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LCMV glycosylation modulates viral fitness and cell tropism.

ABSTRACT: The glycoprotein (GP) of arenaviruses is glycosylated at 11 conserved N-glycosylation sites. We constructed recombinant lymphocytic choriomeningitis virus (rLCMV) featuring either additions or deletions of these N-glycans to investigate their role in the viral life cycle. N-glycosylation at two sites, T87 and S97, were found to be necessary to rescue rLCMV. Three of nine successfully rescued mutants, S116A, T234A, and S373A, under selective pressures in either epithelial, neuronal, or macrophage cells reverted to WT sequence. Of the seven stable N-glycan deletion mutants, five of these led to altered viral fitness and cell tropism, assessed as growth in either mouse primary cortical neurons or bone marrow derived macrophages. These results demonstrate that the deletion of N-glycans in LCMV GP may confer an advantage to the virus for infection of neurons but a disadvantage in macrophages.

SUBMITTER: Bonhomme CJ

PROVIDER: S-EPMC3538765 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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LCMV glycosylation modulates viral fitness and cell tropism.

Bonhomme Cyrille J CJ Knopp Kristeene A KA Bederka Lydia H LH Angelini Megan M MM Buchmeier Michael J MJ

PloS one 20130107 1

The glycoprotein (GP) of arenaviruses is glycosylated at 11 conserved N-glycosylation sites. We constructed recombinant lymphocytic choriomeningitis virus (rLCMV) featuring either additions or deletions of these N-glycans to investigate their role in the viral life cycle. N-glycosylation at two sites, T87 and S97, were found to be necessary to rescue rLCMV. Three of nine successfully rescued mutants, S116A, T234A, and S373A, under selective pressures in either epithelial, neuronal, or macrophage ...[more]

PMID: 23308183

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Project description:BackgroundThe 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 high or low affinity to α-DG and evaluated their properties in vitro and in vivo.MethodsWe pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-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.ResultsIn mice, hepatic α- and β-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 α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-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 β-galactosidase staining.ConclusionsThese 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.AbbreviationsArmstrong 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 β-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).

Project description:HIV-1 is highly variable genetically and at protein level, a property it uses to subvert antiviral immunity and treatment. The aim of this study was to assess if HIV subtype differences were associated with variations in glycosylation patterns and co-receptor tropism among HAART patients experiencing different virologic treatment outcomes.A total of 118 HIV env C2V3 sequence isolates generated previously from 59 Kenyan patients receiving highly active antiretroviral therapy (HAART) were examined for tropism and glycosylation patterns. For analysis of Potential N-linked glycosylation sites (PNGs), amino acid sequences generated by the NCBI's Translate tool were applied to the HIVAlign and the N-glycosite tool within the Los Alamos Database. Viral tropism was assessed using Geno2Pheno (G2P), WebPSSM and Phenoseq platforms as well as using Raymond's and Esbjörnsson's rules. Chi square test was used to determine independent variables association and ANOVA applied on scale variables.At respective False Positive Rate (FPR) cut-offs of 5% (p?=?0.045), 10% (p?=?0.016) and 20% (p?=?0.005) for CXCR4 usage within the Geno2Pheno platform, HIV-1 subtype and viral tropism were significantly associated in a chi square test. Raymond's rule (p?=?0.024) and WebPSSM (p?=?0.05), but not Phenoseq or Esbjörnsson showed significant associations between subtype and tropism. Relative to other platforms used, Raymond's and Esbjörnsson's rules showed higher proportions of X4 variants, while WebPSSM resulted in lower proportions of X4 variants across subtypes. The mean glycosylation density differed significantly between subtypes at positions, N277 (p?=?0.034), N296 (p?=?0.036), N302 (p?=?0.034) and N366 (p?=?0.004), with HIV-1D most heavily glycosylated of the subtypes. R5 isolates had fewer PNGs than X4 isolates, but these differences were not significant except at position N262 (p?=?0.040). Cell-associated isolates from virologic treatment success subjects were more glycosylated than cell-free isolates from virologic treatment failures both for the NXT (p?=?0.016), and for all the patterns (p?=?0.011).These data reveal significant associations of HIV-1 subtype diversity, viral co-receptor tropism, viral suppression and envelope glycosylation. These associations have important implications for designing therapy and vaccines against HIV. Heavy glycosylation and preference for CXCR4 usage of HIV-1D may explain rapid disease progression in patients infected with these strains.

Project description:Influenza virus tissue tropism defines the host cells and tissues that support viral replication and contributes to determining which regions of the respiratory tract are infected in humans. The location of influenza virus infection along the respiratory tract is a key determinant of virus pathogenicity and transmissibility, which are at the basis of influenza burdens in the human population. As the pathogenicity and transmissibility of influenza virus ultimately determine its reproductive fitness at the population level, strong selective pressures will shape influenza virus tissue tropisms that maximize fitness. At present, the relationships between influenza virus tissue tropism within hosts and reproductive fitness at the population level are poorly understood. The selective pressures and constraints that shape tissue tropism and thereby influence the location of influenza virus infection along the respiratory tract are not well characterized. We use mathematical models that link within-host infection dynamics in a spatially-structured human respiratory tract to between-host transmission dynamics, with the aim of characterizing the possible selective pressures on influenza virus tissue tropism. The results indicate that spatial heterogeneities in virus clearance, virus pathogenicity or both, resulting from the unique structure of the respiratory tract, may drive optimal receptor binding affinity--that maximizes influenza virus reproductive fitness at the population level--towards sialic acids with ?2,6 linkage to galactose. The expanding cell pool deeper down the respiratory tract, in association with lower clearance rates, may result in optimal infectivity rates--that likewise maximize influenza virus reproductive fitness at the population level--to exhibit a decreasing trend towards deeper regions of the respiratory tract. Lastly, pre-existing immunity may drive influenza virus tissue tropism towards upper regions of the respiratory tract. The proposed framework provides a new template for the cross-scale study of influenza virus evolutionary and epidemiological dynamics in humans.

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LCMV glycosylation modulates viral fitness and cell tropism.

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LCMV glycosylation modulates viral fitness and cell tropism.

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