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" Alohomora! Unlocking Hantavirus-Receptor Interactions Using Viral Surrogates "
Slough, Megan Merrill
Chandran, Kartik
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Latin Dissertation
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English
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| Record Number
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1057477
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| Doc. No
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TL56594
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| Main Entry
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Slough, Megan Merrill
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| Title & Author
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Alohomora! Unlocking Hantavirus-Receptor Interactions Using Viral Surrogates\ Slough, Megan MerrillChandran, Kartik
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| College
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Albert Einstein College of Medicine
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| Date
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2020
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| Degree
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Ph.D.
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2020
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| Note
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211 p.
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| Abstract
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Rodent-borne hantaviruses are RNA viruses found throughout the world that can cause deadly diseases in humans. These viruses can be classified into two clades, depending on the geographical distribution of their natural hosts and zoonotic human illness. Those primarily located in Europe and Asia are so named Old World hantaviruses (OWH), and those endemic to North and South America are known as New World hantaviruses (NWH). Although both Old and New World pathogenic hantaviruses cause vascular leakage in humans, the principal organs affected differ. OWHs primarily damage the kidneys, causing hemorrhagic fever with renal syndrome (HFRS), and NWHs predominantly impair the lungs, resulting in hantavirus cardiopulmonary syndrome (HCPS). Treatments against these diseases are in need, as mortality rates can reach up to 50%, and there are no hantavirus-specific, Federal Drug Administration (FDA)-approved vaccines or therapeutics available. Unfortunately, hantavirus research is limited due to the requirement for high biocontainment laboratories. To overcome these restrictions and study hantaviruses in a lower, biosafety level 2 (BSL-2) laboratory, I set out to develop viral surrogate systems that faithfully reproduce their entry. I employed these to understand the mechanism of receptor-dependent hantavirus entry and to gain insight into hantavirus assembly and egress. The first study in my thesis (Chapter II) explores the role of the recently identified NWH receptor, protocadherin-1 (PCDH1). PCDH1 is a member of the cadherin superfamily and is important for maintaining cell integrity in the lung endothelium and epithelium. Our lab previously reported that the hantavirus glycoproteins, Gn/Gc, directly interact with PCDH1’s first extracellular cadherin (EC1) repeat. To gain insights into PCDH1’s role in NWH entry, I screened a panel of mutant, soluble PCDH1 proteins, altering the surface-exposed residues within three apical loops of EC1. Using a surrogate system of replication-competent, recombinant vesicular stomatitis viruses (rVSVs) bearing NWH, Andes virus (ANDV) or Sin Nombre virus (SNV), Gn/Gc, I evaluated the mutants’ ability to recognize Gn/Gc, identifying three residues (F83, D85, and I140A) critical for Gn/Gc binding. Infection experiments showed that these residues are required for ANDV and SNV Gn/Gc-mediated entry, as well as for authentic SNV infection in vitro. Interestingly, one of the residues (F83) represented an amino acid variation in the PCDH1 orthologs, between hantavirus-susceptible and non-susceptible species, suggesting F83 as a potential host-determining factor for SNV infection. Among the three residues, two (F83 and D85) are located close to each other, while I140 is located further away on EC1. To evaluate the two nearby residues’ in vivo relevance, Syrian hamster PCDH1 residues, corresponding to human PCDH1’s F83 and D85, were mutated by CRISPR/Cas9-mediated homologous recombination. These hamsters were protected from a lethal ANDV challenge, thus supporting these residues' important role in mediating infection and pathogenesis. Overall, we’ve pinpointed a key set of three residues that can be targeted for the development of therapeutics to block NWH infection. Our findings also provide insight into the residues that determine the cellular host range of NWH and have implications for predicting animal species that might become carriers for these deadly viruses. For my second study (Chapter III), I harnessed the forward genetic capability of the self-replicating rVSVs to identify two-point mutations in the OWH Gn/Gc that afforded the generation of highly infectious rVSV vectors. Although generating replication-competent rVSVs bearing NWH Gn/Gc is relatively straightforward, those for OWH Gn/Gc were quite difficult. An rVSV bearing the prototypical OWH, Hantaan virus (HTNV), Gn/Gc only rescued after three serial passages, over a period of 3-4 weeks in cell culture, and was associated with the acquisition of two-point mutations, I532K located in the cytoplasmic tail of Gn, and S1094L in the stem region of Gc. To decipher if the mutations were the reason for viral growth and successful rescue of the rVSV, I analyzed the mutations' effect on Gn/Gc protein expression, trafficking within the cell, and incorporation into VSV particles. I found these mutations enabled the escape of the Gn/Gc from the Golgi apparatus, the site of assembly for OWHs, to the cell surface where it can be successfully packaged into the budding VSV virions. The level of virion-incorporated Gn/Gc was sufficient to generate replication-competent rVSVs bearing another OWH, Dobrava-Belgrade virus Gn/Gc. This suggests that the enhancement of cell surface expression of viral glycoproteins by cognate mutations could enable the generation of other hard-to-rescue rVSVs. These new surrogates can not only permit the exploration of the elusive host cell receptors for OWHs but can also be utilized as vaccine candidates. Overall, these surrogate systems offer powerful tools to study viral:host interactions, afford forward genetic screens of virus entry inhibitors and neutralization antibodies, and can unlock viral entry mechanisms to further explore what would never have been possible otherwise.
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| Descriptor
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Cellular biology
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Genetics
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Immunology
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Pathology
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Pharmaceutical sciences
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Public health
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Virology
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Chandran, Kartik
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Albert Einstein College of Medicine
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