IFN-α treatment exerted a strong in vitro anti-HIV-1 effect in HIV-1/HTLV-1 co-infected MT-4 cells, demonstrated by the dose-dependent inhibition of the HIV-1-induced CPE and p24 secreted levels upon HIV-1 infection. In contrast, IFN-α treatment did not affect cell viability of HTLV-1-infected cell lines C8166, MT-2 nor MT-4. Furthermore, we demonstrate for the first time that IFN-α treatment did not affect HTLV-1 viral mRNA levels in MT-2 and MT-4 cells, demonstrated by nCounter analysis at various time points. Although these observations suggest the lack of biological activity of IFN-α against HTLV-1, we were able to confirm the previously described post-transcriptional inhibition of HTLV-1 p19 secretion by IFN-α , both in cell lines as well as in ATL patient samples. However, in comparison to the strong dose-dependent inhibition of HIV-1 p24 levels, IFN-α only modestly reduced p19 levels in cell-free supernatant of MT-2 cells and ATL PBMCs. Despite the absence of pronounced antiviral, pro-apoptotic and antiproliferative activity of IFN-α, microarray and nCounter analysis revealed significant transcriptional activation of ISGs and intact IFN-α signaling in MT-2 and MT-4 cells. MT-4 cells appeared to be more responsive to IFN-α treatment in comparison to MT-2 cells, demonstrated by significant up- or down-regulation of 284 vs. 77 genes, respectively, and the early activation of STAT1 and STAT2 genes. Nevertheless, approximately 70% of the IFN-α-up-regulated genes in MT-2 were identical of those up-regulated in MT-4 cells, indicating the similarity and reliability of both cell lines as HTLV-1 in vitro models.
Because of the intact transcriptional activation of ISGs, the absence of distinct antiviral activity of IFN-α against HTLV-1 was not due to a general defect in IFN-α signaling pathways in MT-2 or MT-4 cells. In agreement with our findings, over-expression of a subset of ISGs in chronic HTLV-1 infection has recently been shown to fail to constitute an efficient antiviral host response, but might instead contribute to HAM/TSP pathogenesis . We speculate that IFN-α fails to decrease HTLV-1 mRNA levels due to highly virus-specific retroviral restriction factors, as IFN-α exerted strong anti-HIV-1 yet weak anti-HTLV-1 effects in HIV-1/HTLV-1 co-infected MT-4 cells. Since all known HTLV-1 mRNAs, including antisense HBZ, remain unchanged upon IFN treatment, defective RNAseL activity, downstream of OAS gene activation (reviewed in ), might be hypothesized as a possible HTLV-1 escape mechanism,. Although several OAS family members are IFN-inducible in both HTLV-1 infection (this study, Table I and results not shown) and HIV-1 infection , little is known of downstream RNAse L activation, which occurs at the protein level . In addition, blunting of IFN-α biological activity has been mainly addressed in HTLV-1 mono-infection, but not in HIV-1/HTLV-1 coinfection. HTLV-1 expression has been reported to up-regulate SOCS1 expression, inducing ubiquitination and proteasomal degradation of IRF3, leading to the inhibition of type I interferon production and thus inhibiting activation of IFN-α signaling pathways . Still, up-regulation of SOCS1 mRNA levels was shown in CD4+ T cells isolated from HAM/TSP patients and asymptomatic carriers, but not from ATLL patients . Furthermore, HTLV-1 Tax has been shown to induce SOCS1 expression, leading to the inhibition of RIG-I-dependent antiviral signaling, but not the JAK/STAT signaling pathways . Inhibition of cytoplasmatic pattern recognition receptors such as RIG-I, has been associated with IRF3 inhibition and thus subsequent inhibition of type I interferon production. Consequently, HTLV-1-induced SOCS1 expression could counteract activation of IFN-α signaling via reduced type I interferon production. However, whereas exogenous IFN-α has been shown to increase SOCS1 expression in HeLa-cells, HTLV-1 expressed from an infectious molecular clone reduced IFN-α-induced up-regulation of SOCS1 mRNA levels . Taken together, the precise correlation between HTLV-1 and SOCS1 expression and its effect on IFN-α signaling, remains unclear. Our microarray analysis revealed significant IFN-α-induced up-regulation of suppressors of cytokine signaling SOCS1, SOCS2 and SOCS3 levels in MT-4 cells, but not in MT-2 cells, although IPA pathway analysis revealed strikingly similar IFN signaling in both cell lines. Therefore, an IFN-α-induced increased SOCS1 level is not a generalized finding in HTLV-1 infection in vitro and is not by itself sufficient to define the blunted biological activity of IFN-α. HTLV-1 expression has also been reported to up-regulate IRF4 levels in HTLV-1-transformed cell lines and PBMCs of ATLL patients [59, 60]. IRF4 was shown to negatively regulate type I interferon production and appeared to be associated with AZT + IFN-α antiviral resistance in ATLL patients [59–61]. Our microarray results showed no effect of IFN-α on IRF4 expression in MT-2 or MT-4 cells, although more sensitive nCounter analysis revealed slight IFN-α-induced up-regulation of IFR4 levels, which was significant in MT-4 cells, but not in MT-2 cells (p = 0.049 and p = 0.65, respectively, data not shown). Furthermore, treatment with exogenous IFN-α was able to activate IFN-α signalling to a similar extent in both cell lines (Figure 6A-B and Tables 1
2), although SOCS1 and IRF4 were significantly up-regulated in MT-4 cells only. Nevertheless, our study was limited to the broad antiviral, pro-apoptotic and antiproliferative activities of IFN-α, as well as IFN-α signaling in HTLV-1-infected cells, whereas the precise contribution of cellular factors such as SOCS1 or IRF4 has been investigated in detail in previous studies [50, 58–61]. However, it should be stated that some of these studies [48, 50] investigate de novo infection with HTLV-1 molecular clones, in contrast to stable HTLV-1 infection (this study). The latter might be closer to the in vivo situation, considering the latency of the virus in vivo and its slow molecular evolution, pointing at limited de novo infection . In addition, there is increasing evidence that activation of multiple IFN-α signaling pathways is required to generate the antiviral, pro-apoptotic and immunomodulatory effects of IFN-α . Antiviral and antiproliferative activities of IFN-α have been reported to depend on both STAT- and p38-signaling pathways . Although we observed transcriptional activation of STAT1, STAT2 and downstream ISGs in both MT-2 and MT-4 cells, other important IFN-α signaling pathways, such as p38-signaling, could be affected by HTLV-1 replication, possibly explaining the absence of explicit antiviral or antiproliferative activity against HTLV-1 in vitro.
Altogether, one can assume that the in vitro antiretroviral activity and, possibly, the in vivo therapeutic success of IFN-α for both HIV-1 and HTLV-1 is determined by virus-specific factors including viral life cycle-related factors (replication, virion production) and the balance between factors blunting or stimulating IFN-α signaling pathways. For example, inhibition of HIV-1 assembly and release of virions by IFN-α has been described through the induction of ISG15, an ubiquitin-like protein . Through ISG15 up-regulation, IFN-α could affect HTLV-1 assembly, which could explain the post-transcriptional inhibition of p19 secretion. On the other hand, a recent systematic screen for antiviral activity of 389 ISGs, revealed the ability of ADAR to enhance HIV-1 replication . Unfortunately, no data are available on IFN signaling and/or specific ISGs levels in HIV-1/HTLV-1 co-infection, which is of significant clinical importance since several cohort studies have revealed accelerated clinical progression to AIDS and/or increased mortality in HIV-1/HTLV-1 co-infected versus HIV-1 mono-infected individuals [63–65].