HIV-1 can persist in peripheral blood monocytes in infected individuals receiving antiretroviral therapies, suggesting that monocytes in vivo constitute a continuing source of viral persistence (9
). In addition to CD4+
resting T cells, viral reservoirs, including the monocyte/macrophage lineage, are one of the challenges in the eradication of HIV-1 with highly active antiretroviral therapy (35
). Although HIV-1 latency is multifactorial, restricted viral transcription enables HIV-1 to remain hidden within nondividing cells (18
). Thus, studying HIV-1 transcriptional regulation in monocytes is important in understanding viral pathogenesis and developing more effective interventions against HIV-1 infection.
In this study, we show that HIV-1 gene expression was impaired in infected primary monocytes, in addition to previously identified restrictions in the early viral life cycle. The lack of Tat transactivation of the LTR promoter correlated with the impaired HIV-1 gene expression in monocytes. Heterokaryon experiments suggest that monocytes lack host factors required for Tat transactivation of the LTR promoter. However, transient expression of CycT1 in undifferentiated monocytes could not rescue Tat transactivation, suggesting that CycT1 is not the only limiting factor of HIV-1 infection in undifferentiated monocytes. Macrophage differentiation from monocytes was correlated with enhanced phosphorylation of CDK9 and significantly increased HIV-1 infection.
CDK9 autophosphorylation is required for high-affinity binding of Tat-P-TEFb to transactivation-responsive RNA, suggesting that the state of P-TEFb phosphorylation regulates Tat transactivation in vivo (11
). Moreover, the phosphorylation of CDK9 at Thr186 appears to be crucial for the P-TEFb activity and HIV-1 transcription (1
). A recent study indicated that protein phosphatase 1A, magnesium dependent (PPM1A) regulates phosphorylation of Thr186 in the CDK9 T-loop (45
). Thus, it is conceivable that limited phosphorylation of CDK9 at Thr186 in undifferentiated monocytes may also contribute to impaired HIV-1 gene transcription.
Liou et al. have reported that CycT1 protein expression is transiently induced but shut off around 7 days during the macrophage differentiation from monocytes (21
). This phenotype was observed in monocytes isolated from 45% (18/40) of healthy blood donors (21
). The shutoff of CycT1 expression in late-differentiated macrophages involves proteasome-mediated proteolysis (20
). Using the same protocol to differentiate macrophages as that described by Liou et al. (20
), we did not observe the decrease in CycT1 during macrophage differentiation from monocytes isolated from five different donors (Fig. and data not shown). These data suggest donor-dependent CycT1 induction during macrophage differentiation.
Transient expression of CycT1 protein in undifferentiated monocytes could not rescue Tat-mediated transactivation of the LTR promoter, suggesting that the lack of CycT1 protein in undifferentiated monocytes is unlikely to be the sole limiting factor of Tat transactivation. However, it is currently unknown whether CycT1 expressed in transfected monocytes can properly localize in the nucleus and bind to CDK9 to render Tat-mediated LTR transactivation. Moreover, the half-life of transiently expressed CycT1 might be different from that of the endogenous CycT1 in macrophages. Further studies are required to address these questions. Interestingly, a recent study identified miR-198 as a microRNA that restricts HIV-1 gene expression in primary monocytes, and its mechanism likely involves repression of CycT1 expression (39
In addition to P-TEFb, other transcription factors may be involved in postentry restriction of HIV-1 infection in undifferentiated monocytes. Lewin et al. reported that constitutive expression of NF-κB in primary monocytes is significantly modulated during the macrophage differentiation (19
). Undifferentiated monocytes constitutively express high levels of transcriptionally inactive homodimers, which decrease with time in culture in favor of the transcriptionally active heterodimers (19
). These data suggest that the change in NF-κB components with monocyte differentiation can also contribute to the transcriptional restriction of HIV-1 infection in monocytes. Moreover, a proteolysis-resistant inhibitor of NF-κB efficiently inhibits HIV-1 replication in primary monocytes, indicating a major requirement of NF-κB activation for the optimal replication of HIV-1 in monocytes (30
In summary, our data indicate that impaired HIV-1 gene transcription contributes to the postentry restriction of HIV-1 infection in undifferentiated monocytes, at least in part. When monocytes differentiate into macrophages, they become increasingly susceptible to HIV-1 infection and permissive to viral gene expression and production of infectious viruses. A better understanding of HIV-1 infection and regulation in primary monocytes will provide new insights into HIV-1 molecular pathogenesis.