Type I interferons, particularly interferon-alpha (IFN-α), play a vital role in the host's anti-viral defensesby interfering with viral replication. However, the virus rapidly evolves to exploit the IFN-α response for its replication, spread, and pathogenic function. In this study, we attempted to determine IFN-α susceptibility and productivity of infectious transmitted/founder (TF) (n = 8) and non-transmitted (NT) viruses (n = 8) derived from HIV-1 infected infants. Independent experiments were carried out to determine IFN-α resistance, replication fitness, and viral productivity in CD4 + T cells over a short period. In vitro studies showed that TF viruses were resistant to IFN-α during the very near moment of transmission, but in the subsequent time points, they became susceptible to IFN-α. We did not observe much difference in replicative fitness of the TF viruses in cultures treated with and without IFN-α, but the difference was significant in the case of NT viruses obtained from the same individual. Despite increased susceptibility to IFN-α, NT viruses produced more viral particles than TF viruses. Similar results were also obtained in cultures treated with maraviroc (MVC). The study identified unique characteristics of TF viruses thus prompting further investigation into virus-host interaction occurring during the early stages of HIV infection.
Determination of saturation point for maraviroc. TZM-bl and GHOST (3) CCR5+ (Hi-5) celllines were employed to assess the sensitivity of chimeric viruses to the CCR5 antagonist, maraviroc. Briefly, 10 4 TZM-bl and Hi-5 cells were plated in each well of a 96-well plate 24 hours before infection. Cells were incubated with different dilutions of MVC (10-fold dilutions starting from 10 μM up to 10 −3 nM) overnight at 37 °C before inoculation with chimeric viruses. Virus infected cells cultured in the absence of MVC served as negative control. At 48 hours post-infection, cells were lysed with 1X passive lysis buffer (Bright-Glo luciferase assay system) and luciferase expression was measured using an Infinite M200Pro plate reader (Tecan, Switzerland) as the number of relative light units (RLU). All conditions were tested in triplicate in each of at least two independent experiments.Single round virus replication kinetics assay. For single cycle infectivity assay, virus stocks normalized by TCID 50 and further diluted to 0.01 MOI were used to infect TZM-bl cells (1 × 10 4 cells/well) in triplicates in the presence of DEAE dextran (10 μg/mL). Cells were pre-incubated for 2 hours with different concentrations (0, 250, 500 and 750 IU/mL) of IFN-α at 37 °C before infection with chimeric viruses. 48 hours after infection, luciferase activity was measured using the Bright-Glo luciferase assay system (Promega).
Multiple round virus replication kinetics assay. CD4 + T cells were isolated from buffy coats by deplet-ing CD8 + T cells and stimulated with PHA (20 μg/mL) in the presence of 20 U/mL IL-2 in RPMI medium containing 10% FBS at a concentration of 1 × 10 6 cells per milliliter....