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Role of LAMP1 Binding and pH Sensing by the Spike Complex of Lassa Virus.

ABSTRACT: To effectively infect cells, Lassa virus needs to switch in an endosomal compartment from its primary receptor, α-dystroglycan, to a protein termed LAMP1. A unique histidine triad on the surface of the receptor-binding domain from the glycoprotein spike complex of Lassa virus is important for LAMP1 binding. Here we investigate mutated spikes that have an impaired ability to interact with LAMP1 and show that although LAMP1 is important for efficient infectivity, it is not required for spike-mediated membrane fusion per se Our studies reveal important regulatory roles for histidines from the triad in sensing acidic pH and preventing premature spike triggering. We further show that LAMP1 requires a positively charged His230 residue to engage with the spike complex and that LAMP1 binding promotes membrane fusion. These results elucidate the molecular role of LAMP1 binding during Lassa virus cell entry and provide new insights into how pH is sensed by the spike.

Importance

Lassa virus is a devastating disease-causing agent in West Africa, with a significant yearly death toll and severe long-term complications associated with its infection in survivors. In recent years, we learned that Lassa virus needs to switch receptors in a pH-dependent manner to efficiently infect cells, but neither the molecular mechanisms that allow switching nor the actual effects of switching were known. Here we investigate the activity of the viral spike complex after abrogation of its ability to switch receptors. These studies inform us about the role of switching receptors and provide new insights into how the spike senses acidic pH.

SUBMITTER: Cohen-Dvashi H

PROVIDER: S-EPMC5105667 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Role of LAMP1 Binding and pH Sensing by the Spike Complex of Lassa Virus.

Cohen-Dvashi Hadas H Israeli Hadar H Shani Orly O Katz Aliza A Diskin Ron R

Journal of virology 20161028 22

To effectively infect cells, Lassa virus needs to switch in an endosomal compartment from its primary receptor, α-dystroglycan, to a protein termed LAMP1. A unique histidine triad on the surface of the receptor-binding domain from the glycoprotein spike complex of Lassa virus is important for LAMP1 binding. Here we investigate mutated spikes that have an impaired ability to interact with LAMP1 and show that although LAMP1 is important for efficient infectivity, it is not required for spike-media ...[more]

PMID: 27605678

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Project description:UnlabelledLassa virus is a notorious human pathogen that infects many thousands of people each year in West Africa, causing severe viral hemorrhagic fevers and significant mortality. The surface glycoprotein of Lassa virus mediates receptor recognition through its GP1 subunit. Here we report the crystal structure of GP1 from Lassa virus, which is the first representative GP1 structure for Old World arenaviruses. We identify a unique triad of histidines that forms a binding site for LAMP1, a known lysosomal protein recently discovered to be a critical receptor for internalized Lassa virus at acidic pH. We demonstrate that mutation of this histidine triad, which is highly conserved among Old World arenaviruses, impairs LAMP1 recognition. Our biochemical and structural data further suggest that GP1 from Lassa virus may undergo irreversible conformational changes that could serve as an immunological decoy mechanism. Together with a variable region that we identify on the surface of GP1, those could be two distinct mechanisms that Lassa virus utilizes to avoid antibody-based immune response.ImportanceStructural data at atomic resolution for viral proteins is key for understanding their function at the molecular level and can facilitate novel avenues for combating viral infections. Here we used X-ray protein crystallography to decipher the crystal structure of the receptor-binding domain (GP1) from Lassa virus. This is a pathogenic virus that causes significant illness and mortality in West Africa. This structure reveals the overall architecture of GP1 domains from the group of viruses known as the Old World arenaviruses. Using this structural information, we elucidated the mechanisms for pH switch and binding of Lassa virus to LAMP1, a recently identified host receptor that is critical for successful infection. Lastly, our structural analysis suggests two novel immune evasion mechanisms that Lassa virus may utilize to escape antibody-based immune response.

Project description:UnlabelledArenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Africa and South America. Without effective antiviral therapies or vaccines, these viruses pose serious public health and biodefense concerns. Chemically distinct small-molecule inhibitors of arenavirus entry have recently been identified and shown to act on the arenavirus envelope glycoprotein (GPC) to prevent membrane fusion. In the tripartite GPC complex, pH-dependent membrane fusion is triggered through a poorly understood interaction between the stable signal peptide (SSP) and the transmembrane fusion subunit GP2, and our genetic studies have suggested that these small-molecule inhibitors act at this interface to antagonize fusion activation. Here, we have designed and synthesized photoaffinity derivatives of the 4-acyl-1,6-dialkylpiperazin-2-one class of fusion inhibitors and demonstrate specific labeling of both the SSP and GP2 subunits in a native-like Lassa virus (LASV) GPC trimer expressed in insect cells. Photoaddition is competed by the parental inhibitor and other chemically distinct compounds active against LASV, but not those specific to New World arenaviruses. These studies provide direct physical evidence that these inhibitors bind at the SSP-GP2 interface. We also find that GPC containing the uncleaved GP1-GP2 precursor is not susceptible to photo-cross-linking, suggesting that proteolytic maturation is accompanied by conformational changes at this site. Detailed mapping of residues modified by the photoaffinity adducts may provide insight to guide the further development of these promising lead compounds as potential therapeutic agents to treat Lassa hemorrhagic fever.ImportanceHemorrhagic fever arenaviruses cause lethal infections in humans and, in the absence of licensed vaccines or specific antiviral therapies, are recognized to pose significant threats to public health and biodefense. Lead small-molecule inhibitors that target the arenavirus envelope glycoprotein (GPC) have recently been identified and shown to block GPC-mediated fusion of the viral and cellular endosomal membranes, thereby preventing virus entry into the host cell. Genetic studies suggest that these inhibitors act through a unique pH-sensing intersubunit interface in GPC, but atomic-level structural information is unavailable. In this report, we utilize novel photoreactive fusion inhibitors and photoaffinity labeling to obtain direct physical evidence for inhibitor binding at this critical interface in Lassa virus GPC. Future identification of modified residues at the inhibitor-binding site will help elucidate the molecular basis for fusion activation and its inhibition and guide the development of effective therapies to treat arenaviral hemorrhagic fevers.

Project description:Lassa virus (LASV) is a single-stranded RNA virus that has plagued the Sub-Saharan part of Africa, precisely Nigeria where various pathogenic strains with varied genomic isoforms have been identified. The human lysosomal-associated membrane protein 1 (LAMP1) is alternately required for the micropinocytosis of LASV. Therefore, it is of interest to understand the mechanism of action of the host LAMP1 with LASV protein during infection. The role of ST3 beta-galactoside alpha-2, 3-sialyltransferase 4 (ST3GAL4) in the interaction between LASV (glycoprotein) GP-C and the human LAMP1 is relevant in this context. Deposited curated protein sequences of both LAMP1 and LASV GP-C were retrieved for the study. The ST3GAL4 associated data was constructed and analysed from weighted network analysis to infer the function annotations and molecular mediators that characterize the LASV infection. The gene network shows that glycoprotein sialylation, sialyltransferase enzymatic activities and glycosphingolipid biosynthesis are linked with the ST3GAL4 function. However, the physical interaction of FAM 213A, CD8B molecule and proprotein convertase subtilisin/kexin type 1 inhibitor (PCSK1N) with ST3GAL4 is intriguing in this perspective. There are 11 glycosylated asparagine sequons of the human LAMP1 but only nine were assigned a sialylated glycan cap to mediate the LASV GP-C and LAMP1 interaction having exceeded a recommended glycosylation threshold of 0.5. Therefore, the sialylated glycans of the human LAMP1 are a total of nine and these sialylated glycans mediate the molecular recognition between LASV and LAMP1. This study therefore, predicts that there is a cellular interchange between N-linked glycosylation properties of the human LAMP1 and LASV glycoprotein, and sialylation functions of ST3GAL4 in LASV infectivity. Further studies and the clinical trial of this predictive model on the sialylated glycans of LAMP1 will facilitate the understanding of the LASV micropinocytosis process in host cells.

Dataset Information

Role of LAMP1 Binding and pH Sensing by the Spike Complex of Lassa Virus.

Importance

Publications

Role of LAMP1 Binding and pH Sensing by the Spike Complex of Lassa Virus.

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