Panoply™ Human EPRS Knockdown Stable Cell Line

Host factor tRNA contributes to the replication of retroviruses such as human immunodeficiency virus type 1 (HIV-1). HIV-1 utilizes human tRNALys3 as a primer for reverse transcription, and the assembly of HIV-1 structural protein Gag at the plasma membrane (PM) is regulated by the interaction of its matrix (MA) domain with tRNA. A large, dynamic multi-aminoacyl-tRNA synthetase complex (MSC) exists in the cytoplasm, composed of eight aminoacyl-tRNA synthetases (ARS) and three other cellular proteins. Proteomic studies aimed at identifying interactions between HIV and the host have shown that the MSC is part of the HIV-1 Gag and MA interactome. Here, researchers confirmed that the MA domain of HIV-1 Gag forms a stable complex with the MSC and localized the main interaction site to the linker domain of the bifunctional human glutamyl-prolyl-tRNA synthetase (EPRS), and showed that the MA-EPRS interaction is RNA-dependent. MA mutations that significantly reduce EPRS interaction decrease viral infectivity, and these mutation sites are located on MA residues that interact with phosphatidylinositol-(4,5)-bisphosphate. Overexpression of EPRS or EPRS fragments does not affect susceptibility to HIV-1 infection, while EPRS knockdown reduces the translation of both control reporter genes and HIV-1 proteins. EPRS knockdown leads to a decrease in progeny virus particle production, but this decrease cannot be attributed to a selective effect on viral gene expression, and the specific infectivity of the virus particles remains unchanged.

To further investigate the effect of EPRS on HIV-1 gene expression, researchers infected HEK293T cells overexpressing full-length EPRS or the EPRS linker domain with HIV-Luc/VSV-G virus and measured luciferase activity 24 hours post-infection. The results showed no significant difference in luciferase activity between cells overexpressing full-length EPRS or the EPRS linker domain compared to the control group using the vector alone (Figure 1A). To determine the effect of EPRS knockdown on HIV-1 gene expression, researchers constructed stable EPRS knockdown HEK293T cell lines and infected these cells with HIV-Luc/VSV-G virus encoding firefly (FF) luciferase (Figure 1B). As an internal control, cells were simultaneously transfected with a vector encoding Renilla luciferase (pRL-TK), driven by the HSV thymidine kinase (TK) promoter. In EPRS knockdown cells, HIV-1 gene expression (represented by FF luciferase activity) was reduced fourfold, and a similar reduction was observed in non-HIV gene expression (represented by Renilla luciferase activity) (Figure 1C, D). After normalizing FF luciferase activity to Renilla luciferase activity, HIV-1 gene expression levels were similar in EPRS knockdown cells and control cells (Figure 1E), indicating that the negative effect of EPRS knockdown on gene expression was not HIV-specific. FF luciferase mRNA levels were not different between EPRS knockdown cells and control cells, suggesting that EPRS does not specifically affect the transcription of HIV-1 genes (Figure 1F). Instead, EPRS knockdown affected the translation of FF luciferase, likely due to its overall effect on tRNA aminoacylation.

Figure 1. Effect of EPRS on HIV-1 gene expression. (Jin D, et al., 2023)