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Altering ?-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism.

ABSTRACT: BACKGROUND:The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with ?-dystroglycan (?-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low ?-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to ?-DG and evaluated their properties in vitro and in vivo. METHODS:We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish ?-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals. RESULTS:In mice, hepatic ?- and ?-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized ?-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble ?-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and ?-galactosidase staining. CONCLUSIONS:These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer. ABBREVIATIONS:Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted ?-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50).

SUBMITTER: Dylla DE

PROVIDER: S-EPMC3080791 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism.

Dylla Douglas E DE Xie Litao L Michele Daniel E DE Kunz Stefan S McCray Paul B PB

Genetic vaccines and therapy 20110408

<h4>Background</h4>The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with hig ...[more]

PMID: 21477292

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Project description:BackgroundHendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4) containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP) gene encoding human immunodeficiency virus type-1 (HIV-1) genome in conjunction with the HeV and NiV fusion (F) and attachment (G) glycoproteins.ResultsFunctional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2) peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F.ConclusionsTogether, these results demonstrate that a specific henipavirus entry assay has been developed using NiV or HeV F and G glycoprotein pseudotyped reporter-gene encoding retrovirus particles. This assay can be conducted safely under BSL-2 conditions and will be a useful tool for measuring henipavirus entry and studying F and G glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors.

Dataset Information

Altering ?-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism.

Publications

Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism.

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