Acidic pH Triggers Lipid Mixing Mediated by Lassa Virus GP

Abstract

<jats:p>Lassa virus (LASV) is the causative agent of Lassa hemorrhagic fever, a lethal disease endemic to Western Africa. LASV entry is mediated by the viral envelope glycoprotein (GP), a class I membrane fusogen and the sole viral surface antigen. Previous studies have identified components of the LASV entry pathway, including several cellular receptors and the requirement of endosomal acidification for infection. Here, we first demonstrate that incubation at a physiological temperature and pH consistent with the late endosome is sufficient to render pseudovirions, bearing LASV GP, non-infectious. Antibody binding indicates that this loss of infectivity is due to a conformational change in GP. Finally, we developed a single-particle fluorescence assay to directly visualize individual pseudovirions undergoing LASV GP-mediated lipid mixing with a supported planar bilayer. We report that exposure to endosomal pH at a physiologic temperature is sufficient to trigger GP-mediated lipid mixing. Furthermore, while a cellular receptor is not necessary to trigger lipid mixing, the presence of lysosomal-associated membrane protein 1 (LAMP1) increases the kinetics of lipid mixing at an endosomal pH. Furthermore, we find that LAMP1 permits robust lipid mixing under less acidic conditions than in its absence. These findings clarify our understanding of LASV GP-mediated fusion and the role of LAMP1 binding.</jats:p>

Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins

“…Because the five glycoproteins range in size, (e.g., SARS2 S protein is more than twice the size of VSV G), we wanted to confirm that the delay in luciferase production correlated to the virions fusing with the endosomal membrane. All five of the glycoproteins enter VeroS cells through an endosomal route, and fusion is triggered through low-pH-dependent processes [ 9 , 20 , 25 , 59 , 60 ]. Therefore, we infected VeroS cells at an MOI of 1 and added ammonium chloride (NH 4 Cl), a lysosomotropic agent, that quickly prevents endosomal acidification and should prevent luciferase production if the virions have not undergone fusion.…”

“…While all five viral glycoproteins require low pH for entry, LASV GP and EBOV GP require endosomal receptor binding and EBOV GP requires proteolytic processing [ 15 , 20 ]. Because we observed that rVSV∆G/EBOV entry was slower than rVSV∆G/LASV entry, we examined if EBOV GP proteolytic processing delays rVSV∆G/EBOV entry.…”

“…Once the virion is within the endolysosomal compartment, low pH and Lysosomal Associated Membrane Protein 1 (LAMP1) induce a conformational change in the glycoprotein [ 15 ] resulting in fusion with the endosomal membrane [ 18 ]. While many fusion proteins are triggered by low pH (pH < 6), optimal LASV GP fusion activity requires highly acidic conditions (pH 4.5) ( Figure 1 ) [ 19 , 20 ].…”

Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins

“…SARS2 S protein is more than twice the size of VSV G), we wanted to confirm that the delay in luciferase production correlated to the virions fusing with the endosomal membrane. All five of the glycoproteins enter VeroS cells through an endosomal route, and fusion is triggered through low pH dependent processes [9,20,25,55,56]. Therefore we infected VeroS cells at MOI 1 and added ammonium chloride (NH4Cl), a lysosomotropic agent, that quickly prevents endosomal acidification and should prevent luciferase production if the virions have not undergone fusion.…”

The late endosome-resident lipid bis(monoacylglycero)phosphate is a cofactor for Lassa virus fusion

“…However, some arenaviruses can infect cells lacking the known receptors, albeit less efficiently (29–32). Although LAMP1 promotes LASV entry/fusion, it is not strictly required for the GPC fusion activity (31–33). Several lines of evidence indicate that acidic pH alone is sufficient to trigger LASV GPC conformational changes/functional inactivation (33), including GP1 dissociation from the GP2 (26) and fusion (31, 33) (but see (34) reporting LASV GPC resistance to acid treatment).…”