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Principal Investigator: Brian Geiss
Organization: COLORADO STATE UNIVERSITY
Fiscal Year: 2020
Award: $374,993
Funding agency: National Institute of Allergy and Infectious Diseases
Approximately 2/3rd of the world population is at risk of infection by at least one of the 35 insect-borne
flaviviruses known to cause disease in humans. There are currently few vaccines and no therapeutics available
to treat patients infected by flaviviruses such as Dengue, Zika, and West Nile viruses despite the severe
morbidity and mortality they cause globally each year. The development of improved vaccines and therapeutics
to prevent and treat flavivirus infections requires improved knowledge of the molecular mechanisms these
serious human pathogens use to replicate their genomes. RNA capping of flavivirus genomes has received
increasing attention over the last decade as an antiviral drug target due to its critical roles in maintaining viral
RNA stability, controlling viral protein translation, and innate immune evasion. There is, however, not much
known about how flavivirus RNAs are capped during infection. Therefore, this proposal will define how
flaviviruses cap their RNA genomes during infection and evaluate how capping affects innate immune evasion.
1) The NS5 RNA guanylyltransferase is a novel flavivirus enzyme with no structural or sequence similarities to
any other known nucleotidyltransferase enzyme. It is currently unknown how this important viral enzyme
functions, which is a critical gap in our understanding of flavivirus RNA replication. We are using a
combination mutagenesis and viral replication experiments to define the active site of the flavivirus NS5
guanylyltransferase, providing the first in-depth characterization of this unique viral replication enzyme.
2) The 5' untranslated region (UTR) of the flavivirus genome contains conserved sequence and structural
elements known to be involved in RNA replication, but their role in RNA capping has never been assessed. We
will use a series of mutated 5' UTR RNAs to test the specificity of NS5-mediated RNA capping to define how 5'
UTR terminal sequences and the stem-loop A structure affect binding and capping efficiency. 3) Degradation
of flavivirus genomes by the cellular RNA decay pathway results in the inhibition of RNA decay and RNAi
pathways, altering the immune response to infection. We wish to test the intriguing hypothesis that viral
capping efficiency may be strategically regulated by viruses to produce non-coding RNAs that alter infection
dynamics. Interestingly, we have recently found that uncapped viral RNAs are incorporated into virus particles
and may comprise up to a third of viral RNAs in an infected cell. This surprisingly high level of uncapped RNA
is likely processed by RNA decay factors and leads to high levels of a small sfRNA which has been shown
previously to antagonize RNAi and interferon responses (among other things). We will examine, therefore,
how NS5 capping efficiency affects viral RNA production, the fate of uncapped viral RNAs in cells, and how the
products of these uncapped RNAs influence the dynamics of a flavivirus infection. Overall, this project will
significantly advance our understanding of flavivirus replication mechanisms that we can exploit for antiviral
and vaccine development, and will provide critical new information about how flaviviruses cause disease.
Terms: <3' Untranslated Regions><3' exonuclease><3'UTR><5' Capped RNA><5' Untranslated Regions><5' mRNA Cap Structure><5'UTR><Active Sites><Address><Affect><Antiviral Agents><Antiviral Drugs><Antivirals><Assay><Attention><Binding><Bioassay><Biochemical><Biochemical Reaction><Biologic Assays><Biological Assay><Cell Body><Cells><Cessation of life><Clinical><Conserved Sequence><Culicidae><Data><Death><Dengue><Development><Disease><Disorder><Drug Targeting><EC 2.1.1><EC 2.7.7><Egypt 101 virus><Elements><Enzymatic Reaction><Enzyme Gene><Enzymes><Exonuclease><Exoribonucleases><Family><Flavivirus><Flavivirus Infections><Functional RNA><GTP><GTP mRNA guanylyltransferase><Genetics-Mutagenesis><Genome><Goals><Group B Arbovirus><Guanosine Monophosphate><Guanosine Triphosphate><Guanylic Acids><Human><IFN><Immune Evasion><Immune response><Immunological response><Infection><Innate Immune Response><Insecta><Insects><Insects Invertebrates><Interferons><Knowledge><Mediating><Methyltransferase><Modern Man><Molecular><Molecular Interaction><Morbidity><Morbidity - disease rate><Mosquitoes><Mutagenesis><Mutagenesis Molecular Biology><Mutate><N-terminal><NH2-terminal><Neurologic><Neurological><Non-Coding><Non-Coding RNA><Non-Polyadenylated RNA><Non-translated RNA><Noncoding RNA><Nontranslated RNA><Pathway interactions><Patients><Peptides><Play><Population><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Process><Production><Protein Biosynthesis><Proteins><Public Health><Quelling><RNA><RNA Caps><RNA Decay><RNA Degradation><RNA Gene Products><RNA Interference><RNA Sequences><RNA Silencing><RNA Stability><RNA Viruses><RNA guanylyltransferase><RNA replication><RNAi><Reaction><Ribonucleic Acid><Ribosomal Peptide Biosynthesis><Ribosomal Protein Biosynthesis><Ribosomal Protein Synthesis><Role><Sequence-Specific Posttranscriptional Gene Silencing><Series><Site><Specificity><Structure><Substrate Specificity><Testing><Therapeutic><Translations><Untranslated RNA><Urbanization><Vaccines><Viral><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Genome><Viral Hemorrhagic Fevers><Viral Proteins><Virion><Virus><Virus Diseases><Virus Particle><Virus Replication><WNV><West Nile><West Nile virus><ZIKA><ZIKV><Zika Virus><anti-viral agents><anti-viral drugs><anti-virals><base><climate change><combat><design><designing><develop a vaccine><development of a vaccine><developmental><experiment><experimental research><experimental study><genomic RNA><global climate change><guanylate><guanylyltransferase><hemorrhagic fever><host response><human pathogen><immunoresponse><improved><infection risk><insight><m-RNA Guanylyltransferase><mRNA 5'-guanylyltransferase><mRNA Decay><mRNA Leader Sequences><mRNA capping enzyme><mRNA guanylyltransferase><member><methylase><mortality><mosquito-borne><mosquitoborne><mutant><noncoding><novel><nucleotidyltransferase><pathway><phosphodiesterase II><prevent><preventing><protein synthesis><public health relevance><response><social role><spleen exonuclease><spleen phosphodiesterase><stem><transmethylase><trend><vaccine development><vaccine formulation><viral RNA><viral infection><viral multiplication><viral replication><virus RNA><virus genome><virus infection><virus multiplication><virus protein><virus-induced disease><zikav>