Peste Des Petits Ruminants Virus Nucleocapsid Protein Interacts with Protein Kinase R-Activating Protein and Induces Stress Granules To Promote Viral Replication

Stress granule (SG) formation is a conserved cellular strategy to reduce stress-related damage regulating cell survival. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N interacted with PACT to regulate the assembly of SGs. The pathogenic mechanisms of peste des petits ruminants virus (PPRV) infection remain poorly understood, leaving peste des petits ruminants (PPR) control and eradication especially difficult. Here, we determined that PPRV nucleocapsid (N) protein triggers formation of stress granules (SGs) to benefit viral replication. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N protein interacted with protein kinase R (PKR)-activating protein (PACT), and this interaction was confirmed in the context of PPRV infection. PACT was essential for PPRV replication. Besides, the ectopic expression of N activated the PKR/eIF2 alpha (alpha subunit of eukaryotic initiation factor 2) pathway through induction of PKR phosphorylation, but it did not induce PKR phosphorylation in PACT-deficient (PACT(-/-)) cells. PPRV N interacted with PACT, impairing the interaction between PACT and a PKR inhibitor, transactivation response RNA-binding protein (TRBP), which subsequently enhanced the interaction between PACT and PKR and thus promoted the activation of PKR and eIF2 alpha phosphorylation, resulting in formation of stress granules (SGs). Consistently, PPRV infection induced SG formation through activation of the PKR/eIF2 alpha pathway, and knockdown of N impaired PPRV-induced SG formation. PPRV-induced SG formation significantly decreased in PACT(-/-) cells as well. The role of SG formation in PPRV replication was subsequently investigated, which showed that SG formation plays a positive role in PPRV replication. By using an RNA fluorescence in situ hybridization assay, we found that PPRV-induced SGs hid cellular mRNA rather than viral mRNA. Altogether, our data provide the first evidence that PPRV N protein plays a role in modulating the PKR/eIF2 alpha/SG axis and promotes virus replication through targeting PACT.IMPORTANCE Stress granule (SG) formation is a conserved cellular strategy to reduce stress-related damage regulating cell survival. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N interacted with PACT to regulate the assembly of SGs. N protein inhibited the interaction between PACT and a PKR inhibitor, TRBP, through binding to the M1 domain of PACT, which enhanced the interaction between PACT and PKR and thus promoted PKR activation and subsequent eIF2 alpha phosphorylation as well as SG formation. The regulatory function of N protein was strikingly abrogated in PACT(-/-) cells. SGs induced by PPRV infection through the PKR/eIF2 alpha pathway are PACT dependent. The loss-of-function assay indicated that PPRV-induced SGs were critical for PPRV replication. We concluded that the PPRV N protein manipulates the host PKR/eIF2 alpha/SG axis to favor virus replication.

Automated summary

Stress granule (SG) formation induced by Peste des petits ruminants virus (PPRV) N protein plays a positive role in PPRV replication. PPRV N protein interacts with PKR-activating protein (PACT) to enhance PKR activation and subsequent SG formation. The regulatory function of N protein is dependent on the presence of PACT.