Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean Red Ginseng.

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean Red Ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

Regulation of tyrosine hydroxylase gene expression by retinoic acid receptor.

Retinoic acid (RA), a derivative of vitamin A, critically controls brain patterning and neurogenesis during embryogenesis, and is known to regulate morphological differentiation of catecholaminergic neuronal cells. In this study, we investigated whether the retinoic acid receptor (RAR), a transcription factor specifically activated by all-trans-RA, could directly regulate transcription of tyrosine hydroxylase (TH), the first and rate-limiting step in the catecholamine biosynthesis pathway. First, treating TH-expressing human neuroblastoma SK-N-BE(2)C cells with all-trans RA resulted in an approximately 1.7-fold increase in endogenous TH mRNA expression, as determined by real-time PCR analysis. Second, when SK-N-BE(2)C cells were transiently co-transfected with the TH promoter-luciferase reporter construct, reporter gene expression was prominently activated by RAR in a ligand-dependent manner. Third, we identified a putative RAR responsive cis-regulatory element at - 1500 to - 1487 bp in the TH upstream promoter region by deletional and site-directed mutational analysis. Finally, we demonstrated that this putative motif directly interacts with RAR protein in a sequence-specific manner by means of an electrophoretic mobility shift assay. Taken together, our results indicate that the TH gene may be a direct downstream target of the RA signaling pathway and that RAR is able to activate TH transcription through interaction with an upstream sequence motif residing at - 1500 to - 1487 bp.

Regulation of the transcriptional activity of the tyrosine hydroxylase gene by androgen receptor.

Dopamine and the sex hormone testosterone are important factors regulating male sexual behavior. To investigate the possibility that these two factors are functionally interrelated, we investigated the potential role of the androgen receptor (AR) on transcriptional activity of the tyrosine hydroxylase (TH) gene that encodes the rate-limiting enzyme of the dopamine biosynthesis pathway. In this study, using transient co-transfection assays in TH-positive SK-N-BE(2)C and MN9D cells, we show that AR prominently transactivates TH promoter function in a ligand-dependent manner. Deletional and site-directed mutational analyses have mapped a putative androgen response element (ARE) in a region from -1562 to -1328 base pairs in the upstream TH promoter. We also found that DJ-1, one of recently identified genes whose mutations cause Parkinson's disease, down-regulated AR-dependent TH activation by approximately 50% in SK-N-BE(2)C cells. Based on these data, we propose that AR activates TH gene expression and that DJ-1 may modulate AR activity as a transcriptional co-repressor.