PDCD4 restricts PRRSV replication in an eIF4A-dependent manner and is antagonized by the viral nonstructural protein 9

Abstract

ABSTRACT As obligate parasites, viruses have evolved multiple strategies to evade the host immune defense. Manipulation of the host proteasome system to degrade specific detrimental factors is a common viral countermeasure. To identify host proteins targeted for proteasomal degradation by porcine reproductive and respiratory syndrome virus (PRRSV), we conducted a quantitative proteomics screen of PRRSV-infected Marc-145 cells under the treatment with proteasome inhibitor MG132. The data revealed that the expression levels of programmed cell death 4 (PDCD4) were strongly downregulated by PRRSV and significantly rescued by MG132. Further investigation confirmed that PRRSV infection induced the translocation of PDCD4 from the nucleus to the cytoplasm, and the viral nonstructural protein 9 (Nsp9) promoted PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway. The C-terminal domain of Nsp9 was responsible for PDCD4 degradation. As for the role of PDCD4 during PRRSV infection, we demonstrated that PDCD4 knockdown favored viral replication, while its overexpression significantly attenuated replication, suggesting that PDCD4 acts as a restriction factor for PRRSV. Mechanistically, we discovered eukaryotic translation initiation factor 4A (eIF4A) was required for PRRSV. PDCD4 interacted with eIF4A through four sites (E249, D253, D414, and D418) within its two MA3 domains, disrupting eIF4A-mediated translation initiation in the 5′-untranslated region of PRRSV, thereby inhibiting PRRSV infection. Together, our study reveals the antiviral function of PDCD4 and the viral strategy to antagonize PDCD4. These results will contribute to our understanding of the immune evasion strategies employed by PRRSV and offer valuable insights for developing new antiviral targets. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) infection results in major economic losses in the global swine industry and is difficult to control effectively. Here, using a quantitative proteomics screen, we identified programmed cell death 4 (PDCD4) as a host protein targeted for proteasomal degradation by PRRSV. We demonstrated that PDCD4 restricts PRRSV replication by interacting with eukaryotic translation initiation factor 4A, which is required for translation initiation in the viral 5′-untranslated region. Additionally, four sites within two MA3 domains of PDCD4 are identified to be responsible for its antiviral function. Conversely, PRRSV nonstructural protein 9 promotes PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway, thus weakening the anti-PRRSV function. Our work unveils PDCD4 as a previously unrecognized host restriction factor for PRRSV and reveals that PRRSV develops countermeasures to overcome PDCD4. This will provide new insights into virus-host interactions and the development of new antiviral targets.