The Papain-Like Protease of Porcine Reproductive and Respiratory Syndrome Virus Impedes STING Translocation from the Endoplasmic Reticulum to the Golgi Apparatus by Deubiquitinating STIM1.

Stimulator of interferon (IFN) genes (STING) was recently pinpointed as an antiviral innate immune factor during the infection of RNA viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), the swine arterivirus, is an enveloped RNA virus which has evolved many strategies to evade innate immunity. To date, the interactive network between PRRSV and STING remains to be fully established. Herein, we report that STING suppresses PRRSV replication through type I interferon signaling. However, PRRSV impedes STING trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, leading to the decreased phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). Furthermore, PRRSV nonstructural protein 2 (Nsp2) colocalizes with STING, blocks STING translocation, and disrupts the STING-TBK1-IRF3 complex. Mechanistically, PRRSV Nsp2 retains STING at the ER by increasing the level of Ca2+ sensor stromal interaction molecule 1 (STIM1) protein. Functional analysis reveals that PRRSV Nsp2 deubiquitinates STIM1 by virtue of its papain-like protease 2 (PLP2) deubiquitinating (DUB) activity. Finally, we demonstrate that loss of STIM1 is associated with an elevated IFN response and restricts PRRSV replication. This work delineates the relationship between PRRSV infection and STING signaling and the importance of papain-like proteases (PLPs) in interfering in this axis. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the family Arteriviridae, is responsible for reproductive disorders in pregnant sows and respiratory problems in piglets, resulting in huge losses in the swine industry worldwide. Of note, PRRSV infection causes immunosuppression, of which the mechanism is not completely understood. Here, we demonstrate for the first time that STING, a protein typically associated with the antiviral response in DNA viruses, plays a critical role in controlling PRRSV infection. However, PRRSV utilizes its encoded protein Nsp2 to inhibit STING activity by blocking its translocation from the ER to the Golgi apparatus. In particular, Nsp2 retains STING at the ER by interacting with and further deubiquitinating STIM1. For this process, the activity of the viral PLP2 DUB enzyme is indispensable. The study describes a novel mechanism by which PLP2 plays a critical role in suppressing the innate immune response against arteriviruses and potentially other viruses that encode similar proteases.

Porcine reproductive and respiratory syndrome virus infection triggers autophagy via ER stress-induced calcium signaling to facilitate virus replication.

Calcium (Ca2+), a ubiquitous second messenger, plays a crucial role in many cellular functions. Viruses often hijack Ca2+ signaling to facilitate viral processes such as entry, replication, assembly, and egress. Here, we report that infection by the swine arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV), induces dysregulated Ca2+ homeostasis, subsequently activating calmodulin-dependent protein kinase-II (CaMKII) mediated autophagy, and thus fueling viral replication. Mechanically, PRRSV infection induces endoplasmic reticulum (ER) stress and forms a closed ER-plasma membrane (PM) contacts, resulting the opening of store operated calcium entry (SOCE) channel and causing the ER to take up extracellular Ca2+, which is then released into the cytoplasm by inositol trisphosphate receptor (IP3R) channel. Importantly, pharmacological inhibition of ER stress or CaMKII mediated autophagy blocks PRRSV replication. Notably, we show that PRRSV protein Nsp2 plays a dominant role in the PRRSV induced ER stress and autophagy, interacting with stromal interaction molecule 1 (STIM1) and the 78 kDa glucose-regulated protein 78 (GRP78). The interplay between PRRSV and cellular calcium signaling provides a novel potential approach to develop antivirals and therapeutics for the disease outbreaks.

Autophagy induced by Rab1a-ULK1 interaction promotes porcine reproductive and respiratory syndrome virus replication.

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus from the Nidovirales order, continues to be a threat to the swine industry worldwide causing reproductive failure and respiratory disease in pigs. Previous studies have demonstrated that autophagy plays a positive role in PRRSV replication. However, its mechanism is less clearly understood. Herein, we report first that the protein level of Rab1a, a member of the Ras superfamily of GTPases, is upregulated during PRRSV infection. Subsequently, we demonstrate that Rab1a enhances PRRSV replication through an autophagy pathway as evidenced by knocking down the autophagy-related 7 (ATG7) gene, the key adaptor of autophagy. Importantly, we reveal that Rab1a interacts with ULK1 and promotes ULK1 phosphorylation dependent on its GTP-binding activity. These data indicate that PRRSV utilizes the Rab1a-ULK1 complex to initiate autophagy, which, in turn, benefits viral replication. These findings further highlight the interplay between PRRSV replication and the autophagy pathway, deepening our understanding of PRRSV infection.