Defining mechanisms of PKR activation and evasion during Adenovirus infection

Host cells have evolved an array of sensors to detect pathogen-associated molecular patterns and activate defense responses.

Protein Kinase RNA-activated (PKR) is a key sensor of double-stranded RNA (dsRNA) produced by viruses and is vital for protection against human pathogens. Activated PKR halts global protein translation to limit virus infection. Since many DNA viruses antagonize PKR, it has been presumed that they produce dsRNA.

Indeed, PKR is activated upon infection with mutant viruses lacking a PKR inhibitor. It has been suggested that transcription from both strands of compact DNA virus genomes generates dsRNA. However, there is limited direct evidence this occurs.

This proposal addresses gaps in our understanding of how the critical antiviral sensor PKR is activated during DNA virus infection.

Using the important clinical pathogen human adenovirus (AdV) as our model, we could not detect dsRNA during infection with wildtype (WT) or mutant virus lacking the well-characterized PKR inhibitor VA RNA (f:..VA).

However, infection with ubiquitin ligase-deficient (f:..E4) AdV mutants produced abundant nuclear dsRNA composed of poorly processed viral transcripts and activated PKR despite adequate VA RNA expression.

Among the substrates of the viral ligase is a spliceosome protein hnRNPC, targeted to promote efficient splicing of viral late mRNAs. Knockdown of hnRNPC reduced dsRNA accumulation and PKR activation.

Similarly, my preliminary data reveal a novel role for the host protein NF90 in regulating PKR activation during f:..VA infection. Together, these data suggest PKR may be activated independently of its canonical activator dsRNA during AdV infection.

The objective of the proposal is to define the role that hnRNPC and NF90 play in PKR activation during f:..VA infection. I propose to validate the interaction between these proteins and PKR by co-IP.

Expression and localization of both proteins will be tracked over a time course of f:..VA infection and changes correlated with the timing of PKR activation.

Knockdown (KO) of each protein will be used to examine activation of PKR during f:..VA infection and to check for rescue from defects in viral late mRNA accumulation or splicing. CLIP-qPCR will be used to probe for binding to VA RNA and viral mRNAs. I will also examine impacts on protein translation of viral mRNAs using HPG labeling of nascent proteins.

Results of this proposal will delineate the antiviral function of NF90 during AdV infection, expand our understanding of VA RNA's pro-viral roles, and redefine our understanding of PKR activation during f:..VA infection with broader implications for other nuclear-replicating DNA viruses.

This work will take place in the collaborative and interdisciplinary training environment provided by the Weitzman lab and the integrated research communities of both the Children's Hospital of Philadelphia and the University of Pennsylvania.

I am uniquely positioned to perform these studies in the Weitzman lab where I will gain hands-on training in proteomics, RNA biology, and high-resolution microscopy and image processing. This training award will leave me poised for my future research studying how viruses overcome host antiviral responses.