Determination of the interactome of non-structural protein12 from highly pathogenic porcine reproductive and respiratory syndrome virus with host cellular proteins using high throughput proteomics and identification of HSP70 as a cellular factor for virus replication

Wang

et al. 2018

J Virol

Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating the nucleocytoplasmic translocation of proteins through the NPC. In this study, we found that karyopherin α6 (KPNA6; also known as importin α7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that in mock-infected controls, whereas the KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of the KPNA6 half-life and the elevation of the KPNA6 level in comparison to mock-infected cells. PRRSV nsp12 protein induced KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1β but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6-knockout cells results in restoration of the nuclear translocation of PRRSV nsp1β and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV. Positive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin α6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced an elevation of the KPNA6 protein level due to an extension of the KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced the replication of PRRSV and ZIKV. PRRSV nsp1β depended on KPNA6 to translocate into the nucleus. In addition, exogenous restitution of KPNA6 expression in KPNA6-knockout cells led to the restoration of nsp1β nuclear translocation and ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics.

Section: Discussionmentioning

confidence: 99%

Karyopherin Alpha 6 Is Required for Replication of Porcine Reproductive and Respiratory Syndrome Virus and Zika Virus

Wang

et al. 2018

J Virol

“…Besides nsp9, the protein–protein interaction analyses also identified multiple interaction partners (nsp3, nsp5, nsp7α, nsp7β, nsp8, nsp9, nsp10, nsp11, and nsp12 itself) for nsp12, the structure and function of which is still unknown. Using high throughput proteomics, we have found that PRRSV nsp12 interacts with many cellular proteins with high probability ( Dong et al, 2016 ). Among the cellular proteins, HSP70 is recruited by nsp12 to maintain the protein’s stability and benefit the viral replication.…”

Section: Discussionmentioning

confidence: 99%

The Network of Interactions Among Porcine Reproductive and Respiratory Syndrome Virus Non-structural Proteins

et al. 2018

Self Cite

The RNA synthesis of porcine reproductive and respiratory syndrome virus (PRRSV), a positive-strand RNA virus, is compartmentalized in virus-induced double-membrane vesicles where viral proteins and some cellular proteins assemble into replication and transcription complexes (RTCs). The viral replicase proteins are the major components of the RTCs but the physical associations among these non-structural proteins (nsps) remain elusive. In this study, we investigated the potential interactions between PRRSV nsps by yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and pull-down assays. Our analyses revealed a complex network of interactions involving most of PRRSV nsps. Among them, nsp9 and nsp12 were identified as the hubs of the nsp interactome; transmembrane proteins nsp2 and nsp5 both interacted with nsp3, indicating that the three membrane-bound proteins might bind together to form the scaffold to support the association of RTCs with the intracellular membrane. The PRRSV nsp interactions identified in this study may provide valuable clues for future researches on the RTC formation and function.

“…There are reports that the combination of cysteine 35 and cysteine 79 in NSP12 is required for sgmRNA synthesis [ 60 ]. To explore the function of NSP12, Dong et al [ 61 ] studied the interaction of NSP12 with cell proteins using an immunoprecipitation strategy of a quantitative proteomics-binding NSP12-EGFP fusion protein overexpressed in 293T cells to determine whether HSP70 can interact with NSP12. They found that NSP12 recruits HSP70 to maintain its stability and inhibits viral replication [ 61 ].…”

Section: Nonstructural Proteinsmentioning

confidence: 99%

“…To explore the function of NSP12, Dong et al [ 61 ] studied the interaction of NSP12 with cell proteins using an immunoprecipitation strategy of a quantitative proteomics-binding NSP12-EGFP fusion protein overexpressed in 293T cells to determine whether HSP70 can interact with NSP12. They found that NSP12 recruits HSP70 to maintain its stability and inhibits viral replication [ 61 ]. Porcine galactoctin-3 (GAL3) is a 29 kDa protein encoded by a single gene, LGAS3, located on chromosome 1.…”

Section: Nonstructural Proteinsmentioning

confidence: 99%

Research Progress in Porcine Reproductive and Respiratory Syndrome Virus–Host Protein Interactions

Zhang

Sha

Qin

et al. 2022

Animals

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which has been regarded as a persistent challenge for the pig industry in many countries. PRRSV is internalized into host cells by the interaction between PRRSV proteins and cellular receptors. When the virus invades the cells, the host antiviral immune system is quickly activated to suppress the replication of the viruses. To retain fitness and host adaptation, various viruses have evolved multiple elegant strategies to manipulate the host machine and circumvent against the host antiviral responses. Therefore, identification of virus–host interactions is critical for understanding the host defense against viral infections and the pathogenesis of the viral infectious diseases. Most viruses, including PRRSV, interact with host proteins during infection. On the one hand, such interaction promotes the virus from escaping the host immune system to complete its replication. On the other hand, the interactions regulate the host cell immune response to inhibit viral infections. As common antiviral drugs become increasingly inefficient under the pressure of viral selectivity, therapeutic agents targeting the intrinsic immune factors of the host protein are more promising because the host protein has a lower probability of mutation under drug-mediated selective pressure. This review elaborates on the virus–host interactions during PRRSV infection to summarize the pathogenic mechanisms of PRRSV, and we hope this can provide insights for designing effective vaccines or drugs to prevent and control the spread of PRRS.