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Principal Investigator: Kenneth Harold Dinnon
Organization: ROCKEFELLER UNIVERSITY
Fiscal Year: 2023
Award: $69,080
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY/ABSTRACT
Alphaviruses are arthropod-transmitted viruses that cause acute febrile illness in humans, with some
cases causing long-term skeletomuscular and neurological sequelae. Currently there are no FDA-approved
vaccines or therapeutics for alphavirus infection, and continued global outbreaks of chikungunya and Eastern
equine encephalitis virus highlight the need for medical countermeasures. Effective antiviral treatments target
key viral replication processes without host side-effect are built on the foundational knowledge of viral replication
as evidenced by the rapid development of RNA polymerase and viral protease inhibitors towards SARS-CoV-2
during the COVID-19 pandemic. While alphaviruses have been studied for decades, there are many foundational
gaps in our understanding of their biology. Like all viruses, alphavirus infection of host cells induces a
multifaceted innate immune response, of which one result is the activation of the integrated stress response
(ISR) through viral double-stranded RNA sensing by host protein kinase R (PKR) and subsequent
phosphorylation of translation initiation factor 2 (eIF2a), causing shutoff of global protein translation. However,
alphaviruses have evolved a mechanism by which their structural proteins are uniquely still efficiently translated
from the viral subgenomic RNA (sgRNA) during ISR activation. While many canonical translation initiation factors
have been ruled dispensable for sgRNA translation, the mechanism of sgRNA translation is yet to be described.
Interestingly, the resistance of the sgRNA to the ISR is only observed in infected cells, suggesting viral factors
are necessary. Other work has shown that viral nonstructural protein nsP3 interacts with components of stress
granules, organelles formed during ISR activation to sequester translation machinery until relief of the stress and
return to homeostasis, suggesting a role for nsP3 in modulating translation during alphavirus infection-induced
ISR. Together, this leads to the hypothesis that alphaviruses evade the ISR through recruitment of non-canonical
initiation factors or the ribosome directly, and that ISR resistance is mediated in part through interactions of the
nsP3 and host proteins.
Using Sindbis virus (SINV) as a model system, this proposal seeks to identify how alphaviruses evade
the ISR to maintain translation of viral structural proteins with hopes of identifying druggable targets to prevent
alphavirus replication and spread. Aim 1 seeks to identify translation initiation factors responsible for sgRNA
translation during ISR activation and eIF2a phosphorylation. Aim 2 will address the role of viral factors,
specifically the hypervariable domain (HVD) of nsP3, in modulating the ISR and sgRNA translation through use
of deep mutational scanning methods. Successful completion of this proposed work will provide mechanistic
insight for an under-characterized phenomenon observed for decades in alphavirus research, allowing for novel
therapeutic interventions for alphavirus infections.
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