The unfolded protein response (UPR) relieves endoplasmic reticulum (ER) stress through multiple strategies, including reducing protein synthesis, increasing protein folding capabilities, and enhancing misfolded protein degradation. After a multi-omics analysis, we find that signal recognition particle 14 (SRP14), an essential component of the SRP, is markedly reduced in cells undergoing ER stress. Further experiments indicate that SRP14 reduction requires PRKR-like ER kinase (PERK)-mediated eukaryotic translation initiation factor 2α (eIF2α) phosphorylation but is independent of ATF4 or ATF3 transcription factors. The decrease of SRP14 correlates with reduced translocation of fusion proteins and endogenous cathepsin D. Enforced expression of an SRP14 variant with elongation arrest capability prevents the reduced translocation of cathepsin D in stressed cells, whereas an SRP14 mutant without the activity does not. Finally, overexpression of SRP14 augments the UPR and aggravates ER-stress-induced cell death. These data suggest that translocational attenuation mediated by the PERK-SRP14 axis is a protective measure for the UPR to mitigate ER stress.

SRP14 is a crucial protein subunit of the signal recognition particle (SRP), a ribonucleoprotein complex essential for co-translational translocation to the endoplasmic reticulum. During our investigation of SRP14 expression across diverse cell lines, we observe variations in its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with some cells exhibiting slower migration and others migrating faster. However, the cause of this phenomenon remains elusive. Our research rules out alternative splicing as the cause and, instead, identifies the presence of a P124A mutation in SRP14 (SRP14 P124A) among the faster-migrating variants, while the slower-migrating variants lack this mutation. Subsequent ectopic expression of wild-type SRP14 P124 or SRP14 WT and SRP14 P124A in various cell lines confirms that the P124A mutation indeed leads to faster migration of SRP14. Further mutagenesis analysis shows that the P117A and A121P mutations within the alanine-rich domain at the C-terminus of SRP14 are responsible for migration alterations on SDS-PAGE, whereas mutations outside this domain, such as P39A, Y27F, and T45A, have no such effect. Furthermore, the ectopic expression of SRP14 WT and SRP14 P124A yields similar outcomes in terms of SRP RNA stability, cell morphology, and cell growth, indicating that SRP14 P124A represents a natural variant of SRP14 and retains comparable functionality. In conclusion, the substitution of proline for alanine in the alanine-rich tail of SRP14 results in faster migration on SDS-PAGE, but has little effect on its function.

MicroRNAs (miRNAs) play an important role in the virus-host interaction. Our previous work has indicated that the expression level of miR-10a increased in porcine alveolar macrophages (PAMs) during porcine reproductive and respiratory syndrome virus (PRRSV) infection and further inhibited viral replication through downregulates the expression of host molecule signal-recognition particle 14 (SRP14) protein. However, the molecular mechanism of miR-10a increased after PRRSV infection remains unknown. In the present study, transcription factor interferon regulatory factor 8 (IRF8) was identified as a negative regulator of miR-10a. PRRSV infection decreases the expression level of IRF8 in PAMs, leading to upregulating miR-10a expression to play an anti-PRRSV role. Meanwhile, this work first proved that IRF8 promoted PRRSV replication in an miR-10a-dependent manner. Further, we explained that SRP14, the target gene of miR-10a, promotes the synthesis of the PRRSV genome by interacting with the viral components Nsp2, thus facilitating PRRSV replication. In conclusion, we identified a novel IRF8-miR-10a-SRP14 regulatory pathway against PRRSV infection, which provides new insights into virus-host interactions and suggests potential new antiviral strategies to control PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has rapidly spread to the global pig industry and caused incalculable economic damage since first discovered in the 1980s. However, conventional vaccines do not provide satisfactory protection. Understanding the molecular mechanisms of host resistance to PRRSV infection is necessary to develop safe and effective strategies to control PRRSV. During viral infection, miRNAs play vital roles in regulating the expression of viral or host genes at the posttranscriptional level. The significance of our study is that we revealed the transcriptional regulation mechanism of the antiviral molecule miR-10a after PRRSV infection. Moreover, our research also explained the mechanism of host molecule SRP14, the target gene of miR-10a regulating PRRSV replication. Thus, we report a novel regulatory pathway of IRF8-miR-10a-SRP14 against PRRSV infection, which provides new insights into virus-host interactions and suggests potential new control measures for future PRRSV outbreaks.

HIV-1 Tat protein is essential for virus production. RNA-binding proteins that facilitate Tat production may be absent or downregulated in resting CD4+ T-cells, the main reservoir of latent HIV in people with HIV (PWH) on antiretroviral therapy (ART). In this study, we examined the role of Tat RNA-binding proteins on the expression of Tat and control of latent and productive infection. Affinity purification coupled with mass spectrometry analysis was used to detect binding partners of MS2-tagged tat mRNA in a T cell-line model of HIV latency. The effect of knockdown and overexpression of the proteins of interest on Tat transactivation and translation was assessed by luciferase-based reporter assays and infections with a dual color HIV reporter virus. Out of the 243 interactions identified, knockdown of SRP14 (Signal Recognition Particle 14) negatively affected tat mRNA processing and translation as well as Tat-mediated transactivation, which led to an increase in latent infection. On the other hand, knockdown of HMGB3 (High Mobility Group Box 3) resulted in an increase in Tat transactivation and translation as well as an increase in productive infection. Footprinting experiments revealed that SRP14 and HMGB3 proteins bind to TIM-TAM, a conserved RNA sequence-structure in tat mRNA that functions as a Tat IRES modulator of tat mRNA. Overexpression of SRP14 in resting CD4+ T-cells from patients on ART was sufficient to reverse HIV-1 latency and induce virus production. The role of SRP14 and HMGB3 proteins in controlling HIV Tat expression during latency will be further assessed as potential drug targets.