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Principal Investigator: Christopher F Basler
Organization: WASHINGTON UNIVERSITY
Fiscal Year: 2023
Award: $436,375
Funding agency: National Institute of Allergy and Infectious Diseases
RP02 Project Summary/Abstract for Contributions of Ebola and Marburg virus VP30 and VP24 proteins
to viral RNA synthesis, assembly and egress
Marburg virus (MARV) and Ebola virus (EBOV) are deadly emerging viruses of the filovirus family. Major gaps
remain in our understanding as to how filoviral-filoviral and filoviral-host interactions promote replication and
severe disease. Contributing to these limitations in knowledge is the requirement to study replicating filoviruses
under biosafety level 4 (BSL4) containment. To overcome the BSL4 limitation, we have produced replication
cycle modeling assays, called transcription-replication competent virus-like particle (trVLP) assays, that
recapitulate each of the major steps in the EBOV and MARV replication cycles at BSL2. These tools enable
the efficient analysis of filoviral and host proteins in the context of a replicating system. These systems also
permit the study of manipulations such as lethal mutations or elimination of key host factors that are not
compatible with replication of live virus. With these assays, we have explored the role of MARV VP30 (mVP30)
and MARV VP24 (mVP24) that exhibit distinct functional properties compared to their EBOV homologues.
EBOV VP30 is required for transcription initiation of the EBOV nucleoprotein (eNP) gene and is regulated by
host proteins with PPxPxY motifs. In contrast, mVP30 is reported to not be required for viral transcription in
minigenome assays, yet it is essential for virus growth. Why it is essential has been unclear. EBOV VP24 binds
importin alpha proteins to block interferon signaling and also participates in the maturation of nucleocapsid
formation, modulates viral RNA synthesis, and is critical for filoviral infectivity. In contrast, mVP24 interacts with
host protein Keap1, a regulator of cellular antioxidant responses. Our MARV trVLP data suggests that mVP30
is essential for efficient production of viable virus, due to its requirement for transcription of the mGP gene, and
that mutations disrupting mVP24-Keap1 interaction substantially decrease infectivity. Given these findings, we
will test the hypothesis that mVP30 plays a critical role in mGP transcription initiation; we will characterize
secondary structures that we hypothesize confer mVP30 dependence on the mGP gene; and we will evaluate
how interacting host proteins modulate mVP30 function. We will also test the hypothesis that mVP24
interaction with Keap1 is required for viral growth and define specific replication steps for which mVP24-Keap1
interaction is required. Finally, with our trVLP assays, we will define the mechanisms by which other filovirus-
host interactions identified in Research Projects 1 and 3 affect virus growth. Together, these studies will
substantially enhance the understanding of interactions that play key roles in filovirus replication and
pathogenesis.
Terms: <Address><Affect><Antioxidants><Assay><Basal Transcription Factor><Basal transcription factor genes><Binding><Bioassay><Biologic Assays><Biological Assay><Biophysics><CDC42 Homolog Gene><CDC42-Interacting Protein Gene><CIP4><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><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 technology><Cas nuclease technology><Cell Body><Cell Communication and Signaling><Cell Protection><Cell Signaling><Cells><Clustered Regularly Interspaced Short Palindromic Repeats approach><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><Containment><Cytoprotection><Data><Dependence><Disease><Disorder><EBOV><Ebola virus><Ebola-like Viruses><Exhibits><Family><Filoviridae><Filovirus><Frankfurt-Marburg Syndrome Virus><G25K Gene><GP Gene><Gene Transcription><GeneHomolog><General Transcription Factor Gene><General Transcription Factors><Generalized Growth><Genes><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Goals><Growth><Homolog><Homologous Gene><Homologue><Host Factor><Host Factor Protein><IFN><Importin-alpha><Importin-α><Infection><Integration Host Factors><Interferons><Intracellular Communication and Signaling><Knock-out><Knockout><Knowledge><Length><MGLAP><MGP gene><Marburg><Marburg virus><Marburg-like Viruses><Marburgvirus><Mediating><Modeling><Modification><Molecular><Molecular Interaction><Mutation><NLS Receptor><NLS-Binding Protein><NLSBP><Non-Polyadenylated RNA><Nuclear Family><Nuclear Import><Nuclear Localization Sequence Receptor><Nuclear Localization Signal-Binding Protein><Nucleocapsid><Nucleoproteins><Outcome><Pathogenesis><Pathway interactions><Peptides><Play><Production><Property><Protein Truncation><Proteins><R-Series Research Projects><R01 Mechanism><R01 Program><RNA><RNA Expression><RNA Gene Products><RNA Processing><RNA analysis><RNA and protein interaction><RNA chemical synthesis><RNA synthesis><RNA-Protein Interaction><Recombinants><Regulation><Reporting><Research Grants><Research Project Grants><Research Projects><Ribonucleic Acid><Role><Short interfering RNA><Signal Transduction><Signal Transduction Systems><Signaling><Small Interfering RNA><Structure><System><TRIP10><TRIP10 gene><Testing><Thyroid Hormone Receptor Interactor 10 Gene><Tissue Growth><Transcription><Transcription Factor Proto-Oncogene><Transcription Initiation><Transcription Initiation Site><Transcription Regulation><Transcription Start Site><Transcription factor genes><Transcriptional Control><Transcriptional Regulation><Transfection><Viral><Viral Diseases><Viral Structural Proteins><Virion><Virus><Virus Diseases><Virus Particle><Virus Replication><Virus-like particle><Work><alpha Karyopherins><anti-oxidant><biological signal transduction><biophysical foundation><biophysical principles><biophysical sciences><cytoprotective><ebolavirus><genome mutation><member><mutant><novel><ontogeny><overexpress><overexpression><pathway><recombinant virus><response><siRNA><social role><stem><success><tool><transcription factor><viral RNA><viral infection><viral multiplication><viral replication><virus RNA><virus infection><virus multiplication><virus-induced disease><virus-like nanoparticles><viruslike particle>