Background:  Astrocyte dysregulation correlates with the severity and the rate of HIV-associated dementia, highlighting a pivotal role for astrocytes in HIV neuropathogenesis. Yet, astrocytes limit HIV replication, indicating that they posses an intrinsic molecular mechanism to restrict HIV replication. We previously established that this restriction can be modulated by priming astrocytes with interferon-gamma (IFN-g). We evaluated the mechanism of restrictive HIV replication in astrocytes and how IFN-γ priming regulates this restriction.  

Methods:  To evaluate the role of Wnt signaling in regulating HIV replication in astrocytes, the association between TCF-4, a downstream effector of Wnt pathway, and Tar was evaluated by the chromatin immunoprecipitation (ChIP) using TCF-4 antibody for immunoprecipitation and amplifying for HIV TAR by real-time polymerase chain reaction (PCR) from HIV Bal untreated and IFN-γ-treated astrocytes. To evaluate the role of TCF-4 in restricting HIV replication, human primary fetal astrocytes (HFA) and the astrocytoma cell line U87MG were transfected with TCF-4 dominant negative mutant or GFP construct. The cells were then infected with HIV and HIV expression monitored by p24 ELISA. To evaluate the impact of IFN-γ on Wnt signaling, U87MG were left untreated or treated with IFN-γ for 24 hours then transfected with TCF-4 luciferase reporter (TOPflash) or GFP construct. Transfected cells were then cultured with or without IFN-γ and luciferase activity measured at 24 hours using standard protocol. Data were analyzed by unpaired Student’s t-test or Wilcoxon rank-sum test, as appropriate.

Results:  We demonstrate that active Wnt signaling is associated with restricted HIV replication in astrocytes. Specifically, we show that the downstream effector of Wnt signaling, TCF-4, is part of a transcriptional complex that is immunoprecipitated with HIV TAR in untreated astrocytes, but not in IFN-γ-treated cells. Blocking TCF-4 activity with a dominant negative mutant overcame this restriction by enhancing HIV replication by 3-fold in HFA and U87MG. We also directly demonstrate that Wnt signaling is active in astrocytes and is markedly reduced by IFN-γ treatment.

Conclusions:  These data implicate active Wnt signaling in repressing HIV replication and the ability of IFN-γ to augment this restriction by inhibiting Wnt. Harnessing this pathway to limit HIV replication may emerge as a powerful tool to regulate HIV replication within and outside of the brain.