Simian immunodeficiency virus mac251 infection of astrocytes.

Human immunodeficiency virus type 1 (HIV-1) infection of astrocytes has been demonstrated in the brains of patients with AIDS dementia complex (ADC) and may play an important role in neuropathological pathways of HIV-related encephalopathy. SIVmac-infected monkeys develop an acquired immunodeficiency syndrome (AIDS) with CNS involvement which is quite similar to that seen in human AIDS. We investigated the in vitro infection of primary astrocytes derived from adult macaques with SIVmac251 or an isogenic virus that expresses a non-functional Nef protein (SIVmac251-DeltaNef). In both cases we observed that viral expression was mostly limited to early regulatory genes after a transient phase of late viral gene expression (i.e. env and gag), as reported for HIV-1-infected astrocytes in vivo. Late viral gene expression could be reactivated by TNF-alpha, GM-CSF and IFN-gamma treatment of SIVmac251-infected astrocytes but not by similarly treated SIVmac251-DeltaNef-infected cells. Our findings suggest that Nef is not involved in the restricted expression of SIV in astrocytes, but may be important for astrocytes to function as a viral reservoir in the CNS. In additional experiments, we demonstrated Rev and Nef expression in 17 of 27 primary astrocyte cultures derived from macaques infected by SIVmac251. Nef was located in the cytoplasm of astrocytes infected by SIVmac251 in vivo, but displayed perinuclear localisation after infection in vitro. Attempts to activate late viral gene expression by astrocytes infected in vivo using cytokines or by coculture with human cord blood mononuclear cells were unsuccessful.

[Astrocytes and lentivirus infection in an experimental models of macaque infected with SIVmac251]. / Astrocytes et infection lentivirale dans le modèle expérimental du macaque infecté par le SIVmac251.

The present study demonstrates the susceptibility of astrocytes to infection with SIVmac251. Indeed, primary cultures of astrocytes derived from simian adult brains, can be infected in vitro with the SIVmac251. Results show that SIVmac251 establishes a persistent infection in primary astroglial cultures and that viral replication can be reactivated by TNF-alpha, GM-CSF, IFN-gamma. Viral proteins as Nef, Rev, Vpx and occasionally gp120/160 are evidenced by immunocytochemistry. In vivo SIVmac251 and/or HIV-2 infected astrocytes have been isolated from brains of macaques following ex vivo primary cultures. The whole of these results demonstrated that, in this model, SIV establishes a persistent state of infection of astrocytes, that viral replication can be reactivated by cytokines and moreover suggest strongly an in vivo infection of astrocytes in the brain of these infected macaques.

Obtention and characterization of primary astrocyte and microglial cultures from adult monkey brains.

Simple methods for obtention of primary cultures of isolated astrocytes and microglia from adult simian brain have been developed. Characterization of these two glial cell populations were performed by morphological observations and by immunocytochemistry. The astroglial cultures were obtained by an indirect method. After L-leucine methyl-ester treatment and trypsinizations, more than 99% of cells expressed glial fibrillary acidic protein (GFAP), whereas no macrophages or microglia could be detected. Likely, the 1% remaining cells were immature astrocytes or cells that lost their GFAP expression. Cultured simian astrocytes expressed vimentin, laminin, and fibronectin. We also found a constitutively low expression of major histocompatibility complex (MHC) class II by cultured astrocytes which was significantly enhanced by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or tumor necrosis factor alpha (TNF-alpha) treatments. Microglial cultures were obtained by a direct method of isolation using Percoll gradient separations and compared to simian monocyte-derived macrophages or alveolar macrophages. Microglial cells differed from macrophages by their proliferation upon granulocyte-macrophage colony stimulating factor (GM-CSF) treatment and by their typical morphology when observed by scanning electron microscopy. As macrophages, they expressed in vitro CD68, CD64, CD14, CD11b, MHC class II, and fibronectin. However, contrary to macrophages, simian cultured microglia expressed laminin. This observation suggests that microglia represent a new potential source of this extracellular matrix protein in the brain.