Deciphering the glycosylome of dystroglycanopathies using haploid screens for lassa virus entry

Abstract

Glycosylated α-dystroglycan (α-DG) serves as cellular entry receptor for multiple pathogens, and defects in its glycosylation cause hereditary Walker-Warburg syndrome (WWS). At least eight proteins are critical to glycosylate α-DG, but many genes mutated in WWS remain unknown. To identify modifiers of α-DG, we performed a haploid screen for Lassa virus entry, a hemorrhagic fever virus causing thousands of deaths annually that hijacks glycosylated α-DG to enter cells. In complementary screens, we profiled cells for absence of α-DG carbohydrate chains or biochemically related glycans. This revealed virus host factors and a suite of glycosylation units, including all known Walker-Warburg genes and five additional factors critical for the modification of α-DG. Our findings accentuate the complexity of this posttranslational feature and point out genes defective in dystroglycanopathies.

Fig. 1. Haploid genetic screen for cellular host factors required for rVSV-GP-LASV infection

Significance of enrichment of gene-trap insertions in the virus-selected population Jae, page 7 compared with nonselected control cells is indicated on the y axis. Bubbles represent genes, and bubble size corresponds to the number of independent gene-trap events observed in the virus-selected population. Significant hits are grouped by function horizontally (other genes in random order). Genes carrying the majority of gene-trap insertions in introns were colored if they passed two statistical tests: enrichment of disruptive mutations compared with control cells (one-sided Fisher’s exact test, P ≤ 10⁻⁵) and bias for gene trap–insertion events in the transcriptional orientation of the affected gene (binomial test, P ≤ 0.05). Intron-poor genes were colored if they passed the former criterion using a stricter cut-off (one-sided Fisher’s exact test P ≤ 10⁻³⁰) (13). Data are displayed until −log(P value) = 0.001.

Fig. 2. Cell surface profiling of mutagenized haploid HAP1 cells

(A) Genes enriched for mutations in a cell population depleted for glycosylated α-DG at the cell surface. The cell population enriched for mutants lacking glycosylated α-DG at the cell surface was analyzed and depicted as described in Fig. 1. (B) A mutant cell population selected for diminished cell surface heparan sulfate was obtained as described above. Data were analyzed as previously, except that, for intron-rich genes, the cut-off for disruptive mutations compared with control cells was adjusted (one-sided Fisher’s exact test, P ≤ 10⁻²¹) (13).

Fig. 3. TALEN-induced mutations in identified genes affect susceptibility to rVSV-GP-LASV

(A) HAP1 cells transfected with TALENs display frameshift mutations and/or introduce premature stop codons in targeted genes. Sequences recognized by the TALENs are displayed in red and blue. (B) The HAP1 cell lines with TALEN-induced mutations in the corresponding genes and wild-type control cells were infected with rVSV-GP-LASV [infected cells express enhanced green fluorescent protein (eGFP)].

Fig. 4. TMEM5 and SGK196 mutations found in patients with WWS and MEB lacking mutations in known WWS genes

(A) Pedigree structure of consanguineous, respectively first and second cousins, families 43 and 56 segregating a TMEM5 mutation. Family 43 has an affected male with features of WWS and a stillbirth, without available clinical records. Family 56 has two affected females with clinical features reminiscent of MEB (13). A nonsense mutation in exon 6 was identified in family 43. Family 56 harbors a frameshift mutation in exon 1. Both mutations were homozygously present in the patient(s) and heterozygously in the parents. The unaffected boy in family 43 is heterozygous for the mutation. IC, intracellular domain; TM, transmembrane domain; EC, extracellular domain; EF, exostosin family domain. (B) Cranial MRI of the oldest affected female of family 56 at the age of 1 year; sagittal cut (T1-weighted image): atrophy of pons and cerebellum; axial cut (flair image): fronto-parietal pachygyria, enlarged ventricles, and abnormal white matter. (C) Compound heterozygosity of mutant SGK196 in an affected patient. Both nonconsanguineous parents are heterozygous carriers of either mutation. KL, kinase-like domain (D) HAP1 cells with TALEN-induced disruption of endogenous TMEM5 or SGK196 were complemented with cDNAs encoding the mutant variants observed in patients and analyzed for presence of the α-DG laminin-binding epitope using flow cytometry.