Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin

Song, Tao; Fang, Liurong; Wang, Dan; Zhang, Ruoxi; Zeng, Songlin; An, Kang; Chen, Huanchun; Xiao, Shaobo

doi:10.1016/j.jprot.2016.05.009

Cited by 36 publications

(34 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The luciferase reporter plasmids IFN-β-Luc, 4×PRDIII/I-Luc (referred to as IRF-3-Luc), and 4×PRDII-Luc (referred to as NF-κB-Luc), and molecules in RLR signaling pathway including RIG-I, MDA5, IPS-1, TKB1, IKKε, IRF3, IRF3-5D have been described previously [38], as has the plasmid containing the N protein of PRRSV, pCAGGS-Flag-PRRSV-N [87]. pCAGGS-Flag-PEDV-N-encoded PEDV N protein was constructed with specific primers and amplified from pCAGGS-HA-PEDV-N [38].…”

Section: Plasmidsmentioning

confidence: 99%

The nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same IFN-β antagonizing mechanism: attenuation of PACT-mediated RIG-I/MDA5 activation

et al. 2017

Self Cite

View full text Add to dashboard Cite

Coronaviruses (CoVs) are a huge threat to both humans and animals and have evolved elaborate mechanisms to antagonize interferons (IFNs). Nucleocapsid (N) protein is the most abundant viral protein in CoV-infected cells, and has been identified as an innate immunity antagonist in several CoVs, including mouse hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV. However, the underlying molecular mechanism(s) remain unclear. In this study, we found that MHV N protein inhibited Sendai virus and poly(I:C)-induced IFN-β production by targeting a molecule upstream of retinoic acid-induced gene I (RIG-I) and melanoma differentiation gene 5 (MDA5). Further studies showed that both MHV and SARS-CoV N proteins directly interacted with protein activator of protein kinase R (PACT), a cellular dsRNA-binding protein that can bind to RIG-I and MDA5 to activate IFN production. The N–PACT interaction sequestered the association of PACT and RIG-I/MDA5, which in turn inhibited IFN-β production. However, the N proteins from porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV), which are also classified in the order Nidovirales, did not interact and counteract with PACT. Taken together, our present study confirms that both MHV and SARS-CoV N proteins can perturb the function of cellular PACT to circumvent the innate antiviral response. However, this strategy does not appear to be used by all CoVs N proteins.

show abstract

Section: Plasmidsmentioning

confidence: 99%

The nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same IFN-β antagonizing mechanism: attenuation of PACT-mediated RIG-I/MDA5 activation

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, we missed the previously reported self-interactions for nsp1␤ and nsp11 (40,55), which were mainly characterized by in vitro assays, suggesting that fusion domains likely have an adverse effect on the interaction. Overall, among the 29 identified interactions, 5 interaction pairs (nsp1␣-nsp1␣, nsp12-nsp12, nsp2-nsp1␣, nsp2-nsp1␤, and nsp7␣-nsp9) have been reported in PRRSV (44,45,56,57). However, these interactions were mainly characterized by using in vitro assays and have not been tested within mammalian cells.…”

Section: Resultsmentioning

confidence: 99%

“…9E). A recent study has identified nsp1␣ and nsp1␤ as the binding partners for nsp2 (45), but they were missing in our Y2H screen. We confirmed these interactions by co-IP assay ( Fig.…”

Section: Figmentioning

confidence: 96%

“…As a critical step toward understanding this process, it is necessary to investigate the interaction network among PRRSV nsps. Information in this regard has been scarce, with only a limited number of interactions identified (e.g., nsp2-nsp1␣, nsp2-nsp1␤, and nsp9-nsp7) (44,45), and the picture for a comprehensive map remained unavailable. Yeast two-hybrid (Y2H) screening, glutathione S-transferase (GST) pulldown, and coimmunoprecipitation (co-IP) are the most commonly used methods for investigating protein-protein interactions (PPI) of nidoviruses (e.g., coronaviruses) (46)(47)(48)(49).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus

Song

Liu

Gao

et al. 2018

J Virol

View full text Add to dashboard Cite

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positivestranded RNA virus belonging to the family Arteriviridae. Synthesis of the viral RNA is directed by replication/transcription complexes (RTC) that are mainly composed of a network of PRRSV nonstructural proteins (nsps) and likely cellular proteins. Here, we mapped the interaction network among PRRSV nsps by using yeast two-hybrid screening in conjunction with coimmunoprecipitation (co-IP) and cotransfection assays. We identified a total of 24 novel interactions and found that the interactions were centered on open reading frame 1b (ORF1b)-encoded nsps that were mainly connected by the transmembrane proteins nsp2, nsp3, and nsp5. Interestingly, the interactions of the core enzymes nsp9 and nsp10 with transmembrane proteins did not occur in a straightforward manner, as they worked in the co-IP assay but were poorly capable of finding each other within intact mammalian cells. Further proof that they can interact within cells required the engineering of N-terminal truncations of both nsp9 and nsp10. However, despite the poor colocalization relationship in cotransfected cells, both nsp9 and nsp10 came together with membrane proteins (e.g., nsp2) at the viral replication and transcription complexes (RTC) in PRRSV-infected cells. Thus, our results indicate the existence of a complex interaction network among PRRSV nsps and raise the possibility that the recruitment of key replicase proteins to membrane-associated nsps may involve some regulatory mechanisms during infection. IMPORTANCE Synthesis of PRRSV RNAs within host cells depends on the efficient and correct assembly of RTC that takes places on modified intracellular membranes. As an important step toward dissecting this poorly understood event, we investigated the interaction network among PRRSV nsps. Our studies established a comprehensive interaction map for PRRSV nsps and revealed important players within the network. The results also highlight the likely existence of a regulated recruitment of the PRRSV core enzymes nsp9 and nsp10 to viral membrane nsps during PRRSV RTC assembly. Downloaded fromframe 1a (ORF1a) and ORF1b (Fig. 1A), which specify replicase nonstructural proteins (nsps) important for viral RNA synthesis and for antagonizing host antiviral immunity (8,9). Upon virus entry, ORF1a is translated from the incoming genome to produce replicase polyprotein pp1a, which is further matured into at least 10 nsps (e.g., nsp1␣, nsp1␤, nsp2 to nsp6, nsp7␣, nsp7␤, and nsp8) by virus-encoded proteases within nsp1␣, nsp1␤, nsp2, and nsp4 (7, 10, 11). In addition, there exist isoforms for nsp2, and one of them (nsp2TF) is made via a Ϫ2 frameshift mechanism (12, 13). On the other hand, translation of ORF1b, an open reading frame that specifies replicase proteins nsp9, nsp10, nsp11, and nsp12, involves a Ϫ1 ribosome frameshift (14-16).Synthesis of PRRSV RNA within host cells depends on the efficient and correct assembly of replication and transcription complexes (RTC) that coordinate the tran-FI...

show abstract

“…4A). Previous studies have demonstrated that PRRSV nsp2 interacts with many mitochondrial proteins (31)(32)(33), and our laboratory has also observed that nsp2 localizes to the mitochondria (unpublished data), so we tested the exact intracellular localizations of nsp2 and nsp2-Δtm. To this end, HeLa cells were transfected with plasmids encoding nsp2 or nsp2-Δtm together with the pDsRed2-Mito plasmid, which expresses the Mito-DsRed2 fusion protein and targets it to the mitochondria.…”

Section: Prrsv Infection Induces Translation Suppression In Cellsmentioning

confidence: 99%

Porcine Reproductive and Respiratory Syndrome Virus Infection Induces both eIF2α Phosphorylation-Dependent and -Independent Host Translation Shutoff

Fang

Zhou

et al. 2018

J Virol

Self Cite

View full text Add to dashboard Cite

Porcine reproductive and respiratory syndrome virus (PRRSV) is an that has caused tremendous economic losses in the global swine industry since it was discovered in the late 1980s. Inducing host translation shutoff is a strategy used by many viruses to optimize their replication and spread. Here, we demonstrate that PRRSV infection causes host translation suppression, which is strongly dependent on viral replication. By screening PRRSV-encoded nonstructural proteins (nsps), we found that nsp2 participates in the induction of host translation shutoff and that its transmembrane (TM) domain is required for this process. nsp2-induced translation suppression is independent of protein degradation pathways and the phosphorylation of eukaryotic initiation factor 2α (eIF2α). However, the overexpression of nsp2 or its TM domain significantly attenuated the mammalian target of rapamycin (mTOR) signaling pathway, an alternative pathway for modulating host gene expression. PRRSV infection also attenuated the mTOR signaling pathway, and PRRSV-induced host translation shutoff could be partly reversed when the attenuated mTOR phosphorylation was reactivated by an activator of the mTOR pathway. PRRSV infection still negatively regulated the host translation when the effects of eIF2α phosphorylation were completely reversed. Taken together, our results demonstrate that PRRSV infection induces host translation shutoff and that nsp2 is associated with this process. Both eIF2α phosphorylation and the attenuation of the mTOR signaling pathway contribute to PRRSV-induced host translation arrest. Viruses are obligate parasites, and the production of progeny viruses relies strictly on the host translation machinery. Therefore, the efficient modulation of host mRNA translation benefits viral replication, spread, and evolution. In this study, we provide evidence that porcine reproductive and respiratory syndrome virus (PRRSV) infection induces host translation shutoff and that the viral nonstructural protein nsp2 is associated with this process. Many viruses induce host translation shutoff by phosphorylating eukaryotic initiation factor 2α (eIF2α). However, PRRSV nsp2 does not induce eIF2α phosphorylation but attenuates the mTOR signaling pathway, another pathway regulating the host cell translational machinery. We also found that PRRSV-induced host translation shutoff was partly reversed by eliminating the effects of eIF2α phosphorylation or reactivating the mTOR pathway, indicating that PRRSV infection induces both eIF2α phosphorylation-dependent and -independent host translation shutoff.

show abstract

Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin

Cited by 36 publications

References 68 publications

The nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same IFN-β antagonizing mechanism: attenuation of PACT-mediated RIG-I/MDA5 activation

The nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same IFN-β antagonizing mechanism: attenuation of PACT-mediated RIG-I/MDA5 activation

Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome Virus Infection Induces both eIF2α Phosphorylation-Dependent and -Independent Host Translation Shutoff

Contact Info

Product

Resources

About