New World arenavirus clade C, but not clade A and B viruses, utilizes alpha-dystroglycan as its major receptor

Abstract

Alpha-dystroglycan (alpha-DG) has been identified as a major receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa virus, two Old World arenaviruses. The situation with New World arenaviruses is less clear: previous studies demonstrated that Oliveros virus also exhibited high-affinity binding to alpha-DG but that Guanarito virus did not. To extend these initial studies, several additional Old and New World arenaviruses were screened for entry into mouse embryonic stem cells possessing or lacking alpha-DG. In addition, representative viruses were further analyzed for direct binding to alpha-DG by means of a virus overlay protein blot assay technique. These studies indicate that Old World arenaviruses use alpha-DG as a major receptor, whereas, of the New World arenaviruses, only clade C viruses (i.e., Oliveros and Latino viruses) use alpha-DG as a major receptor. New World clade A and B arenaviruses, which include the highly pathogenic Machupo, Guanarito, Junin, and Sabia viruses, appear to use a different receptor or coreceptor for binding. Previous studies with LCMV have suggested the need for a small aliphatic amino acid at LCMV GP1 glycoprotein amino acid position 260 to allow high-affinity binding to alpha-DG. As reported herein, this requirement appears to be broadly applicable to the arenaviruses as determined by more extensive analysis of alpha-DG receptor usage and GP1 sequences of Old and New World arenaviruses. In addition, GP1 amino acid position 259 also appears to be important, since all arenaviruses showing high-affinity alpha-DG binding possess a bulky aromatic amino acid (tyrosine or phenylalanine) at this position.

FIG. 1.

New World arenavirus infection of mouse ES cells expressing or not expressing α-DG. Virus-specific immunofluorescence staining of mouse ES cells infected with representative viruses of the different clades of the New World arenaviruses. Cells expressing α-DG (A, C, E, and G) are labeled α-DG^+/+. α-DG null mutant cells (B, D, F, and H) are labeled α-DG^−/−. Viruses: Amapari (A and B) as a clade B representative, Parana (G and H) as a clade A representative, and Latino (C and D) and Oliveros (E and F) as members of clade C. As would be expected, the percentages of virus-positive cells in α-DG^+/+ versus α-DG^−/− cells per optical field for α-DG-binding viruses, Oliveros and Latino, were 97 and 98%, respectively, and 47 and 52% for the non-α-DG-binding viruses, Amapari and Parana, respectively.

FIG. 2.

Comparison of the binding affinities for α-DG between Old and New World arenaviruses. α-DG purified from rabbit skeletal muscle was separated by SDS-PAGE and blotted to nitrocellulose. The Old World arenaviruses LCMV WE54, LCMV ARM53b, and Lassa virus (LFV) (A) were applied at 10⁷ PFU/ml and detected by the monoclonal anti-LCMV GP2 antibodies 33.6 and 86.6, using an HRP-conjugated anti-mouse IgG secondary antibody and ECL substrate. The New World arenaviruses Oliveros, Latino, Amapari, and Parana (B), each shown in a duplicate lane and used at a concentration of 10⁷ PFU/ml, were detected by a 1:1,000 dilution of mouse hyperimmune serum against New World arenaviruses and an HRP-conjugated secondary antibody as for panel A.

FIG. 3.

Detection of Oliveros, Latino, Amapari, and Parana viruses by dot blot assay. Five microliters (upper row) and 2.5 μl (lower row) of 10⁷-PFU/ml Oliveros, Latino, Amapari, and Parana viruses were immobilized in nitrocellulose and detected with a 1:1,000 dilution of mouse hyperimmune serum against New World arenaviruses, using a peroxidase-conjugated secondary antibody and ECL substrate for detection.

FIG. 4.

Arenavirus phylogenetic analysis. An arenavirus phylogenetic tree was generated based on maximum-parsimony analysis of the sequence differences present among an aligned 637-nucleotide region of the virus genome S segments (6, 21). Analysis using the heuristic search option and a 3:1 weighting of transversions over transitions generated a single most-parsimonious tree. Horizontal distances represent nucleotide step differences (see bar scale), while vertical branches are for visual clarity only. The arenavirus S segment sequences included the following viruses: LCMV WE54 (GenBank accession number M22017), LCM Armstrong (M20869), Lassa AV (Af246121), Lassa Josiah (J04324), Lassa 803213 (x52400), Lassa LP (af181854), Lassa GA391 (af181853), Mobala (af012530), Mopeia (m33879), Ippy (u80003), White Water Arroyo (af228063), Tamiami (u43690), Allpahuayo (ay012687), Pichinde (k02734), Parana (u43689), Flexal (u43687), Pirital (af277659), Oliveros (u34248), Latino (u43688), Guanarito (u43686), Amapari (u43685), Machupo (x62616), Junin (d10072), Tacaribe (m20304), and Sabia (u41071). Two major clades are seen, corresponding to the Old and New World arenaviruses. The New World viruses form three major clades: A, B, and C. The high- or low-affinity α-DG binding of each virus tested is indicated adjacent to the virus label. ND, not done.

FIG. 5.

Possible sequence requirements on the GP1 for high-affinity binding to α-DG. Arenaviruses using α-DG as their major receptor have the small aliphatic amino acid leucine or isoleucine at position 260 (position numbering relative to LCMV) and a bulky aromatic amino acid, phenylalanine or tyrosine, at position 259 (presented in boldface). The consensus sequence cleavage site (tetrapeptide) between GP1 and GP2 of each arenavirus is shown underlined in italics.