Herpes simplex virus type 2 infection. Part 2
Thus, in order to analyze whether the low susceptibility to HSV-2 infection displayed by U937 cells could be related to their differentiation level, the cells were induced to differentiate by treatment with TPA (50 ng/ml). After twelve hours of incubation, U937 cells were washed twice with PBS and cultured for additional twenty-four hours in TPA-free medium, in order to avoid possible effects of residual TPA. The percentage of cells positive for CD14 surface expression, a marker of macrophage differentiation, was then determined by Fluorescence-Activated Cell Sorting (FACS). Briefly, 1 × 106 cells were harvested and directly incubated for one hour in cold PBS containing 1:100 (v/v) of an anti-human CD14 primary antibody (Li StarFISH). A fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin G antibody (Santa Cruz) was employed as secondary antibody and the fluorescence was evaluated by FACS analysis (FACScalibur, Beckton Dickinson). As reported in Figure 1B, after TPA treatment the percentage of CD14-positive U937 cells is significantly increased. Differentiated U937 cells were infected with HSV-2 (MOI of 1 PFU/cell). Infectious virus yields, which peaked approximately three days post-infection, appear to be significantly higher than those obtained from undifferentiated U937 cells. Thus, our data suggest that HSV-2 replication efficiency is dependent on the differentiated phenotype of U937 cells along the monocytic pathway. Interestingly, while untreated U937 cells did not display a significant HSV-2 induced cytopathic effect (CPE), TPA-differentiated U937 cells were fully susceptible to viral CPE (data not shown).
Next, infection of primary monocyte-derived macrophages (MDMs) was performed. MDMs were obtained from at least three pooled buffy coats of HIV-1 seronegative healthy blood donors, by Ficoll-Hystopaque gradient, followed by plastic adherence of PBMCs for sixteen hours in complete RPMI. Non-adherent cells were removed, and adherent cells were extensively washed with PBS and grown in complete RPMI supplemented with Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF, 500 U/ml). The cells were cultured for one week, before evaluating the preparation purity by measuring the percentage of CD14-positive cells through FACS analysis, as described above. The cut-off employed to accept the purity of MDM preparation was a CD14-positive percentage higher than 90%. Purified MDMs (2 × 106) were infected with HSV-2 strain G at two different MOIs, 10 PFU/cell and 1 PFU/cell, following the same experimental procedure described for the U937 cell line. After viral adsorption, the infected MDMs were maintained in complete RMPI medium supplemented with GM-CSF. Our data show that MDMs support HSV-2 replication. As expected, at the MOI of 10 PFU/cell the infection appears to be more efficient, achieving a peak in the viral titre seventy-two hours post-infection. No clear viral induced CPE could be observed at any time post-infection (data not shown). Next, we analyzed the effect of HSV-2 infection on HIV-1 receptor and coreceptors expression on macrophage cell surface. Indeed, HIV-1 entry into target cells involves the interaction of the viral envelope glycoproteins with the host cell receptors, CD4, and one of two coreceptors CCR5 or CXCR4. The chemokine receptors CXCR4 and CCR5 represent indeed the coreceptors for T-cell-tropic (X4-tropic) and macrophage-tropic HIV-1 (R5-tropic) strains, respectively. Dual/mixed-tropic populations are also present in HIV infected patients and evidence has been collected in antiretroviral-naive patients suggesting that the majority of viruses within these dual/mixed-tropic populations use CCR5. Moreover, natural history studies of HIV-1 infection have shown that most patients harbour R5-tropic virus populations soon after infection and through the asymptomatic phase.