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Principal Investigator: MING LUO
Organization: GEORGIA STATE UNIVERSITY
Fiscal Year: 2022
Award: $560,443
Funding agency: National Institute of Allergy and Infectious Diseases
The devastating impact on public health, global economy and social stability incurred by the COVID-19 pandemic
in the last two years has highlighted the importance of basic research into zoonotic pathogens. This application
describes structural and functional studies into the rodent-borne human pathogen lymphocytic choriomeningitis
virus (LCMV), a member of the Arenaviridae family in the Bunyavirales order. Like other members of the same
family, LCMV has a negative sense, bi-segmented genome consisting of a large (L) and a small (S) segment.
The L segment encodes the RNA-dependent RNA polymerase (L RdRp) protein and the multi-functional matrix
protein (Z). The S segment encodes the viral nucleoprotein (NP) and the glycoprotein (GP) precursor of the
glycoprotein complex (GPC) that is later cleaved into a stable signal peptide (SSP), GP1, and GP2. In the virion,
nucleocapsids of NP coated L and S segments associated with the L protein are copackaged through interactions
with membrane-associated Z proteins, which also interact with GPs embedded in the membrane envelope.
Although structures of individual proteins from AVs have been solved by x-ray crystallography or cryo electron
microscopy (cryoEM), the architectural organization of these proteins in the virion and the assembly mechanism
of NP and RNA into the nucleocapsid are poorly understood. We hypothesize that NP interacts with genomic
RNA segments and L RdRp to form a nucleocapsid, which is recruited to GP-decorated membrane patches
through Z for budding of virions. The proposed structural and functional studies aim to test this hypothesis of
LCMV virion and nucleocapsid assembly with techniques just established by our team in the collaborative studies
of vesicular stomatitis virus (VSV), another negative sense RNA virus. Specifically, cryo electron tomography
(cryoET) will be used to reconstruct the first 3D model of the LCMV virion at molecular resolution and atomic
models of individual proteins will be fitted into the virion tomogram to establish the architectural framework of the
virion and to unveil molecular interactions among GP, Z, NP and L proteins (Aim 1). Near-atomic resolution with
novel sub-particle reconstruction method will be used to image fully assembled nucleocapsids consisting of NP
protein and genomic RNA segment to define the protein-RNA interactions at atomic details. The nucleocapsid
structure will be used to guide sub-particle reconstruction workflow and be complemented by in situ structures
of nucleocapsids from virions (Aim 2). In both Aims, structure-guided functional studies will be performed to test
hypotheses of assembly mechanisms of LCMV nucleocapsid and virion. Structure-function relationship relevant
to viral RNA synthesis will also be explored. Overall, the anticipated results will provide new insights into the
mechanism of virion assembly and viral RNA synthesis, not only for LCMV but also for Arenaviruses in general.
The proposed studies harness cutting-edge technologies in structural biology and will generate new knowledge
of viral structures currently unavailable to any of Arenaviruses. As such, the innovative studies shall make unique
contributions by accelerating discoveries of antiviral agents and vaccines to control future AV outbreaks.
Terms: <3-D modeling><3-D structure><3-dimensional structure><3D modeling><3D structure><Animals><Antiviral Agents><Antiviral Drugs><Antivirals><Architecture><Arenaviridae><Arenavirus><Arenavirus group><Argentine hemorrhagic fever virus><Argentinian Hemorrhagic Fever Virus><Basic Research><Basic Science><Binding><Binding Proteins><Bunyavirales><COVID crisis><COVID epidemic><COVID pandemic><COVID-19 crisis><COVID-19 epidemic><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 pandemic><COVID-19 public health crisis><COVID19 crisis><COVID19 epidemic><COVID19 global health crisis><COVID19 global pandemic><COVID19 health crisis><COVID19 pandemic><COVID19 public health crisis><Cell Body><Cell Communication and Signaling><Cell Signaling><Cell membrane><Cells><Collaborations><Complement><Complement Proteins><Complex><Cryo-electron Microscopy><Cryo-electron tomography><Cryoelectron Microscopy><Crystallization><Crystallographies><Crystallography><Cytoplasmic Membrane><Data><Density Gradient Centrifugation><Density Gradient Fractionation><Deposit><Deposition><Disease Outbreaks><EC 2.7.7.48><Electron Cryomicroscopy><Engineering / Architecture><Family><Future><GP1><GP2><GP2 gene><GTP-Binding Protein 1><GTPBP1><GTPBP1 gene><Gene Transcription><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Genome><Genomic Segment><Glycoprotein 2, Zymogen Granule Membrane><Glycoproteins><Human><Image><In Situ><Individual><Intracellular Communication and Signaling><Joints><Junin><Junin virus><Knowledge><LCM Viruses><LCM-Lassa Complex Viruses><LCMV><Lassa fever virus><Lassa virus><Ligand Binding Protein><Ligand Binding Protein Gene><Lymphocyte Function><Lymphocytic Choriomeningitis-Lassa Complex Viruses><Lymphocytic choriomeningitis virus><Measures><Membrane><Messenger RNA><Methods><Modeling><Modern Man><Molecular><Molecular Interaction><Mutation><Non-Polyadenylated RNA><Nucleocapsid><Nucleoproteins><Old World Arenaviruses><Outbreaks><Peptide Signal Sequences><Pichinde><Pichinde virus><Plasma Membrane><Polymerase><Protein Binding><Proteins><Public Health><RNA><RNA Expression><RNA Gene Products><RNA Replicase><RNA Sequences><RNA Viruses><RNA chemical synthesis><RNA synthesis><RNA-Dependent RNA Polymerase><RNA-Directed RNA Polymerase><RNA-Protein Interaction><Resolution><Ribonucleic Acid><Rodent><Rodentia><Rodents Mammals><SARS-CoV-2 epidemic><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 pandemic><SARS-CoV2 epidemic><SARS-CoV2 pandemic><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><Series><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 pandemic><Signal Peptide><Signal Sequences><Signal Transduction><Signal Transduction Systems><Signaling><Single Crystal Diffraction><Structure><Structure-Activity Relationship><System><TOMO><Techniques><Technology><Testing><Tomogram><Transcription><Ultracentrifugation><VSV><Vaccines><Vesicular Stomatitis Virus><Vesicular stomatitis Indiana virus><Viral><Viral Gene Products><Viral Gene Proteins><Viral Hemorrhagic Fevers><Viral Packaging><Viral Proteins><Virion><Virus><Virus Assembly><Virus Packagings><Virus Particle><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><Zoonoses><Zoonotic><Zoonotic Infection><Zymogen Granule Membrane Glycoprotein 2><anti-viral agents><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-virals><antiviral compound><antiviral medication><antiviral therapeutic><base><biological signal transduction><bound protein><chemical structure function><corona virus disease 2019 epidemic><corona virus disease 2019 pandemic><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><coronavirus disease 2019 global health crisis><coronavirus disease 2019 global pandemic><coronavirus disease 2019 health crisis><coronavirus disease 2019 pandemic><coronavirus disease 2019 public health crisis><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><coronavirus disease-19 global pandemic><coronavirus disease-19 pandemic><cryo-EM><cryo-EM tomography><cryoEM><cryoEM tomography><cryoelectron tomography><density><density gradient ultracentrifugation><design><designing><develop a vaccine><develop vaccines><development of a vaccine><electron cryo-tomography><electron crystallography><experiment><experimental research><experimental study><genome mutation><genomic RNA><genomic region><hemorrhagic fever><human pathogen><image construction><image generation><image reconstruction><imaging><innovate><innovation><innovative><insight><mRNA><member><membrane structure><new approaches><novel><novel approaches><novel strategies><novel strategy><pandemic><pandemic disease><particle><pathogen><plasma protein Z><plasmalemma><protein Z><protein signal sequence><prototype><recombinant virus><reconstruction><recruit><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><social><structural biology><structure function relationship><three dimensional structure><three-dimensional modeling><vaccine development><viral RNA><viral assembly><virus RNA><virus protein>