Mechanisms of Protein Kinase PKR-Mediated Amplification of Beta Interferon Induction by C Protein-Deficient Measles Virus

Abstract

The measles virus P gene products V and C antagonize the host interferon (IFN) response, blocking both IFN signaling and production. Using Moraten vaccine strain-derived measles virus and isogenic mutants deficient for either V or C protein production (V ko and C ko , respectively), we observed that the C Measles virus (MV), a member of the genus Morbillivirus of the Paramyxoviridae, causes an acute febrile illness. Despite an effective vaccine, measles continues to cause extreme morbidity and mortality worldwide (10), and recently, there has been a resurgence of measles in industrialized countries, where a lack of adherence to vaccine recommendations is an increasing problem (5, 12). The need for improved MV vaccines (11), together with the potential for use of engineered MV vaccine strains with defined mechanisms of attenuation as oncolytic viruses for cancer therapy (4), further justify ongoing efforts to gain an enhanced understanding of the host response to MV infection at the molecular level.The 15.9-kb negative-stranded RNA genome of MV consists of six genes (N, P/V/C, M, F, H, and L). The P gene is polycistronic, encoding the V and C nonstructural proteins in addition to P, a structural phosphoprotein and essential cofactor for the viral polymerase (2, 3, 10). The V protein shares its N-terminal 231 amino acids with P, but the C-terminal 68 amino acids are unique because of the pseudotemplated G insertion that causes a frameshift in V mRNA, whereas the C protein is synthesized by an alternative translation initiation AUG codon positioned 22 nucleotides downstream of the P/V translation start codon (3, 11). V and C are accessory proteins that serve a variety of functions including the modulation of the host innate immune response to MV infection (8, 9, 27). Isogenic MV mutants that are defective for the expression of either V or C have been generated. Strong adaptive immune responses, but dysregulated innate responses, are seen with these mutants (4,8,35).An important component of the host innate antiviral response is the interferon (IFN) response. IFNs are proinflammatory cytokines that possess antiviral activity (27,31). IFN action involves IFN binding to cognate receptors and subsequent prototypical JAK-STAT signal transduction that leads to the expression of IFN-stimulated genes, whose products inhibit virus growth. IFN production involves the recognition of pathogen-associated molecular patterns including viral RNAs by retinoic acid-inducible protein (RIG-I)-like cytosolic receptors (RLRs) and membrane-associated Toll-like receptors (TLRs). The RLR and TLR3 sensors signal through cognate adapter proteins, including IPS-1 and TRIF, respectively, to transcriptionally activate IFN expression (36,40). In the case of the IFN-␤ gene, IFN regulatory factor 3 (IRF-3) and nuclear factor B (NF-B) are activated by RLR or TLR signaling, enter the nucleus, and function together with activating transcription factor 2 (ATF-2)/c-jun to constitute the IFN-␤ enhanceosome that drives IFN-␤ transcription (22).The antago...

Protein Kinase PKR Amplification of Interferon β Induction Occurs through Initiation Factor eIF-2α-mediated Translational Control

“…For transient knockdown experiments, a double transfection strategy with siRNA was utilized (27). HeLa cells in 60-mm dishes at ϳ60% confluency were transfected with 50 nM of siRNA with Lipofectamine 2000 on day 1 and again on day 3.…”

“…Protein fractionation and immunoblot analysis were as described (27,40) using the following primary antibodies: PKR and IB-␣ (Santa Cruz Biotechnology); phosphorylated-PKR threonine 446 (Epitomics); eIF-2␣ (Cell Signaling Technology) and phosphorylatedeIF-2␣ serine 51 (Cell Signaling Technology or Epitomics) and ␣-tubulin (Sigma). Antibody against MV H protein was generously provided by Dr. R. Cattano (Mayo Clinic, Rochester, MN).…”

“…Random-primed reverse transcription was carried out using 500 ng of RNA and SuperScript II (Invitrogen) according to the manufacturer's protocol. For qPCR reactions, the GAPDH and IFN-␤ primer pairs were as previously described (27). qPCR reactions were performed in duplicate or triplicate with each RT template, using IQ SYBR Green Supermix (Bio-Rad) and a Bio-Rad MyIQ multicolor real-time qPCR instrument (3-min hot start followed by 30 s at 95°C, 45 s at 58°C, 45 s at 72°C, for 40 to 45 cycles).…”

“…Because NF-B is one of the transcription factors required for IFN-␤ induction and the activation of NF-B and induction of IFN-␤ are enhanced by PKR in measles virus-infected and RNA-transfected cells (24,27,43) we hypothesized that differential NF-B signaling may be responsible in part for the differences in IFN-␤ expression triggered by the different-sized dsRNAs. We previously observed that the inhibitor of NF-B signaling, IB-␣, was degraded following Pds50 and pIpC transfection (24).…”

“…Transcriptional induction of IFN-␤ is known to require the activation of both IRF and NF-B transcription factors downstream of the TLRs and RLRs (25,26). We earlier established that while PKR is not required for IRF3 activation following virus infection or RNA transfection, PKR is required for maximal NF-B activity (24,27). The NF-B family members involved in type I IFN signaling include the p65 (RelA) and p50 subunits.…”