3h dttp

HIV-1 stocks were produced and quantified as previously described [42 (link)]. HIV-1 infection of primary cells followed the procedure as referenced [57 (link)]. Viral reverse transcriptase activity was measured to determine the amounts of virus in culture supernatants. Briefly, 10 μl of culture supernatant was mixed with 40 μl of reaction buffer containing 0.5 unit/ml poly(rA)-oligo(dT) (Midland Certified Reagent Co.) and 0.1 mCi/ml [³H]dTTP (Perkin-Elmer). After a 3-h incubation at 37 °C, reactions were terminated by the addition of 10 % trichloroacetic acid (TCA). The precipitated oligonucleotides were collected by filtering the reaction mixtures through Millipore MultiScreen Glass Fiber FC plates (Millipore). After two washes with 10 % TCA and one wash with ethanol, levels of ³H that were retained on the filters were scored in a liquid scintillation counter (Perkin-Elmer).

The experiments were performed as previously described [15 (link)]. Briefly, 10 μL virions containing culture supernatants were incubated with 40 μL of reaction mixture at 37°C for 3 h. The mixture contains 50 mM Tris-HCl pH 7.9, 5 mM MgCl2, 0.5 mM EGTA, 0.05% Triton X-100, 2% (V/V) ethylene glycol, 150 mM KCL, 5 mM DTT, 0.3 mM GSH (reduced glutathione), 0.5 U/mL poly (rA) oligo (dT), and 0.1 μCi/μL 3H dTTP (Perkin-Elmer). The reaction was stopped by adding 10% (V/V) cold Trichloroacetic Acid (TCA) at 4°C for 30 min, and the mixture was transferred to Millipore MultiScreen Glass Fiber FC plate. After being washed twice with cold 10% (V/V) TCA and cold ethanol, the membranes were inserted into Beta Gamma vials and read in Microbeta counter (Beckman Coulter).

The inhibitory potential of biosurfactant on HCV NS5B (Creative biolabs, USA) polymerase activity was performed using the modified method of Bellecave et al.^{63 (link)}. Briefly, serial concentrations of biosurfactant were added to a mixture reaction of 150 nM NS5B, 86 nM RNA template, 1 mM dithiothreitol (DTT), 5 mM MgCl₂, 40 mM NaCl, 20 mM Tris pH 7.5 and 0.5 mM each of nucleotides including 10 µCi radiolabeled [³²P-UTP, PerkinElmer LAS, UK]. After 2 h, the newly synthesized DNA was then transferred to the filter paper disks and precipitated with trichloroacetic acid before being measured with a scintillation counter. For DNA polymerase of HSV, serial concentrations of biosurfactant and such enzyme were incubated with reaction mixture of 8 mM MgCl₂, 0.5 mM DTT, 50 mM Tris–HCl pH 8, 100 mM ammonium sulfate, 0.5 μg/mL albumin and 100 μM deoxynucleotides with 1 µCi radiolabeled [³H-dTTP, PerkinElmer LAS, UK]. After 1 h incubation at 37 °C, reaction was terminated by acid precipitation then radioactivity of newly synthesized DNA was measured using scintillation counter^{64 (link),65 (link)}.