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Literature Sharing |S146 and M148 within the mature chain domain of PSMB4 are crucial for degrading PRRSV nsp1α
Release time:
2025-04-18
Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded positive-sense RNA virus with an envelope. It-encoded non-structural protein 1α (nsp1α) plays a key role in evading host immune responses. Exploring the interaction between host factors and PRRSV nsp1α is crucial for understanding the mechanism of virus immune escape and virus control. Here, we constructed a cDNA library using porcine lung tissues and identified 33 potential host proteins interacting with viral nsp1α using yeast two-hybrid (Y2H) screening. These interactions were further analyzed using Gene Ontology and KEGG pathway analysis. Confocal microscopy revealed that proteasome subunit beta type-4 (PSMB4), carnosine dipeptidase 2 (CNDP2) and poly(rC) binding protein 1 (PCBP1) colocalized with viral nsp1α. The interaction between PSMB4 and nsp1α was further confirmed by Y2H and co-immunoprecipitation. PRRSV infection did not affect PSMB4 expression in both Marc-145 cells and porcine alveolar macrophages (PAMs). Overexpression of PSMB4 reduced nsp1α protein levels in a dose-dependent manner and decreased the accumulation of both viral N and nsp1α proteins in the context of PRRSV infection, while its knockdown promoted PRRSV replication. These data suggest that PSMB4 is a host restriction factor for PRRSV. Structure prediction and truncated mutant assays found that S146 and M148 within the mature chain domain of PSMB4 were crucial for binding and degrading nsp1α. These findings suggest that PRRSV nsp1α interacts with host proteins, with PSMB4 specifically binding to degrade nsp1α, thereby inhibiting PRRSV replication.
To identify host proteins that interact with nsp1α, the classical Y2H method was utilized in the screening assays. Nsp1α is translated from a single open reading frame (ORF) as a polyprotein, which is then self-cleaved to produce distinct proteins. The full-length sequence of nsp1α was inserted into the library bait vector pGBKT7. Self-activation test results indicated that co-transforming pGBKT7-nsp1α with pGADT7-T yielded a few colonies on the TDO/X medium, but no yeast growth occurred on the QDO/X/A solid media (Fig. 1A). Under these conditions, the Y2H screening assays using pGBK-nsp1α as bait were performed on the most stringent QDO/X/A nutritional deficiency screening medium (Fig. 1B).
Fig. 1 Detection of self-activation of pGBKT7-nsp1α bait and screening host proteins interacting with nsp1α in Y2H assay.
To maximize the screening of host proteins interacting with nsp1α, co-transformed clones were cultured on QDO/X/A medium plates. In the nsp1α screening experiment, a total of 128 clones turned blue, of which 60 were randomly selected for successful sequencing (including 9 sequencingdoublets).
The GO molecular function analysis revealed that the majority of proteins interacting with nsp1α are involved in cell adhesion molecular binding, catalytic activity, and kinase binding activity (Fig. 2A). The biological process analysis showed significant enrichment in processes such as extracellular matrix organization, regulation of cellsubstrate adhesion, response to abiotic stimuli, hemopoiesis regulation, and development (Fig. 2B). The cellular component analysis highlighted the presence of these interacting proteins in collagen-containing extracellular matrices, postsynaptic sites, and membrane rafts (Fig. 2C). Additionally, KEGG pathway analysis indicated that these proteins are primarily associated with viral infectious diseases, transport and catabolism, the host immune system, and signal transduction (Fig. 2D). Furthermore, several proteins were found to regulate channel activity, receptor activity, and enzyme activity. In summary, the interaction of PRRSV nsp1α with various host proteins suggests that these viral proteins may facilitate immune evasion by modulating diverse cellular signals and pathways.
Fig. 2 The analysis of GO and KEGG enrichment.
To further determine whether nsp1α interacts with the potentially identified proteins, we selected three functionally diverse proteins, including PSMB4, carnosine dipeptidase 2 (CNDP2) and poly(rC) binding protein 1 (PCBP1). Their coding sequences from Chlorocebus sabaeus were cloned into the expression vector pcDNA3.1, fused with a Myc tag at the C-terminus. HEK293T cells were separately co-transfected with nsp1α-mCherry and each of the four host proteins. After 24 h post-transfection, an indirect immunofluorescence assay (IFA) was performed using a Myc antibody, and the cell nuclei were stained with DAPI. Confocal fluorescence microscopy revealed that nsp1α- mCherry and mCNDP2-Myc exhibited overlapping yellow fluorescence in the cytoplasm. Conversely, nsp1α-mCherry and mPCBP1-Myc displayed overlapping yellow fluorescence in the cell nucleus (Fig. 3A).
Fig. 3 Confocal fluorescence microscopy confirmed that nsp1α colocalizes with host proteins CNDP2 and PCBP1.
Among the screened proteins, PSMB4 stands out as an important factor involved in regulating the structure and function of the 26 S proteasome. To better understand the interaction between nsp1α and host proteins, we then focused on PSMB4. The PSMB4 sequence from pigs was cloned into the pGBKT7 vector, and its interaction with nsp1α was tested in yeast assays (Fig. 4A). Additionally, HEK293T cells were co-transfected with pPSMB4-Myc or mPSMB4-Myc and nsp1α-mCherry plasmids for 24 h, and anti-Myc or anti-mCherry immunoprecipitation assays were performed. Western blot analysis detected both nsp1α and PSMB4 in the precipitates (Fig. 4B, C, E & F). Notably, the endogenous interaction between nsp1α and monkey PSMB4 was confirmed by Co-IP in PRRSV-infected Marc-145 cells (Fig. 4H).
Fig. 4 Interactions between nsp1α and pPSMB4 or mPSMB4 were evaluated using Co-IP and co-localization assays both in vivo and in vitro
To assess whether PSMB4 expression is influenced by PRRSV, we evaluated the accumulation of PSMB4 mRNA and protein levels during PRRSV infection. Marc-145 cells,cultured on 12-well plates to 100% confluency, were infected with PRRSV at a multiplicity of infection (MOI) of 0.5 for 0, 6, 12, 24 to 36 h. Uninfected cells served as controls. Both total RNA and protein were analyzed using qPCR and western blot. The results indicated that the accumulation of PSMB4 mRNA and protein was consistent across PRRSVinfected time points from 0 to 36 h and in uninfected groups (Fig. 5A, B & C). Additionally, varying PRRSV titers (0, 0.05, 0.5, and 5.0 MOI) were used to infect Marc-145 cells. At 24 h post-infection (hpi), there were no differences in endogenous PSMB4 mRNA and protein levels despite the gradual accumulation of N protein (Fig. 5D & E). Meanwhile, porcine alveolar macrophages were infected with PRRSV for 0, 6, 12, 24 and 36 h. Western blot analysis of PSMB4 protein levels showed no significant difference at these time points (Fig. 5F). Furthermore, increasing doses of nsp1α did not affect PSMB4 protein levels either by increase or decrease (Fig. 5G). These data demonstrate that PRRSV infection does not influence PSMB4 expression.
Fig. 5 PRRSV infection does not affect PSMB4 mRNA and protein expression
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