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Principal Investigator: Lok-Yin Roy Wong
Organization: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
Fiscal Year: 2024
Award: $248,999
Funding agency: National Institute of Allergy and Infectious Diseases
Project summary/Abstract
SARS-CoV, MERS-CoV and SARS-CoV-2 belong to the genus Betacoronavirus and encode sets of specific
accessory proteins. Accessory proteins encoded by coronaviruses are not essential for the viral life cycle but are
important regulators that mediate immune evasion for optimal virus replication and propagation. One unique
feature of Betacoronavirus that is not seen in other genera of the family Coronaviridae is the presence of a small
accessory protein (I) encoded by the +1 open reading frame (ORF) relative to and within the ORF encoding the
nucleocapsid (N) gene. The internal (I) proteins of SARS-CoV (ORF9b), MERS-CoV (ORF8b) and SARS-CoV-
2 (ORF9b) have not been extensively characterized. However, in vitro experiments suggest that the I proteins of
these viruses have a role in suppressing IFN-I expression, which could potentially contribute to pathogenesis. In
this application, we hypothesize that the I protein is a virulence factor with functions specific to each virus. The
goal of this project is to study the roles of I proteins in pathogenesis and determine if I proteins possess functions
specific to viruses within the genus Betacoronavirus. We generated mutant MERS-CoV and SARS-CoV-2 with
deletions of I protein expression without altering the coding sequence of the N protein by reverse genetics and
found that MERS-CoV lacking I protein expression showed increased virulence in mice, while the absence of
the SARS-CoV-2 I protein resulted in attenuation. It is intriguing that the absence of the I protein resulted in such
disparate changes in the virulence of the two related CoVs. In Aim 1, we will investigate the virus-specifc
functions of the I proteins by inserting the I protein of MERS- and SARS-CoV-2 into mouse hepatitis virus (MHV),
another betacoronavirus. In addition, we will interrogate the role of I proteins involved in regulating virus
production by interfering with the virus/host machinery required for virus replication. This will be performed by
analyzing the I protein interactome by mass spectrometry, detecting the presence of I protein in virions, which
would be consistent with a role in virion assembly and release, and comparing the viral life cycle in mutant vs.
parental virus-infected mice. In Aim 2, we will assess the role of the I protein in regulating immune responses by
comparing mice infected with mutant viruses or parental viruses. We will interrogate the role of IFN-I signaling
in the altered virulence of mutant viruses, as I proteins have been shown to suppress IFN-I induction in vitro. In
addition, changes in immune responses will be investigated by measuring inflammatory cytokines in the blood,
bronchoalveolar lavage (BAL) and the lungs of mice infected with mutant or parental viruses. Immune profiling
of mice infected with mutant viruses will be performed and compared to mice infected with parental viruses by
scRNA-seq. The training and experiments proposed in this career development award will not only offer
invaluable opportunities for me to acquire new skills and techniques required for developing my own independent
research in viral immunology and pathogenesis but also address important questions regarding how specific
viral proteins act as virulence factors in coronavirus-infected cells.
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corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Signal Transduction><Signal Transduction Systems><Signaling><Subcellular Process><System><Techniques><Training><Up-Regulation><Upregulation><Viral><Viral Gene Products><Viral Gene Proteins><Viral Genes><Viral Proteins><Virion><Virulence><Virulence Factors><Virulent><Virus><Virus Particle><Virus Replication><Virus-like particle><Visualization><Wuhan coronavirus><attenuate><attenuates><attenuation><beta CoV><beta coronavirus><betaCoV><betacoronavirus><biological signal transduction><bronchopulmonary lavage therapy><corona virus><coronavirus disease 2019 virus><coronavirus disease-19 virus><cytokine><deficiency of protein><disease prevention><disorder prevention><experiment><experimental research><experimental study><experiments><hCoV19><health economics><host response><human CoV><human corona virus><human coronavirus><immune evasive><immune modulation><immune regulation><immune system response><immunologic reactivity control><immunomodulatory><immunoregulation><immunoregulatory><immunoresponse><in vivo><interferon alpha receptor><interferon α receptor><interventional strategy><life course><lung injury><member><membrane structure><mouse model><murine hepatitis coronavirus><murine model><mutant><nCoV2><pandemic><pandemic disease><parent><protein expression><protein function><pulmonary><pulmonary damage><pulmonary injury><pulmonary tissue damage><pulmonary tissue injury><recombinant virus><reverse genetics><scRNA-seq><severe acute respiratory syndrome-CoV><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><skills><social role><viral RNA><viral multiplication><viral replication><virus RNA><virus multiplication><virus protein><virus-like nanoparticles><viruslike particle><β CoV><β coronavirus><βCoV>