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Principal Investigator: Joyce Jose
Organization: PENNSYLVANIA STATE UNIVERSITY, THE
Fiscal Year: 2024
Award: $196,044
Funding agency: National Institute of Allergy and Infectious Diseases
Mosquito-borne alphaviruses such as chikungunya, Mayaro, and Eastern equine encephalitis viruses cause high
morbidity and mortality in their mammalian hosts. Alphaviruses are globally distributed arthropod-borne viruses
that are enzootic in nature with the potential to disseminate to new geographical regions due to vector
adaptations causing new outbreaks. A 2019 outbreak of 38 human cases of Eastern equine encephalitis virus
occurred in the United States, raising concerns about its reemergence. In 2005–2006, a chikungunya outbreak
started in the Indian Ocean Island of Réunion had spread around the world, infecting millions of people.
Chikungunya fever is characterized by debilitating joint pain that can last up to 2–3 years, causing arthritis-like
conditions. No effective antiviral strategies or vaccines are available against any of these pathogens. Studying
these viruses to gain a molecular understanding of their lifecycle is essential to discovering novel targets for
therapeutic intervention. Specifically, the poorly understood intracellular mechanisms that drive alphavirus
assembly and budding represent promising antiviral targets. We reported for the first time that the alphavirus-
encoded ion channel protein 6K plays an essential part in virus budding by enabling the formation of cytopathic
vacuoles-II and envelope spike protein transport to the plasma membrane. The defects due to the deletion of 6K
can be restored to varying levels by the expression of a functional HIV-1 Vpu and influenza A virus M2 ion
channel. We also demonstrated that ion channel inhibitors could be utilized as antivirals. Building on these
observations, in Aim 1, we will characterize chikungunya and Sindbis virus 6K ion channels and their activity by
reverse genetics, transport assays in proteoliposomes and fractionated intracellular membrane vesicles, and
live-confocal imaging of virus-infected cells with ion-specific fluorescent probes. In Aim 2, by expressing virus-
encoded ion channels, including the SARS-CoV-2 E protein in cells and from an alphavirus, we will determine if
ion channel-based Golgi remodeling is a standard mechanism used by enveloped RNA viruses for membrane
modification and efficient virus budding. With new reverse genetics tools and CRISPR-Cas9 methods, we will
investigate the functional involvement of host-encoded ion channels in alphavirus budding. By completing these
aims, we will define a novel mechanism by which ion channel proteins modify the secretory pathway for virus
budding and how this process can be exploited as an antiviral target. We will also generate new reverse genetics
tools that will be useful to the scientific community. The critical knowledge gaps we will address are 1) what are
the ions transported by alphavirus 6K? 2) how does 6K participate in virus budding? and 3) an understanding of
a general mechanism involving virus and host ion channels utilized by alphaviruses for efficient budding.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Acute><Address><Affect><Alpha Virus><Alphavirus Infections><Americas><Amiloride><Anti-viral Agents><Arthralgia><Arthritis><Assay><Autoregulation><Bioassay><Biological Assay><Brain Inflammation><CHIKV><CHIKV fever><COVID-19 virus><COVID19 virus><CRISPR approach><CRISPR based approach><CRISPR library><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based library><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 library><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Function><Cell Membrane Permeability><Cell Physiology><Cell Process><Cell membrane><Cell surface><Cells><Cellular Function><Cellular Physiology><Cellular Process><Chikungunya fever><Chikungunya virus><Chimera><Chimera organism><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats library><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><CoV-2><CoV2><Communities><Confocal Microscopy><Cytoplasmic Membrane><DNA Molecular Biology><Defect><Disease Outbreaks><Drug Design><Drugs><E protein><EEE Virus><Eastern Equine Encephalitis Virus><Eastern Equine Encephalomyelitis Virus><Encephalitis><Envelope Protein><Family><Fluorescence Activated Cell Sorting Fractionation><Fluorescence-Activated Cell Sorting><Fluorescence-Activated Cell Sortings><Fluorescent Probes><Foundations><Genetic Alteration><Genetic Change><Genetic defect><Geographic Area><Geographic Locations><Geographic Region><Geographical Location><Glycoproteins><Golgi><Golgi Apparatus><Golgi Complex><Group A Arboviruses><HCV><HIV-1><HIV-I><HIV1><Hepatitis C virus><Homeostasis><Human><Human Immunodeficiency Virus Type 1><Human immunodeficiency virus 1><Immunoblotting><Immunofluorescence><Immunofluorescence Immunologic><In Vitro><Indian Ocean Islands><Influenza A><Influenza A virus><Influenza Viruses Type A><Influenzavirus A><Integral Membrane Protein><Intracellular Membranes><Intrinsic Membrane Protein><Ion Channel><Ion Channel Protein><Ion Channel Protein Gene><Ion Transport><Ionic Channels><Ions><Joint Pain><Knock-out><Knockout><Knowledge><Life Cycle><Life Cycle Stages><Mediating><Medication><Membrane><Membrane Channels><Methods><Microscopy><Mission><Modern Man><Modification><Molecular><Molecular Biology><Morbidity><Morbidity - disease rate><Morphogenesis><Mutation><NIAID><National Institute of Allergy and Infectious Disease><Nature><Orthomyxovirus Type A><Outbreaks><Pathway interactions><Persons><Pharmaceutical Preparations><Physiological Homeostasis><Plaque Assay><Plasma Membrane><Play><Polyarthritides><Process><Protein Trafficking><Proteins><Public Health><Publishing><Quantitative RTPCR><Quantitative Reverse Transcriptase PCR><RNA Viruses><Reporting><Reunion Island><Risk><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Secretory Granules><Secretory Vesicles><Series><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Sindbis Virus><Structural Protein><Structure><Subcellular Process><Surface><Testing><Therapeutic Intervention><Time><Togaviridae><Togaviruses><Transmembrane Protein><Transmembrane Protein Gene><Type A Influenza><United States><Vacuole><Vesicle><Viral><Virion><Virus><Virus Particle><Western Blotting><Western Immunoblotting><Work><Wuhan coronavirus><access to vaccination><access to vaccines><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-virals><arthritic><arthropod transmission><arthropod transmitted><arthropod-borne><arthropodborne><chikungunya><chimeras><confocal imaging><coronavirus disease 2019 virus><coronavirus disease-19 virus><drug repositioning><drug repurposing><drug/agent><emergent outbreak><emerging outbreak><env Antigens><env Gene Products><env Polyproteins><env Protein><enzootic><experiment><experimental research><experimental study><experiments><genome mutation><geographic site><global health><hCoV19><inhibitor><intervention therapy><knowledge of results><life course><member><membrane permeability><membrane structure><morphogenetic process><mortality><mosquito-borne><mosquitoborne><mutant><nCoV2><new drug target><new druggable target><new outbreak><new pharmacotherapy target><new therapeutic target><new therapy target><novel><novel drug target><novel druggable target><novel outbreak><novel pharmacotherapy target><novel therapeutic target><novel therapy target><pathogen><pathway><plasmalemma><polyarthritis><premature><prematurity><prevent><preventing><protein blotting><protein transport><proteoliposomes><qRTPCR><repurposing agent><repurposing medication><reverse genetics><social role><stable cell line><tool><trafficking><vaccination access><vaccination availability><vaccine access><vaccine availability><vector>