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TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus.

ABSTRACT: 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 ?-dystroglycan (?DG). However, cells lacking ?DG 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 ?DG-independent entry. Here, we demonstrate that another PtdSer receptor, TIM-1, mediates LASV glycoprotein (GP)-pseudotyped virion entry into ?DG-knocked-out HEK 293T and wild-type (WT) Vero cells, which express ?DG 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 ?DG, 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 ?-dystroglycan (?DG), the high-affinity cell surface receptor for LASV. Our studies shed light on LASV receptor utilization and explain why previous studies performed with ?-DG-expressing cells did not find that LASV pseudovirions utilize PtdSer receptors for virus uptake.

SUBMITTER: Brouillette RB

PROVIDER: S-EPMC6069209 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus.

Brouillette Rachel B RB Phillips Elisabeth K EK Patel Radhika R Mahauad-Fernandez Wadie W Moller-Tank Sven S Rogers Kai J KJ Dillard Jacob A JA Cooney Ashley L AL Martinez-Sobrido Luis L Okeoma Chioma C Maury Wendy W

Journal of virology 20180731 16

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 Ax ...[more]

PMID: 29875238

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Project description:Lassa virus (LASV) is an enveloped RNA virus endemic to West Africa and responsible for severe cases of hemorrhagic fever. Virus entry is mediated by the glycoprotein complex consisting of a stable-signal peptide, a receptor-binding subunit, GP1, and a viral-host membrane fusion subunit, GP2. Several cellular receptors can interact with the GP1 subunit and mediate viral entry, including alpha-dystroglycan (?DG) and lysosome-associated membrane protein 1 (LAMP1). In order to define the regions within GP1 that interact with the cellular receptors, we implemented insertional mutagenesis, carbohydrate shielding, and alanine scanning mutagenesis. Eighty GP constructs were engineered and evaluated for GP1-GP2 processing, surface expression, and the ability to mediate cell-to-cell fusion after low-pH exposure. To examine virus-to-cell entry, 49 constructs were incorporated onto vesicular stomatitis virus (VSV) pseudoparticles and transduction efficiencies were monitored in HAP1 and HAP1-?DAG1 cells that differentially produce the ?DG cell surface receptor. Seven constructs retained efficient transduction in HAP1-?DAG1 cells yet poorly transduced HAP1 cells, suggesting that they are involved in ?DG utilization. Residues H141, N146, F147, and Y150 cluster at the predicted central core of the trimeric interface and are important for GP-?DG interaction. Additionally, H92A-H93A, 150HA, 172HA, and 230HA displayed reduced transduction in both HAP1 and HAP1-?DAG1 cells, despite efficient cell-to-cell fusion activity. These mutations may interfere with interactions with the endosomal receptor LAMP1 or interfere at another stage in entry that is common to both cell lines. Insight gained from these data can aid in the development of more-effective entry inhibitors by blocking receptor interactions.IMPORTANCE Countries in which Lassa virus is endemic, such as Nigeria, Sierra Leone, Guinea, and Liberia, usually experience a seasonal outbreak of the virus from December to March. Currently, there is neither a preventative vaccine nor a therapeutic available to effectively treat severe Lassa fever. One way to thwart virus infection is to inhibit interaction with cellular receptors. It is known that the GP1 subunit of the Lassa glycoprotein complex plays a critical role in receptor recognition. Our results highlight a region within the Lassa virus GP1 protein that interacts with the cellular receptor alpha-dystroglycan. This information may be used for future development of new Lassa virus antivirals.

Project description:UnlabelledT-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members were recently identified as phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance entry of Ebola virus (EBOV) and other viruses by binding PtdSer on the viral envelope, concentrating virus on the cell surface, and promoting subsequent internalization. The PtdSer-binding activity of the immunoglobulin-like variable (IgV) domain is essential for both virus binding and internalization by TIM-1. However, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry, indicating that other domains of TIM proteins are functionally important. Here, we investigate the domains supporting enhancement of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer-binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs.ImportanceT-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members are recently identified phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance virus entry by binding the phospholipid, PtdSer, present on the viral membrane. While it is known that the PtdSer binding is essential for the PVEER function of TIM-1, TIM-3 shares this binding activity but does not enhance virus entry. No comprehensive studies have been done to characterize the other domains of TIM-1. In this study, using a variety of chimeric proteins and deletion mutants, we define the features necessary for a functional PVEER. With these features in mind, we generated a TIM-1 mimic using functionally similar domains from other proteins. This mimic, like TIM-1, effectively enhanced transduction. These studies provide insight into the key features necessary for PVEERs and will allow for more effective identification of unknown PVEERs.

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TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus.

Publications

TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus.

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