Arenaviruses are a family of single-stranded, enveloped, bisegmented RNA viruses that include the Old World arenaviruses lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus, and the New World arenaviruses Machupo, Junin, and Guanarito. LCMV glycoproteins can pseudotype retroviral and HIV-based lentiviral vectors facilitating studies of virus biology and gene transfer [1–3]. Encoded by the small RNA fragment, the arenavirus glycoprotein precursor (GP-C) is post-translationally cleaved to yield GP1 and GP2. GP1 is believed to be responsible for receptor binding followed by a pH-dependent fusion step mediated by GP2 [4, 5]. Several Old World arenaviruses utilize α-dystroglycan (α-DG) as a viral receptor . LCMV is the prototypic Old World arenavirus, with different strains displaying either high or low affinity for α-DG. Alpha-DG expression is developmentally regulated, displaying the highest expression levels in developing tissues involved in basement membrane assembly . Expression rises during embryonic stages, peaks in the newborn, and diminishes in adult tissues [8, 9]. Here we develop and investigate the use of LCMV envelope glycoproteins with high or low α-DG affinity for lentiviral gene transfer applications.
α-DG is a ubiquitously expressed, versatile, evolutionarily conserved cell surface receptor that links the extracellular matrix with the cytoskeleton, making it an ideal target for pathogen binding [7, 10]. The dystroglycan complex is transcribed as a precursor peptide that undergoes post-translational cleavage to produce α- and β-DG. Noncovalently linked, α- and β-DG act as peripheral and transmembrane proteins, respectively. Interestingly, α-DG usage correlates with persistent infection, disease kinetics, and tropism . The immunosuppressive LCMV isolates WE54, LCMV Cl13, and Traub efficiently target antigen presenting cells (dendritic cells) in the spleen and perturb their ability to present antigen to T cells and B cells, resulting in a generalized immunosuppression of the host allowing viral persistence. These three LCMV strains bind to α-DG with high affinity, unlike non-immunosuppressive LCMV variants Armstrong 53b, CD4-1, CD8-4 and WE2.2, which demonstrate low affinity. LCMV Arm53b and WE2.2 replicate primarily in the splenic red pulp and infection is rapidly resolved. The tropism differences between LCMV strains, as well as significant infection of DG-/- ES cells by non-immunosuppressive LCMV variants  suggest that an alternate and currently unidentified viral receptor is utilized by LCMV strains with low affinity for α-DG .
LCMV Arm53b and clone13 are nearly genetically identical with the exception of two amino acid changes, one occurring in the viral polymerase encoded by the large RNA fragment and the other at position 260 of GP1 . LCMV WE54 and WE2.2 are also genetically similar with only one amino acid difference at position 153 of GP1. Genetic and phenotypic comparison of the New World arenavirus and LCMV variants led to the observation that amino acid 260 of GP1 plays an important role in their ability to utilize α-DG [12, 15]. Spiropoulou et al. generalized that a leucine or isoleucine residue at position 260 was required for high α-DG affinity, while bulky aromatic residues such as phenylalanine or tyrosine generally resulted in low affinity . A similar change in affinity was observed between WE54 and WE2.2, the result of a serine to phenylalanine mutation at position 153 of GP1. Proper glycosylation, specifically O-mannosylation , and modifications of LARGE glycosyltransferase  also play important roles in receptor recognition by LCMV. However, LCMV isolates can transduce α-DG-/- mouse ES cells with reduced efficacy, indicating that α-DG dependence is not absolute [3, 11, 19]. Utilization of an alternate receptor cannot be ruled out as a possibility.
Among the factors potentially limiting the utility of pseudotyped vectors are low vector titers, envelope instability following ultracentrifugation, glycoprotein cytotoxicity, and limited tropism. Able to withstand ultracentrifugation, LCMV glycoproteins yield MLV and HIV vector titers similar to the widely used amphotropic and VSV-G envelopes [1–3]. Stable cell lines constitutively expressing the WE54 envelope have been generated, demonstrating that the LCMV envelope exhibits little cytotoxicity in comparison to the VSV-G envelope . Cannon and colleagues previously demonstrated successful pseudotyping of MLV-based retroviral vectors with the Armstrong 53b (Arm53b) envelope glycoprotein, and generated the F260L mutation in the Arm53b GP1 to generate a clone 13-like envelope with high α-DG affinity . They successfully used these and other pseudotypes to investigate receptor use and α-DG affinity among several Old and New World arenavirus envelopes in vitro . In this report we show that LCMV envelopes efficiently pseudotype a non-primate FIV lentiviral vector and maintain the entry properties seen in wild-type arenaviruses. Furthermore, we modified the WE54 LCMV envelope with the GP1 mutation L260F and altered vector α-DG affinity. Here we investigate the expression of α- and β-DG in the liver at pre- and postnatal time points and document the in vivo tissue tropisms of these LCMV pseudotypes in neonatal and adult mice. We hypothesized that a FIV vector pseudotyped with a LCMV envelope glycoprotein with low α-DG binding affinity would yield unique in vivo tissue tropism and enhance gene transfer efficiency in neonatal animals.