells, to clone and identify this receptor. The cloned cDNA, denoted RDR, is an allele of the previously cloned neutral amino acid transporter ATB0 (SLC1A5). Both RDR and ATB0 serve as retrovirus receptors and both show specific transport of neutral amino acids. We have localized the receptor by radiation hybrid mapping to a region of about 500-kb pairs on the long arm of human chromosome 19 at q13.3. Infection of cells with RD114/type D retroviruses results in impaired amino acid transport, suggesting a mechanism for virus toxicity and immunosuppression. The identification and functional characterization of this retrovirus receptor provide insight into the retrovirus life cycle and pathogenesis and will be an important tool for optimization of gene therapy using vectors derived from RD114/type D retroviruses.

Figure 1

Amino acid sequence of human RDR. The amino terminus is predicted to be intracytoplasmic and the carboxyl terminus is predicted to be extracellular. Predicted transmembrane regions (underlined) and N-glycosylation sites (bold) are indicated.

Figure 2

Retrovirus vector titers on polyclonal NIH 3T3 cells transfected with the RDR expression vector pL(RDR)SN or the empty expression vector pLXSN. Both SNV and BaEV pseudotypes represent replication-competent virus, whereas RD114, GALV, and VSV-G were helper-free. The titer of replication-competent viral stocks used in these experiments was 4.1 × 10⁴ AP⁺ ffu/ml for SNV and 3.2 × 10⁴ lacZ⁺ ffu/ml for BaEV when assayed on D17 cells. The titer of LAPSN(GALV) was 5.3 × 10⁵ AP⁺ ffu/ml assayed on human cells. Each bar represents the mean ± SD of triplicate measurements, except for VSV-G, which represents the mean of duplicates. A representative experiment is shown; each determination was performed at least twice.

Figure 3

Reduced amino acid transport in cells infected with viruses that use hATB⁰ for entry. Uptake of glutamine in HT-1080 cells (A) and alanine in HeLa cells (B) was examined in cells cultured after exposure to replication-competent virus indicated on the x axis. The presence of replication competent virus was demonstrated by marker rescue of HT-1080 and HeLa cells exposed to RD114, MPMV, BaEV, and AM-MLV, but was not detected in either cell type exposed to SNV or nil. (Bar represents mean ± SD of triplicate measurements.) Statistical significance of P ≤ 0.01 (∗) and of P ≤ 0.001 (∗∗) was demonstrated by ANOVA in which each result was compared with the result in nil. This experiment was performed twice with similar results.