Three human brain‐derived cell lines (including two of astrocytic origin) were exposed in vitro to the human immunodeficiency virus (HIV), the etiologic agent of immunodeficiency in AIDS. In all three lines, HIV transcripts were detected by in situ hybridisation in 20–30% of cells 48 h afterinfection. Synthesis of virus gag gene products p24 and p55 was demonstrated by immunoblotting. No cytopathic effects typical of HIV‐infected human T lymphocytes were observed. Our data indicate that HIV is neurotropic, and support the hypothesis that this virus may infect astrocytes in the brain.

The nature of the interaction between human immunodeficiency virus (HIV) and human cells of astrocytic origin was studied in vitro with cultured glial cells and intact HIV or infectious molecular clones of the virus. Infection of glial cells with intact HIV was characterized by low-level expression of viral transcripts as detected by Northern blotting and in situ hybridization (<10 copies of HIV RNA per cell), transient virus replication, absence of viral antigens detectable by immunofluorescence, and complete lack of cytopathic effects. However, the HIV-infected glial cells persistently expressed HIV tatlll gene activity as detected by a chloramphenicol acetyltransferase assay, and HIV transcripts could be detected by in situ hybridization in 20 to 30% of cells up to 4 months after infection, suggesting that the lack of cytopathicity in HIV-exposed cells was not due to transient viral infection. To evaluate whether increased expression and replication of HIV in glial cells would have any effect on cell growth and viability, we established HIV-positive glial cell lines by cotransfection of cells * Corresponding author. MATERIALS AND METHODS Cells and viruses. Human glial cell lines U-251MG (H80) (4) and U-373MG (38) were obtained from D. D. Bigner and the American Type Culture Collection (Rockville, Md.), 3774 on September 28, 2020 by guest http://jvi.asm.org/ Downloaded from INFECTION OF HUMAN GLIAL CELLS BY HIV AND HIV CLONES 3775 respectively, and were cultivated in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. The CD4-positive T-cell line CEM (16) was received from L. Montagnier, and the HIV lysis-resistant subclone of CEM, CR10, was established in our laboratory (6). The suspension cultures were maintained in RPMI 1640 medium supplemented with 5% fetal bovine serum. The NiT isolate of HIV (32) was propagated in CEM or CR10 cells. Glial cells were infected with cell-free, virus-containing supernatants as described previously (11). Plasmids. Plasmid pSV2neo (37) was obtained from Bethesda Research Laboratories (Gaithersburg, Md.). The murine glial fibrillary acidic protein (GFAP) cDNA (28) was donated by M. Shelanski and subcloned into the plasmid pGEM4 (Promega Biotec, Inc., Madison, Wis.) to allow the synthesis of both sense-and antisense-orientation RNAs. The HIV TAT region probe, pTBtat, was prepared similarly with the 0.7-kilobase (kb) EcoRI-KpnI fragment of HIV NiT proviral DNA (32). The infectious HIV DNA clone pNlT-E2 was prepared as described elsewhere (K. Sakai, S. Dewhurst, C. Meier, and D. J. Volsky, submitted for publication). The nearly full-length HIV DNA probe used for Southern blot analysis was prepared as an 8.9-kb Sacl-Sacl fragment from the HIV N1G-G clone (32). Plasmid X-TAT was donated by S. Rhode, and plasmid RSV CAT (20) was obtained from the American Type Culture Collection. Chemicals and radiochemicals. Photobiotin was purchased from Bresa Ltd. (Adelaide, South Australia). Fluorochromeconjugated or native streptavidin and RNases A and Ti were purchased from ...

Quick-blot, a method for selectively immobilizing either mRNA or DNA on nitrocellulose, is described in detail. Essential elements of the procedure for immobilizing DNA include tissue lysis, proteinase K treatment, solubilization of nucleic acids in hot 12.2 molal NaI, passage through a nitrocellulose filter, and acetylation of residual protein with acetic anhydride. Advantages include speed, quantitative recovery, low background, and elimination of the usual baking step. Essential elements of the procedure for selectively immobilizing mRNA include dissolving cells in Brij-35 and desoxycholate, proteinase K treatment, solubilizing nucleic acids in room temperature 12.2 molal NaI, filtration through nitrocellulose, and acetylation of residual protein. Advantages include selective immobilization of mRNA but not tRNA, rRNA, or DNA, and the maintenance of biological activity of the immobilized mRNA. Control experiments to optimize the procedures and examples of their application are shown.

A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100%o match with B. polymyxa 16S rRNA but only a 60%v match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a * Corresponding author.