Aurintricarboxylic acid increases yield of HSV-1 vectors

Pechan, Peter; Ardinger, Jeffery; Ketavarapu, Jyothi; Rubin, Hillard; Wadsworth, Samuel C; Scaria, Abraham

In this study, we have shown for the first time that micromolar concentrations of ATA increase titers of certain HSV-1 strains in various cell lines. In rHSV-1-based rAAV manufacturing protocol, the yield of rAAV is limited by the maximal titer of helper rHSV-1 vectors, and in addition, the presence of residual ATA in rHSV-1 stocks did not negatively influence rAAV yield. Thus, these findings are important both for large-scale rHSV-1 vector production and rAAV vector production. Previously, several groups were investigating possibilities to increase HSV-1 titers by changing the propagation conditions or by using reagents capable of decreasing host defense. 13-16 HSV-1 is known to both induce and partially evade host antiviral responses. 26 Both dexamethasone and valproic acid have been shown to increase HSV-1 yield by either inhibition of cellular defense against viral propagation or induction of interferon (IFN)-responsive antiviral genes. 15, 16 ATA was reported to reduce inducible nitric oxide synthase (iNOS) expression, to inhibit JAK-STAT signaling, or to prevent IFNmediated transcriptional activation. 27,28 ATA has also previously been shown to increase adenovirus type 5 titer in HEK-293 cells. 25 HSV-1 infection activates innate immune system by inducing intracellular signaling pathways that lead to the expression of proteins with proinflammatory and microbicidal activities, including cytokines and IFNs. [29] [30] [31] IFN signaling is one of the most important cellular defense mechanism for viral clearance; 32, 33 however, both Vero and HEK-293 cells have a dysfunctional intracellular antiviral signaling pathways. [34] [35] [36] [37] [38] Vero cells have inability to produce IFN-β, while HEK-293 cells do not produce IFN-α, which can explain their permissive nature to viral production. [34] [35] [36] [37] [38] ATA is known as an activator of Raf/MEK/MAPK pathway, insulinlike growth factor-1 receptor, and protein kinase C signaling. 39, 40 It has been reported that ATA has a survival-promoting effect transducted via activation of the insulin-like growth factor-1 receptor signaling pathway and Akt, MAP, and p42/p44 mitogenactivated protein kinases (Erk-1 and -2). 39, 41 We have observed that ATA treatment delayed HSV-1 plaque formation, cell lysis in V27 cell monolayers, and CPE, which may suggest its antiapoptotic properties. ATA has also been shown to prevent apoptotic cell death in a variety of cell types including breast cancer MDA-231 and MCF-7 cells, macrophage RAW 264.7 cells, and rat pheochromocytoma PC12 cells. 28, [41] [42] [43] Interestingly, ATA in millimolar and higher concentrations is known as an antiviral agent. [20] [21] [22] [23] In Vero, V27, and HEK-293 cells, we Figure 2 The importance of fetal bovine serum (FBS) in ATA-HSV protocol. Following experiments were conducted to determine optimal conditions, and the effect of the presence or absence of 10% FBS on HSV titer in the ATA@step2 protocol. In this example, 20 µmol/l ATA and 10% FBS were either added or omitted in step 2 (ATA@step2). When ATA was added without presence of 10% FBS, the DNase resistant particles per milliliter (DRP/ml) titer of HSV-1 d27-1 vector was reduced, and the plaque-forming units per milliliter (PFU/ml) titer was below detection limit. The HSV-1 d27-1 titers (d27-1) are expressed as mean values + SD of DRP/ml, shown as black bars, and as mean values + SD of PFU/ml, shown as white bars. Results are representative of two independent expe