Modulation of the Innate Immune Response to Measles Virus Infection by PKR and ADAR1
Final Report Abstract
Recombinant measles virus (MV) lacking expression of C protein (CKO) is a potent activator of the doublestranded RNA (dsRNA) dependent protein kinase (PKR). Activation of PKR mediates downstream signaling events including eIF2a phosphorylation, IFNp induction and stress granule formation. I have shown in this project that significant amounts of dsRNA accumulate during CKO virus infection, but not by infection with isogenic MV expressing C protein. Subcellular localization, kinetics of expression and drug-sensitivity tests suggested that the dsRNA was a viral replication product. PKR autophosphorylation and stress granule formation correlated with the kinetics of appearance of dsRNA. Protein and RNA quantification revealed that the CKO mutation did not affect the steady-state balance of viral protein compared to viral RNA. RNA deep-sequencing established a steeper transcription gradient in CKO virus-infected cells compared to cells infected with the parental virus. Using a PCR-based method to specifically amplify 5’-copyback defective interfering RNA (DI-RNA) I found a correlation between accumulation of DI-RNAs in CKO virus-infected cells and the dsRNA/PKR-mediated cellular response. No DI-RNAs were detected in parental virus-infected cells. These results indicated that the lack of C expression led to decreased processivity of the viral polymerase and thereby to increased accumulation of DI-RNAs activating PKR and downstream signaling. In order to study the generation of DI-RNAs by MV and the role of the C protein in preventing this event I documented the de novo generation of copyback DI-RNAs from a series of independent rescue events for vaccine (vac2) and wild-type (IC323) MV as early as passage 1 after virus rescue. CKO viruses exhibited a higher frequency of generated DI-RNAs than parental viruses, supporting the hypothesis that C is a processivity factor of the viral polymerase. I obtained nucleotide sequences of 65 individual DI-RNAs and from this information predicted their structural features, breakpoints, re-initiation sites and length. Interestingly, several DI-RNAs showed clusters of A-to-G or U-to-C transitions. Sequences flanking mutation sites were characteristic of those favored by adenosine deaminase acting on RNA 1 (ADAR1), which catalyzes the C6 hydrolytic deamination of adenosine to produce inosine in dsRNA, referred to as A-to-I-editing. The CKO-phenotype was dominant: in interferon-incompetent lymphatic cells DI-RNAs derived from CKO virus suppressed replication of parental virus, as shown by co-infections with viruses expressing different fluorescent reporter proteins and quantification of their expression levels. In contrast, co-infection with a C protein-expressing virus did not rescue the suppressive phenotype of DI-RNAs. I was able to characterize the PAMP activating PKR during MV infection: 5’-copyback DI-RNAs. Direct binding of DI-RNAs to PKR could not be shown. However, the PKR-mediated cellular response was found highly synchronized with the expression of DI-RNAs and formation of intracellular dsRNA. I found evidence of ADAR1-editing of the DI-RNAs, supporting the hypothesis that ADAR1 is able to antagonize PKR activation through an editing-dependent mechanism leading to the destabilization of dsRNA structures. The results obtained in this project also gave novel insights into the function of the viral C protein in MV replication, acting as a processivity factor of the viral polymerase. My ongoing research tries to identify the molecular mechanism of the C protein function.
Publications
- Measles virus C protein impairs production of defective copyback double-stranded viral RNA and activation of protein kinase R. J Virol. 2014 Jan;88(1):456-68. Epub 2013 Oct 23
Pfaller CK, Radeke MJ, Cattaneo R, Samuel CE
(See online at https://doi.org/10.1128/JVI.02572-13) - Measles virus defective interfering RNAs are generated frequently and early in the absence of C protein and can be destabilized by adenosine deaminase acting on RNA-1-like hypermutations. J Virol. 2015 Aug 1;89(15):7735-47. Epub 2015 May 13
Pfaller CK, Mastorakos GM, Matchett WE, Ma X, Samuel CE, Cattaneo R
(See online at https://doi.org/10.1128/JVI.01017-15) - Reverse genetics of Mononegavirales: How they work, new vaccines, and new cancer therapeutics. Virology. 2015 May;479-480:331-44. Epub 2015 Feb 18
Pfaller CK, Cattaneo R, Schnell MJ
(See online at https://doi.org/10.1016/j.virol.2015.01.029)
