Antiviral Effects of Artesunate on Polyomavirus BK Replication in Primary Human Kidney Cells

Abstract

dPolyomavirus BK (BKV) causes polyomavirus-associated nephropathy (PyVAN) and hemorrhagic cystitis (PyVHC) in renal and bone marrow transplant patients, respectively. Antiviral drugs with targeted activity against BKV are lacking. Since the antimalarial drug artesunate was recently demonstrated to have antiviral activity, the possible effects of artesunate on BKV replication in human primary renal proximal tubular epithelial cells (RPTECs), the host cells in PyVAN, were explored. At 2 h postinfection (hpi), RPTECs were treated with artesunate at concentrations ranging from 0.3 to 80 M. After one viral replication cycle (approximately 72 hpi), the loads of extracellular BKV DNA, reflecting viral progeny production, were reduced in a concentrationdependent manner. Artesunate at 10 M reduced the extracellular BKV load by 65%; early large T antigen mRNA and protein expression by 30% and 75%, respectively; DNA replication by 73%; and late VP1 mRNA and protein expression by 47% and 64%, respectively. Importantly, the proliferation of RPTECs was also inhibited in a concentration-dependent manner. At 72 hpi, artesunate at 10 M reduced cellular DNA replication by 68% and total metabolic activity by 47%. Cell impedance and lactate dehydrogenase measurements indicated a cytostatic but not a cytotoxic mechanism. Flow cytometry and 5-ethynyl-2=-deoxyuridine incorporation revealed a decreased number of cells in S phase and suggested cell cycle arrest in G 0 or G 2 phase. Both the antiproliferative and antiviral effects of artesunate at 10 M were reversible. Thus, artesunate inhibits BKV replication in RPTECs in a concentration-dependent manner by inhibiting BKV gene expression and genome replication. The antiviral mechanism appears to be closely connected to cytostatic effects on the host cell, underscoring the dependence of BKV on host cell proliferative functions.

Antiviral Effects of Artesunate on JC Polyomavirus Replication in COS-7 Cells

“…Interestingly, the effect on total metabolic activity was not reversed up to 96 hpt. The explanation for this apparent discrepancy is probably linked to a cell cycle arrest in G 0 or G 2 , as previously demonstrated in other cells (39,(54)(55)(56). When the effect of artesunate waned, a high proportion of the synchronized cells moved into the S phase and incorporated BrdU in their DNA, whereas the metabolic activity was still reduced due to reduced cell numbers.…”

“…Artesunate affects a very early stage of herpesvirus infection, but the mechanism seems to be the inhibition of cellular pathways (50). In BKPyV-infected RPTECs, artesunate inhibited early gene expression but also viral DNA replication, late gene expression, and the release of progeny virus (39). Since COS-7 cells constitutively express SV40 LTag, which can initiate viral DNA replication from the JCPyV origin (53), and no specific antibody for JCPyV LTag was available, the effects on early protein expression and of the later connected steps in the replication cycle were not investigated.…”

“…In short, 6,000 cells per 0.32 cm 2 well were seeded, and the next day, the cells were either mock infected or JCPyV infected by incubation with 80 l of the JCPyV infectious supernatant for 2 h. Next, the surplus infectious JCPyV was removed, and the cells were treated with increasing concentrations of artesunate. At 24,48,72, and 96 h posttreatment (hpt), cellular DNA replication was quantified by a colorimetric measurement of 20 h bromodeoxyuridine (BrdU) incorporated into DNA using the cell proliferation enzyme-linked immunosorbent assay (ELISA) and BrdU assay (Roche), and the total metabolic activity was monitored by the colorimetric measurement of 3 h of reduction of resazurin dye by mitochondrial, microsomal, and cytosolic enzymes using the TOX-8 assay (Sigma), as described earlier (39,46). As an alternative measure of the total cellular metabolic activity, the ATP content of both the mock-infected and JCPyV-infected cells was measured at 96 hpt by the CellTiter-Glo luminescent cell viability assay (Promega), according to the manufacturer's instructions.…”

“…Since very little toxicity was observed in the artesunate-treated cells, a CC 50 (Ϯ95% CI) of 48.2 Ϯ 12.0 M (P Ͻ 0.001) was calculated based on cellular DNA replication of mock-infected cells at 96 hpt (Fig. 4A), as previously described (39,45,46) (Fig. 6).…”

“…Apparently, it has been successfully used to treat four transplant patients with recurrent multidrug-resistant cytomegalovirus (CMV) infection (34,35) and one child with human herpesvirus 6 infection (36), but it did not give satisfactory results in other patients (35,37,38). Recently, we reported that artesunate has antiviral activity against BKPyV in human primary renal proximal tubular epithelial cells (RPTECs) and that the antiviral effect is connected to transient cytostatic effects without cytotoxicity (39). Encouraged by this and the good safety profile of artesunate, with a low incidence of side effects found in numerous studies (reviewed in reference 32), we investigated its effects on JCPyV replication.…”