These properties of the GPs do not necessarily reflect a tropism of the pathogenic viruses for human T cells, since viral tropism is influenced by many factors and T cells are not a target for JUNV replication in vivo (3,22,25). == Generation of chimeric GPs. direct entry into host cells, and retroviral vectors pseudotyped with GP recapitulate the entry pathway of these viruses (5,13,24,31). GP is a class I fusion protein comprising two subunits, GP1 and GP2, cleaved from the precursor protein GPC (4,14,16,18,21). GP1 contains the receptor binding domain (19,28), while GP2 contains structural elements characteristic of viral membrane fusion proteins (8,18,20,38). The N-terminal stable signal peptide (SSP) remains associated with the mature glycoprotein after cleavage (2,39) and plays a role in transport, maturation, and pH-dependent fusion (17,35,36,37). The New World arenaviruses are divided into clades A, B, and C based on phylogenetic relatedness (7,9,11). Clade B contains the human pathogenic viruses Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia, and Chapare, which cause severe hemorrhagic fevers in ESI-05 South America (1,10,15,26,34). Clade B also contains the nonpathogenic viruses Amapari (AMAV), Cupixi, and Tacaribe (TCRV), although mild disease has been reported for a laboratory worker infected with TCRV (29). Studies with both viruses and GP-pseudotyped retroviral vectors have shown that the pathogenic clade B arenaviruses use the human transferrin receptor type 1 (hTfR1) to gain entry into human cells (19,30). In contrast, GPs Pfdn1 from nonpathogenic viruses, although capable of using TfR1 orthologs from other species (1), cannot use hTfR1 (1,19) and instead enter human cells through as-yet-uncharacterized hTfR1-independent ESI-05 pathways (19). In addition, human T-cell lines serve as useful tools to distinguish these GPs, since JUNV, GTOV, and MACV pseudotyped vectors readily transduce CEM cells, while TCRV and AMAV GP vectors do not (27; also ESI-05 unpublished data). These properties of the GPs do not necessarily reflect a tropism of the pathogenic viruses for human T cells, since viral tropism is influenced by many factors and T cells are not a target for JUNV replication in vivo (3,22,25). == Generation of chimeric GPs. == At present, there is limited information about the determinants of receptor binding within the arenavirus GP1. Previous studies using GP1 immunoadhesins (IMs) have suggested that the N-terminal 22 amino acids and C-terminal 10 amino acids of clade B GP1s are dispensable for receptor binding (1,19,27,30). To further map the regions involved in the interactions that are characteristic of the pathogenic clade B viruses, we generated a series of chimeric GPs based on the exchange of GP1 protein segments between closely related pathogenic and nonpathogenic viruses from the same sublineage, specifically the B1 viruses JUNV and TCRV and the B2 viruses GTOV and AMAV (9,11). Sequence ESI-05 positioning of representative New World GP1 proteins using the Clustal X software (12) exposed four completely conserved cysteine residues (Fig.1A) that will also be conserved in the Old World arenaviruses. The cysteines were used to delineate 5 segments within GP1, which were swapped to generate the chimera series T(J), comprising JUNV sequences put into a TCRV GP backbone, and the GTOV/AMAV chimeras A(G) and G(A). The crystal structure of a truncated GP1 from MACV (residues 87 to 239) has recently been resolved (6), revealing that conserved cysteines 1 and 4 and conserved cysteines 2 and 3 form disulfide bonds (Fig.1B). Given the close evolutionary relatedness between the GPs of the clade B arenaviruses and the similarity in the patterns of expected secondary constructions across GP1 (data not demonstrated), the structural info available for the MACV GP1 is likely to be a useful blueprint for the JUNV and GTOV GP1 constructions, although as mentioned by Bowden and coworkers (6), there is considerable variance in the location of expected N-glycosylation sites, which suggests that structural variance will exist in the carbohydrate-rich regions of the protein. == FIG. 1. == (A) Sequence positioning of GP1 sequences from representative arenaviruses in New World clades A, B, and C. The positions of four completely conserved cysteines (C1 to C4) residues are demonstrated, which were used to delineate five unique segments in GP1. The sequences used were Pichinde disease (PICV) (GenBank accession no.P31840), GTOV (GenBank accession no.Q8AYW1), AMAV (GenBank accession no.YP_001649208), and Oliveros disease (OLVV) (GenBank accession no.Q84168). Gaps introduced to maximize positioning are indicated by dashes. (B) Locations of segments within MACV GP1 ESI-05 crystal structure. Schematic of MACV.