Herpes simplex virus type 2 infection. Part 4
Twelve hours post-transfection, 293T cells were washed and cultured in RPMI supplemented with 10% FBS. Conditioned medium containing recombinant viruses was harvested and filtered (0.45-μm-pore-size filter) twenty-four hours later. Recombinant viral particles were quantified by reverse transcription (RT) assay. Briefly, virions were precipitated from 1 ml of the filtered supernatants by centrifugation at 13,000 rpm for sixty minutes at 4°C. The precipitate was resuspended in 10 μl of a buffer containing 50 mM Tris-HCl pH 7.5, 1 mM dithiothreitol (DTT), 20% glycerol, 250 mM KCl and 0.25% (v/v) Triton X-100, transferred in dry ice and lysed through three cycles of freezing and thawing. The sample was added to a reaction mixture containing 50 mM Tris-HCl pH 7.5, 7.5 mM MgCl2, 0.05% (v/v) Triton X-100, 5 mM DTT, 100 μg/ml polyA, 10 μg/ml oligo-dT and 2 μCi of 3H-dTTP (43 Ci/mmole) in a final volume of 50 μl. The reaction was incubated for one hour at 37°C and then transferred on Whatman filters. Filters were immediately washed three times in SSC 2× (0.3 M NaCl, 0.03 M sodium citrate pH 7.2) for 10 minutes each, twice in absolute ethanol for ten seconds each and then dried. The radioactivity was measured by using a scintillator (Rackbeta 1214 Wallac) and expressed in counts per million (cpm). In parallel, 1.5 × 106 of purified MDMs were cultured in complete RPMI containing GM-CSF in six-well plates for one week, before being infected with HSV-2 at the MOI of 10 PFU/cell, as previously described. Seventy-two hours later, the cells were transduced with 100,000 H3 cpm RT units of the different HIV-1 recombinant particles previously generated and expressing the CAT reporter gene. Seventy-two hours post-transduction, the MDMs were harvested, lysed in 150 μl of 250 mM Tris-HCl pH 7.5 and then assayed for CAT activity, as previously described. The different forms of acetylated chloramphenicol were separated by thin layer chromatography (TLC) and visualized with an autoradiografic exposure of twelve hours (Kodak Biomax films). The quantitative evaluation was obtained by cutting the TLC paper at the level of the corresponding spots, and by performing a quantification of the spots at the scintillator. The percentage of conversion in the acetylated forms was calculated as follows:
% of conversion = (mono- + di-acetylated forms)/(non acetylated + mono- + di-acetylated forms). Calculated with the above formula, the percentage of conversion is linear for values up to 50%. As expected, the X4-tropic virus (HXBc2 env) does not infect efficiently MDMs, while the dual tropic strain (89.6 env) displays the higher efficiency of infection in all the conditions tested. Our data demonstrate that, following HSV-2 infection, the ability of HIV-1 to superinfect target cells is overall enhanced. The effect is statistically significant (p < 0.05) when R5-tropic recombinant viral particles are examined, correlating this observation, at least partially, with the HSV-2 related increase in CCR5 expression levels on MDMs surface. Not surprisingly, the 89.6 dual tropic strain was extremely efficient in macrophage infection and the observed slight increase in HIV-1 entry upon HSV-2 infection was not statistically significant (p > 0.05).