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Ecotropic murine leukemia virus (MuLV) receptor / Cationic amino acid transporter 1 (CAT-1)

Natural function

The cationic amino acid transporter 1 (CAT-1) is a part of the CAT family which is a subfamily of the solute carrier family 7 (SLC7). They are expressed ubiquitously and build the main entry gate for amino acids such as histidine, arginine or ornithin in mammalian cells. They enable the influx of their substrate in a Na⁺ independent way and also under certain circumstances the eflux. Additionally it was shown that absence of CAT-1 leads to non-viable mice pubs [1][2].

Structure and virus recognition

CAT-1 is a 66 kDa membrane protein. It's built up of 622 amino acids contains 14 transmembrane domains which resolutes in seven extracellulare and eight intracellulare domains. There are two sites for N-glycosylation in the third extracellulare loop. The glycosyled position is very important for virus' entry. The murine leukeamia virus is only able to enter the cell, when it detects the CAT-1 sugar-bound moieties[3].

Scheme of mCAT-1. Members of the CAT family are predicted to have 14 transmembrane domains with intracellular N- and C-termini. Two asparagine residues in the third extracellular loop (indicated as branched lines) have been shown to be glycosylated [7].

Also a comparison between CAT-1 sequences from different species like rats or hamsters shows that this region doesn't include conserved amino acids making a virus infection impossible [4]. The mouse CAT-1 was originally identified by Albritton in 1989 as the receptor for murine ecotropic leukemia viruses (MuLV) [5]. It was shown that in the presence of mCAT-1 on the surface of mouse cells, these cells could be infected by the MuLV. However, human cells acquire the susceptibility to infection by MuLV only if the cells express mCAT-1 ectopically. Studies of Albritton et al. have shown that amino acids in the extracellular loop three of mCAT-1 are critical for virus binding [6].

Receptor expression

To provide optimal conditions for viral infection, we determined the best timepoint for transduction with most presenting receptor on the cell surface. Therefore we transfected HEK-293T cells with CAT-1 fused with a HA-tag. Cells expressing CAT-1 were analyzed after distinct incubation times.</p>

Expression time of the receptor that was transfected into HEK-293T cells. After transfection with mCAT-1-HA cells were lysed with RIPA buffer at distinct time points. A Western blot was performed using an anti-HA antibody.

We found that the expression of the receptor peaked at 24 h after transfection. In later experiments we used this time point for viral infections.

Localisation of CAT-1

We wanted to detect the localisation of CAT-1 and fused mCherry to its N-terminus. Using confocal microscopy we varified not only the protein on the cell surface but showed in a spatial way via several sectional planes how the receptore is expressed by HEK-293T cells.

Confocal pictures were taken with a 20x plan apo objective. Nuclear staining (DAPI) is shown in blue and the mCAT-1-mCherry in red.

Please click on the link below to view a spatial resolution of CAT-1 in a HEK-293T cell watching the presence of CAT-1 on the surface or inside the cells, like Golgi apparatus or Endoplasmic reticulum

https://static.igem.org/mediawiki/parts/6/6b/Freiburg2014_confocal_HEK293T_mCAT1.mp4

References

[1] Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. J. W. Kim, E. I. Closs, L. M. Albritton, J. M. Cunningham Nature. 1991 August 22; 352(6337): 725–728.
[2] Anemia and perinatal death result from loss of the murine ecotropic retrovirus receptor mCAT-1. Perkins CP, Mar V, Shutter JR, del Castillo J, Danilenko DM, Medlock ES, Ponting IL, Graham M, Stark KL, Zuo Y, Cunningham JM, Bosselman RA. Genes Dev. 1997 Apr 1;11(7):914-25.
[3] Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses. Albritton LM, Kim JW, Tseng L, Cunningham JM. J Virol. 1993 Apr;67(4):2091-6.
[4] Second site mutation in the virus envelope expands the host range of a cytopathic variant of Moloney murine leukemia virus, John Ferrarone, Ryan C. Knoper, Randolph Li, Christine A. Kozak, Virology. 2012 November 10; 433(1): 7–11
[5] Naturally Occurring Polymorphisms of the Mouse Gammaretrovirus Receptors CAT-1 and XPR1 Alter Virus Tropism and Pathogenicity,Christine A. Kozak, Adv Virol. 2011
[6] Albritton LM, Tseng L, Scadden D, Cunningham JM (1989). A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57:659-666.
[7] Louis J. Ignarro, Nitric Oxide: Biology and Pathobiology (2009).

Sequence and Features