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. 2001 Sep;75(17):7925-33.
doi: 10.1128/jvi.75.17.7925-7933.2001.

Highly productive infection with pseudotyped human immunodeficiency virus type 1 (HIV-1) indicates no intracellular restrictions to HIV-1 replication in primary human astrocytes

M Canki  1 , J N Thai, W Chao, A Ghorpade, M J Potash, D J Volsky
Affiliations

Highly productive infection with pseudotyped human immunodeficiency virus type 1 (HIV-1) indicates no intracellular restrictions to HIV-1 replication in primary human astrocytes

M Canki et al. J Virol. 2001 Sep.

Abstract

Human astrocytes can be infected with human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo, but, in contrast to T lymphocytes and macrophages, virus expression is inefficient. To investigate the HIV-1 life cycle in human fetal astrocytes, we infected cells with HIV-1 pseudotyped with envelope glycoproteins of either amphotropic murine leukemia virus or vesicular stomatitis virus. Infection by both pseudotypes was productive and long lasting and reached a peak of 68% infected cells and 1.7 microg of viral p24 per ml of culture supernatant 7 days after virus inoculation and then continued with gradually declining levels of virus expression through 7 weeks of follow-up. This contrasted with less than 0.1% HIV-1 antigen-positive cells and 400 pg of extracellular p24 per ml at the peak of astrocyte infection with native HIV-1. Cell viability and growth kinetics were similar in infected and control cells. Northern blot analysis revealed the presence of major HIV-1 RNA species of 9, 4, and 2 kb in astrocytes exposed to pseudotyped (but not wild-type) HIV-1 at 2, 14, and 28 days after infection. Consistent with productive infection, the 9- and 4-kb viral transcripts in astrocytes infected by pseudotyped HIV-1 were as abundant as the 2-kb mRNA during 4 weeks of follow-up, and both structural and regulatory viral proteins were detected in infected cells by immunoblotting or cell staining. The progeny virus released by these cells was infectious. These results indicate that the major barrier to HIV-1 infection of primary astrocytes is at virus entry and that astrocytes have no intrinsic intracellular restriction to efficient HIV-1 replication.

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Figures

FIG. 1
FIG. 1
GFAP expression by human fetal astrocytes and IF detection of HIV-1 antigens in infected astrocytes. (A and B) Uninfected human fetal astrocytes (16 weeks of gestational age, third passage) were grown on glass chamber slides and stained with anti-GFAP antibody (A) or irrelevant control antibody (B). (C to E) VSV/NL4-3-infected astrocytes from the experiment described in the legend to Fig. 2 were harvested, fixed, and stained for HIV-1 antigens by IF 7 days (C) and 28 days (D) after infection; cells harvested 7 days after infection, stained with an irrelevant serum, are also shown (E). Photographs were taken using an Olympus BH-2 fluorescence microscope. Magnification, ×100.
FIG. 2
FIG. 2
Expression of HIV-1 antigens, viability, and growth kinetics in fetal astrocytes infected with native and pseudotyped HIV-1. (A) Astrocyte cultures were infected with VSV/NL4-3, MLV/NL4-3, or NL4-3 as indicated and monitored for HIV-1-specific antigens expression by IF staining with AIDS sera and fluorescein isothiocyarate-conjugated second antibody (left panel) or by production of p24 in cell supernatants (right panel). The proportion of IF-positive cells was determined by counting at least 200 cells each in three different fields under ×20 magnification, using an Olympus BH-2 fluorescence microscope. (B) At the indicated times after infection, total cell numbers and total viable-cell numbers per system (×1,000) were determined as described in Materials and Methods.
FIG. 2
FIG. 2
Expression of HIV-1 antigens, viability, and growth kinetics in fetal astrocytes infected with native and pseudotyped HIV-1. (A) Astrocyte cultures were infected with VSV/NL4-3, MLV/NL4-3, or NL4-3 as indicated and monitored for HIV-1-specific antigens expression by IF staining with AIDS sera and fluorescein isothiocyarate-conjugated second antibody (left panel) or by production of p24 in cell supernatants (right panel). The proportion of IF-positive cells was determined by counting at least 200 cells each in three different fields under ×20 magnification, using an Olympus BH-2 fluorescence microscope. (B) At the indicated times after infection, total cell numbers and total viable-cell numbers per system (×1,000) were determined as described in Materials and Methods.
FIG. 3
FIG. 3
Northern blot analysis of HIV-1 RNA expression in astrocytes infected with pseudotyped HIV-1. Astrocytes were infected with VSV/NL4-3, MLV/NL4-3, or NL4-3 and analyzed for HIV-1 RNA by Northern blotting 2, 14, and 28 days after infection as described in Materials and Methods. A total of 20 μg of total-cell RNA was loaded per lane. Samples from day 2 and 14 were exposed for 24 h, and samples from day 28 were exposed for 14 days. NL4-3-infected H9 cells were analyzed in parallel as positive controls; the samples were exposed for 24 h.
FIG. 4
FIG. 4
Western blot analysis of Gag, gp120, and Nef proteins by infected astrocytes. Astrocytes were infected with HIV-1 as indicated and tested for HIV-1 proteins by immunoblotting as described in Materials and Methods. All systems were normalized first by measurement of the α-tubulin content. (A and D) Gag levels 7 and 35 days after infection, respectively. (B and C) gp120 and Nef protein levels 7 days after infection. Panel D contains four times more protein per lane than does panel A. Mock indicates uninfected astrocytes, and infected and uninfected PBL are shown for comparison. The results are representative of three experiments.
FIG. 5
FIG. 5
Expression of HIV-1 PLAP marker after pseudotyped HIV-1 infection. Astrocytes were infected with MLV/NL-P1 and evaluated at the indicated times by IF staining with AIDS sera or anti-PLAP antibody as described in Materials and Methods. Each time point represents counts of 200 cells each in three different fields.
FIG. 6
FIG. 6
HIV-1-infected astrocytes produce infectious progeny virus. Astrocytes were infected with VSV/NL4-3 or MLV/NL4-3 as described in the text, and culture supernatants were collected 14 and 21 days after infection, filtered through 0.45-μm-pore-size filters, and tested for the presence of infectious virus in the MAGI assay. Values represent means and standard deviations from three different experiments.
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