Cell experiment:

Human breast cancer cell lines MCF-7 and MDA-MB-435, glioblastoma U87MG, and embryonic kidney cells 293T are cultured at 37℃ in a humidified atmosphere containing 5% CO2 in Iscove’s modified Dulbecco medium or Leibovitz’s L-medium and 5% fetal bovine serum (FBS). The assays are performed with slight modifications. In brief, cells are seeded into 24-well plates (5×104 cells/well) and infected 48 h later with 103 particles per cell of unmodified virus (Adtk), PEGylated virus (PEG-Adtk), or RGD-PEG-modified virus (RGD-PEG-Adtk) in triplicates in culture medium with 2% FBS and incubated for 4 h at 37℃. The incubation medium is then replaced by normal medium and cells are further incubated for 48 h. Cells are harvested and lysed with 500 μL of TK lysis buffer that contained 0.5% Nonidet P-40 (NP-40), 20 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES) (pH 7.6), 2 mM Mg(OAc)2, 1 mM dithiothreitol, and 50 μM thymidine. The supernatant is collected after centrifugation. The samples are kept at -80℃ until use. The modified and unmodified adenovirus protein concentrations are determined by the Micro BCA assay. One microgram of cell extract is incubated with HSV1-tk substrate 8-3H-Penciclovir (8-3 H-PCV). The phosphorylated tracer is separated from unphosphorylated 8-3 H-PCV with DE-81 filters. TK activity is expressed as the percentage of conversion of substrate per minute per microgram protein[2].

Penciclovir is an inhibitor of HSV-1 DNA synthesis [1].

Penciclovir is a guanine derivative and is used as a drug for the treatment against herpesviruses. Penciclovir has a similar spectrum of activity with acyclovir. In the plaque reduction assay, penciclovir shows potent inhibitory activities against HSV-1, HSV-2 and VZV with IC50 values of 0.4μg/ml, 1.5μg/ml and 3.1μg/ml, respectively. It also shows minimal activity against CMV. In MRC-5 cells, penciclovir inhibits the replication of HSV-1 with IC99 value of 0.4μg/ml. Moreover, penciclovir exerts persistent antiviral activity against HSV-1and HSV-2 in Vero cells. Besides that, penciclovir is found to be phosphorylated to the triphosphate in HSV-1-infected MRC-5 cells. The penciclovir triphosphate is the metabolite that actually inhibits herpesvirus replication. Penciclovir inhibits HSV-1 DNA synthesis with IC50 value of 0.16μM without effect on cellular DNA synthesis [1, 2].

References:
[1] Boyd M R, Bacon T H, Sutton D, et al. Antiherpesvirus activity of 9-(4-hydroxy-3-hydroxy-methylbut-1-yl) guanine (BRL 39123) in cell culture. Antimicrobial agents and chemotherapy, 1987, 31(8): 1238-1242.
[2] Hodge R A, Perkins R M. Mode of action of 9-(4-hydroxy-3-hydroxymethylbut-1-yl) guanine (BRL 39123) against herpes simplex virus in MRC-5 cells. Antimicrobial agents and chemotherapy, 1989, 33(2): 223-229.