Identification of α-Dystroglycan as a Receptor for Lymphocytic Choriomeningitis Virus and Lassa Fever Virus

C. Peters, M. Buchmeier, P. Rollin, T. Ksiazek, in Fields Virology, B. Fields, Ed. (Lippincott-Raven, Philadelphia, 1996), vol. 2, pp. 1521–1551.

Fabiyi A., Bull. Pan Am. Health Organ. 10, 335 (1976);

Salas R., et al., Lancet 338, 1033 (1991).

Frame J., Baldwin J. J., Gocke D., Troup J., Am. J. Trop. Med. Hyg. 19, 670 (1970);

McCormick J., Webb P., Krebs J., Johnson K., Smith E., J. Infect. Dis. 155, 437 (1987).

P. Borrow and M. B. A. Oldstone, in Viral Pathogenesis, N. Nathanson, Ed. (Lippincott-Raven, Philadelphia, 1997), pp. 593–627;

Zinkernagel R. M., Science 271, 173 (1996);

; Scand. J. Immunol.46, 421 (1997);

Oldstone B. A., Fujinami R. S., Lampert P. W., Prog. Med. Virol. 26, 45 (1980);

Rowe W. P., Nav. Med. Res. Inst. Rev. Rep. 12, 167 (1954) .

Borrow P., Oldstone M., J. Virol. 66, 7270 (1992).

Burns J., Buchmeier M., Virology 183, 620 (1991).

Modified from (5). A goat antibody to mouse immunoglobulin G (anti-mouse IgG) conjugated with horseradish peroxidase (Pierce) [1:5000 dilution in phosphate-buffered saline (PBS)] was used to substitute rabbit anti-mouse IgG and I125–protein A. Afterward, an enhanced chemiluminescence assay was performed with the SuperSignal Chemiluminescent substrate (Pierce).

Monolayers of MC57 cells were dissociated with PBS and 5 mM EDTA. Cells were solubilized in a buffer containing 20 mM tris (pH 7.4), 0.15 M NaCl, 1.5% octyl-glucoside, 0.5 mM phenylmethylsulfonyl fluoride, and 1× Complete protease inhibitor cocktail (Boehringer Mannheim) for 60 min on ice. The sample was then spun for 15 min at 2500g, and the supernatant was centrifuged again for 60 min at 100,000g at 4°C. The cleared sample was loaded onto a Mono Q HR5/5 column (Pharmacia) and eluted with 1 M NaCl. Aliquots of the protein fractions were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) on 6% polyacrylamide gels for VOPBA analysis to locate the fractions enriched for LCMV-binding activity. The peak fractions were combined and passed through a 10-ml lentil-Sepharose column that did not retain the putative receptor of LCMV. Flow-through material was then loaded directly onto a 10-ml wheat germ agglutinin–Sepharose column, from which the protein was eluted with 0.5 M N-acetylglucosamine. Subsequently, VOPBA-positive fractions were combined and put through a 5-ml jacalin-Sepharose column. After the column was washed with 0.2 M melibiose, 1 M NaCl, 3 M KSCN, and 1% SDS to remove contaminating proteins, the whole column was disrupted and the Sepharose was boiled in SDS sample buffer with 2.5% β-mercaptoethanol for 5 min. The denatured sample was then concentrated fivefold by UltraFree-MC centricons (MilliPore) before resolved on a 6% SDS-polyacrylamide gel.

The Coomassie blue–stained protein band with VOPBA binding activity was excised from the SDS-polyacrylamide gel and submitted for peptide sequencing analysis. After in-gel digestion with trypsin, the sequences of five peptides were determined at the Harvard Microchemistry Facility by collisionally activated dissociation on a Finnigan TSQ7000 Triple Quadrupole Mass Spectrometer. GT384 (AGDPAPVVNDIHK) (24), GT441 (GGLSAVDAFEIHVHK), and GT417 (IPSDTFYDNEDTTTDKLK) were homologous to regions of DG precursor. GT429 (AFDDGAFTGIR) was homologous to amino acids 3 to 13 of jacalin and GT348 (KLERKLREK) to amino acids 31 to 39 of U1 small nuclear ribonucleoprotein.

Ibraghimov-Beskrovnaya O., et al., Nature 355, 696 (1992).

Henry M., Campbell K., Curr. Opin. Cell Biol. 8, 625 (1996).

Durbeej M., Henry M., Ferletta M., Campbell K., Ekblom P., J. Histochem. Cytochem. 46, 449 (1998).

Williamson R., et al., Hum. Mol. Genet. 6, 831 (1997).

P. Borrow, E. V. Ravkov, C. J. Peters, M. B. A. Oldstone, S. T. Nichol, in preparation.

Yamada H., et al., J. Neurochem. 66, 1518 (1996).

The α2 subunit of the dihdropyridine receptor complex was purified from rabbit skeletal muscle as described in

Jay S. D., et al., J. Biol. Chem. 266, 3287 (1991).

W. Cao et al., unpublished results.

We plated 1 × 10⁵ of 3T6 cells per well into a 24-well plate the day before the experiment. Aliquots of 1.6 × 10⁵ plaque-forming units of LCMV strain Cl 13 and VSV Indiana strain were incubated for 20 min on ice with either bovine serum albumin (BSA) or purified α-DG protein from rabbit skeletal muscle at concentrations of 0, 9 pM, 90 pM, 0.9 nM, 9 nM, and 90 nM in a final volume of 150 μl. One aliquot of treated virus was then incubated with cells for 30 min at 37°C before being replaced with fresh growth medium. Sixteen hours later, virus titers from culture supernatant were determined by plaque assay on Vero cells. Cells were fixed with acetone and analyzed by immunofluorescence staining with a monoclonal antibody (mAb) 1-1-3 to detect LCMV nucleoprotein or mAb I1 to detect VSV glycoprotein. A fluorescein isothiocyanate–conjugated goat anti-mouse IgG diluted 1:20 with PBS was used as the secondary antibody in the staining.

M. D. Henry and K. P. Campbell, Cell, in press. Briefly, DG^−/− ES cells were isolated from two independently derived parental DG^+/− ES cell clones TD354 and TD556 generated during the initial DG gene targeting effort by selection with active G418 (1.0 mg/ml). Surviving clones were analyzed by Southern (DNA) blot with a Hind III–Pst I fragment from intron 1 of the mouse DG gene as a probe. The DG adenovirus was constructed by subcloning a rabbit cDNA into the pAdRSVpA shuttle vector and then incorporated into an adenovirus vector through standard methods of homologous recombination. The loss of DG protein expression in clones exhibiting homozygous loss of the wild-type DG allele and expression of DG in DG null cells as a result of adenovirus infection were confirmed by protein immunoblot and immunofluorescence analysis.

We plated 2 × 10⁵ of ES cells per well into a 24-well plate the day before 2 × 10⁹ adenovirus particles carrying rabbit DG cDNA (estimated MOI of 5) was added in the culture medium. Two days later, aliquots of LCMV Cl 13 virus in 200 μl were added to each well. After incubation for 45 min at 37°C, the inoculum was replaced by 1 ml of fresh ES growth medium. Sixteen hours later, cells were fixed with acetone and immunostained with mAb 1-1-3 to detect the LCMV nucleoprotein.

J. Clegg, in The Arenaviridae, M. Salvato, Ed. (Plenum, New York, 1993), pp. 175–185.

Bowen M., Peters C., Nichol S., Virology 219, 285 (1996).

Fazakerley J., Southern P., Bloom F., Buchmeier M., J. Gen. Virol. 72, 1611 (1991).

Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

We thank W. Lane at Harvard Microchemistry facility for peptide sequencing; Y. Lu and H. Lewicki for technical assistance; J. C. de la Torre, M. Manchester, and J. Paulson for experimental advice and consultation; and J. C. de la Torre, M. Manchester, D. Naniche, I. Novella, S. Huang, C. Evans, and C. Lebakken for helpful discussions and comments on the manuscript. Supported by a research grant AI 09484 for M.B.A.O. and a training grant AG 00080 for W.C. from the NIH. M.D.H. was supported by a National Research Service Award (DK09712) and by the Iowa Cardiovascular Research Fellowship (HL071221). H.Y. was supported by American Heart Association Fellowship (IA-97-FW-25) and by the Mizutani Foundation. K.P.C. is an investigator of the Howard Hughes Medical Institute.