Background:  Promyelocytic leukemia protein (PML) is responsible for subnuclear organization of transcription-related proteins. It is an interferon-α- and -β-stimulated protein and contributes to their antiviral effects. In several viral infections, PML represses viral transcription and replication. The objective of this study was to determine the role of PML in the non-permissive nature of HIV infection in astrocytes.

Methods:  PML was quantified in primary human astrocytes, and an astrocytic cell line, SVGA, by immunostaining and Western blots and compared with levels in lymphocytes and monocytes. SVGA cells were infected with HIV_NL4-3-YFP following treatment with 4 µM arsenic trioxide (As₂O₃) to degrade PML or short interfering RNA (siRNA) to block translation. Fluorescent cells were quantitated to determine infection rates. HIV p24 levels were analyzed by ELISA. As₂O₃ effects were assessed either pre- or post-infection. The effect of PML on LTR transactivation by Tat was determined by transfecting SVGA LTR-GFP cells with tat₁₀₁ in the presence and absence of As₂O₃. Co-immunoprecipitation determined direct interactions of Tat and PML. All experiments were done in triplicate, expressed as mean ± SEM, and analyzed by Student’s t-test.

Results:  PML protein was maximally expressed in astrocytes as compared with monocytes and lymphocytes. The number of HIV-infected SVGA cells was significantly enhanced by pre-treatment, but not post-treatment, with As₂O₃ or transfection with PML siRNA, which was confirmed by p24 ELISA. The infected astrocytes developed into foci that expanded at a similar rate to that of uninfected SVGA cells. No new astrocytes became infected in the cultures after the initial infection once PML levels had recovered, although the astrocytes produced infectious HIV as determined by infection of lymphocytes by culture supernatants. Degradation of PML led to an increase in the level of LTR activity in astrocytes. Immunostaining for PML in SVGA Tat-GFP cells demonstrated an association of Tat with PML bodies. Co-immunoprecipitation revealed a direct interaction of Tat and PML.

Conclusions:  Endogenously high levels of PML in astrocytes as compared with permissive cell types contribute to the non-permissive nature of HIV infection in astrocytes. This is accomplished at both the pre-integration level and by repressing LTR activity, likely by interfering with Tat transactivation.

Keywords: Astrocyte; HIV; Latency