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

ABSTRACT: Glycosylated ?-dystroglycan (?-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 ?-DG, but many genes mutated in WWS remain unknown. To identify modifiers of ?-DG, we performed a haploid screen for Lassa virus entry, a hemorrhagic fever virus causing thousands of deaths annually that hijacks glycosylated ?-DG to enter cells. In complementary screens, we profiled cells for absence of ?-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 ?-DG. Our findings accentuate the complexity of this posttranslational feature and point out genes defective in dystroglycanopathies.

SUBMITTER: Jae LT

PROVIDER: S-EPMC3919138 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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

Jae Lucas T LT Raaben Matthijs M Riemersma Moniek M van Beusekom Ellen E Blomen Vincent A VA Velds Arno A Kerkhoven Ron M RM Carette Jan E JE Topaloglu Haluk H Meinecke Peter P Wessels Marja W MW Lefeber Dirk J DJ Whelan Sean P SP van Bokhoven Hans H Brummelkamp Thijn R TR

Science (New York, N.Y.) 20130321 6131

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 ...[more]

PMID: 23519211

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Project description:Lassa virus (LASV) is an Old World arenavirus responsible for hundreds of thousands of infections in West Africa every year. LASV entry into a variety of cell types is mediated by interactions with glycosyltransferase LARGE-modified O-linked glycans present on the ubiquitous receptor α-dystroglycan (αDG). However, cells lacking αDG are permissive to LASV infection, suggesting that alternative receptors exist. Previous studies demonstrated that the phosphatidylserine (PtdSer)-binding receptors Axl and Tyro3 along with C-type lectin receptors mediate αDG-independent entry. Here, we demonstrate that another PtdSer receptor, TIM-1, mediates LASV glycoprotein (GP)-pseudotyped virion entry into αDG-knocked-out HEK 293T and wild-type (WT) Vero cells, which express αDG lacking appropriate glycosylation. To investigate the mechanism by which TIM-1 mediates enhancement of entry, we demonstrate that mutagenesis of the TIM-1 IgV domain PtdSer-binding pocket abrogated transduction. Furthermore, the human TIM-1 IgV domain-binding monoclonal antibody ARD5 blocked transduction of pseudovirions bearing LASV GP in a dose-dependent manner. Finally, as we showed previously for other viruses that use TIM-1 for entry, a chimeric TIM-1 protein that substitutes the proline-rich region (PRR) from murine leukemia virus envelope (Env) for the mucin-like domain served as a competent receptor. These studies provide evidence that, in the absence of a functional αDG, TIM-1 mediates the entry of LASV pseudoviral particles through interactions of virions with the IgV PtdSer-binding pocket of TIM-1.IMPORTANCE PtdSer receptors, such as TIM-1, are emerging as critical entry factors for many enveloped viruses. Most recently, hepatitis C virus and Zika virus have been added to a growing list. PtdSer receptors engage with enveloped viruses through the binding of PtdSer embedded in the viral envelope, defining them as GP-independent receptors. This GP-independent entry mechanism should effectively mediate the entry of all enveloped viruses, yet LASV GP-pseudotyped viruses were previously found to be unresponsive to PtdSer receptor enhancement in HEK 293T cells. Here, we demonstrate that LASV pseudovirions can utilize the PtdSer receptor TIM-1 but only in the absence of appropriately glycosylated α-dystroglycan (αDG), the high-affinity cell surface receptor for LASV. Our studies shed light on LASV receptor utilization and explain why previous studies performed with α-DG-expressing cells did not find that LASV pseudovirions utilize PtdSer receptors for virus uptake.

Dataset Information

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

Publications

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

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