Date of Degree
PhD (Doctor of Philosophy)
Wendy J. Maury
First Committee Member
Second Committee Member
Third Committee Member
Fourth Committee Member
The family Arenaviridae consists of over 30 members, some of which can infect humans and cause severe hemorrhagic fever. The three arenaviruses studied in this thesis, Machupo virus (MACV), Junín virus (JUNV), and Lassa virus (LASV), are causative agents of Bolivian hemorrhagic fever, Argentine hemorrhagic fever, and Lassa fever, respectively. Epidemics of these diseases can carry high rates of morbidity and mortality, and due to a lack of available countermeasures, all three viruses are considered category A priority pathogens by the CDC.
Arenavirus glycoproteins (GPCs) are considered class I viral fusion proteins, but in multiple regards, they are quite unusual for viral envelope proteins. The GP precursor is translated as a polypeptide that is proteolytically processed within the secretory pathway by two sequential cleavage events to produce a tripartite complex (heterotrimers) that assemble into homotrimers to form heterononamers. The three distinct units of the GPC structure are a receptor binding domain, GP1, and fusion domain, GP2, and most peculiarly, a stable signal peptide (SSP) that traverses the membrane twice and associates with the GP2.
The glycoprotein complex (GPC) of MACV encodes nine putative sites for N-linked glycosylation. As N-glycans have been shown to be important for a multitude of factors in glycoprotein biology, we sought to investigate the potential roles of the N-glycans on MACV GPC expression, function, antigenicity, and immunogenicity. To do so, we used MACV GPC-VSVΔG-eGFP pseudovirions (MACV-VSV) to study the effects of both individual and combinatorial N-glycan losses. Our results demonstrate that loss of N-glycans at sites N178, N370, or N378 resulted in a loss of GPC proteolytic processing, while the accumulation of multiple N-glycans reduced total GPC expression and function. Replacement of the native proteolytic cleavage motif with those from LCMV or LASV or from furin reduced or eliminated such processing. While individual N-glycans did not themselves affect total GPC expression levels or translocation to the cell surface, those that allowed for efficient GP1-GP2 cleavage led to the favored incorporation of properly processed GPC, which strongly correlated with high virion production and transduction competence. Separate from these findings, we also discovered that loss of N-glycans dramatically increased the antigenicity of subsequent pseudovirions, and while N-glycan mutants retained immunogenicity, the resultant antisera was limited to autologous targets and was not able to inhibit WT MACV-VSV nor JUNV-VSV transduction.
The GPC of arenaviruses is the only antigen correlated with antibody-mediated neutralization, but despite strong cross-reactivity of convalescent antisera between related species, weak or no cross-neutralization occurs. Two closely related arenaviruses, MACV and JUNV, have near identical overall GPC architecture and share a host receptor, transferrin receptor 1. Given their extensive likeness, it is not clear how these two viruses avoid cross-neutralization. To address this, a series of MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (mAbs). All mAbs targeted the GP1, and those that neutralized JUNV-VSV transduction competed with each other for binding to the receptor binding site (RBS), specifically. Interestingly, these mAbs did not recognize MACV GPC in its native conformation, despite detecting the protein in western blots.
Mouse α-JUNV and α-MACV antisera were also evaluated for neutralization of the wild-type and chimeric JUNV- and MACV-VSV. These antisera neutralized pseudovirions containing the autologous wild-type GP1; however, removal of an RBS-adjacent small disulfide bonded loop unique to MACV GPC was sufficient to increase cross-neutralization with α-JUNV antisera. Our studies provide evidence that this additional loop in MACV GP1 is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV.
To enter cells, LASV binds to O-linked glycans present on the cell surface receptor, α-dystroglycan (αDG). While αDG is ubiquitously expressed, glycosylation patterns on some cells restrict the ability of LASV to use this receptor. Multiple studies have suggested that alternative receptors exist on these cell types and have provided evidence that the phosphatidylserine (PtdSer)-binding receptor Axl, along with C-type lectins, play a role in DG-independent entry. In studies presented here, we demonstrate that TIM-1 functions as a receptor for LASV-VSV entry, mediating virion uptake in a PtdSer-dependent, mucin-like domain-independent, manner despite previous reports of the inability of TIM-1 to enhance LASV-VSV transduction. In cases of TIM-1-dependent entry, Ebola virus GP-VSV or LASV-VSV responded differentially to early entry inhibitors, Compound C and EIPA. This provides evidence that mechanism of virus internalization is viral glycoprotein-dependent and cell surface receptor-independent.
Arenaviruses are small viruses composed of two RNA fragments enveloped by a sphere of protein and lipids. Over 30 arenavirus species are known, but only a few can infect humans and lead to severe hemorrhagic fever. Three arenaviruses are studied in this thesis: Machupo virus (MACV), Junín virus (JUNV), and Lassa virus (LASV). Outbreaks of these viruses can carry high rates of morbidity and mortality.
Recovery from MACV or JUNV infection relies on the host‟s production of antibodies, particularly ones that can prevent viruses from entering cells. Such antibodies are called neutralizing antibodies and work by binding the viruses‟ external sugar-coated proteins called glycoproteins. Even though MACV and JUNV proteins are similar, few antibodies can neutralize both species. Through the experiments conducted herein, I learned that the sugars are not targets of neutralizing antibodies but are important shields against them. Also, most, if not all, MACV- or JUNV-neutralizing antibodies function by mimicking the host cellular receptor. MACV escapes binding of JUNV-neutralizing antibodies through an additional loop in the protein that limits access to the binding site.
LASV usually uses the host protein, alpha dystroglycan, as a cellular receptor. This route of virus internalization relies on the viral glycoprotein. An alternative route for enveloped viruses is through receptors that engage with the viral envelope, yet others identified LASV as unresponsive this glycoprotein-independent route. In this thesis, I demonstrate that LASV is not an anomaly among enveloped viruses; use of this route simply relies on the absence of functional alpha dystroglycan.
xxii, 261 pages
Includes bibliographical references (pages 236-261).
Copyright © 2017 Rachel Bottjen Brouillette
Brouillette, Rachel Bottjen. "Arenaviruses: mechanisms of antibody-mediated neutralization and an alternative route of entry." PhD (Doctor of Philosophy) thesis, University of Iowa, 2017.