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Human immunodeficiency virus type 1 efficiently binds to human fetal astrocytes and induces neuroinflammatory responses independent of infection.

Li J, Bentsman G, Potash MJ, Volsky DJ - BMC Neurosci (2007)

Bottom Line: Despite extensive binding, only about 1% of HFA were detectably infected by HIV-RevGFP or HIV-NefGFP, but this proportion increased to the majority of HFA when the viruses were pseudotyped with vesicular stomatitis virus envelope glycoprotein G, confirming that HFA impose a restriction upon HIV-1 entry.Our results indicate that HIV-1 binding to human astrocytes, although extensive, is not generally followed by virus entry and replication.Astrocytes respond to HIV-1 binding by rapidly increased cytokine production suggesting a role of this virus-brain cell interaction in HIV-1 neuropathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Virology Division, St. Luke's-Roosevelt Hospital Center and Columbia University, New York, NY 10019, USA. JiLi@coh.org <JiLi@coh.org>

ABSTRACT

Background: HIV-1 infects human astrocytes in vitro and in vivo but the frequency of infected cells is low and its biological significance is unknown. In studies in vitro, recombinant gp120 alone can induce profound effects on astrocyte biology, suggesting that HIV-1 interaction with astrocytes and its functional consequences extend beyond the limited levels of infection in these cells. Here we determined the relative efficiencies of HIV-1 binding and infection in human fetal astrocytes (HFA), mainly at the single cell level, using HIV-1 tagged with green fluorescence protein (GFP)-Vpr fusion proteins, termed HIV-GFP, to detect virus binding and HIV-1 expressing Rev and NefGFP fusion proteins to detect productive infection.

Results: Essentially all HFA in a population bound HIV-GFP specifically and independently of CCR5 and CXCR4. The dynamics of this binding at 37 degrees C resembled binding of an HIV fusion mutant to CD4-positive cells, indicating that most of HIV-GFP arrested infection of HFA at the stage of virus-cell fusion. Despite extensive binding, only about 1% of HFA were detectably infected by HIV-RevGFP or HIV-NefGFP, but this proportion increased to the majority of HFA when the viruses were pseudotyped with vesicular stomatitis virus envelope glycoprotein G, confirming that HFA impose a restriction upon HIV-1 entry. Exposure of HFA to HIV-1 through its native proteins rapidly induced synthesis of interleukin-6 and interleukin-8 with increased mRNA detected within 3 h and increased protein detected within 18 h of exposure.

Conclusion: Our results indicate that HIV-1 binding to human astrocytes, although extensive, is not generally followed by virus entry and replication. Astrocytes respond to HIV-1 binding by rapidly increased cytokine production suggesting a role of this virus-brain cell interaction in HIV-1 neuropathogenesis.

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Extent and specificity of HIV-1 binding to HFA. Primary HFA were exposed for 1 h at 4°C to NL4-3 carrying GFP-Vpr alone (first row), in the presence of excess unlabeled NL4-3 (second row) or in the presence of soluble CD4 (third row). Cells were stained with anti-GFAP and a Texas Red-labeled secondary antibody to label astrocytes and with DAPI to visualize nuclei. Images of red and green fluorescence were superimposed (third column).
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Figure 1: Extent and specificity of HIV-1 binding to HFA. Primary HFA were exposed for 1 h at 4°C to NL4-3 carrying GFP-Vpr alone (first row), in the presence of excess unlabeled NL4-3 (second row) or in the presence of soluble CD4 (third row). Cells were stained with anti-GFAP and a Texas Red-labeled secondary antibody to label astrocytes and with DAPI to visualize nuclei. Images of red and green fluorescence were superimposed (third column).

Mentions: In previous studies we inferred that HIV-1 interacts with the majority of HFA because exposure to intact or UV-inactivated virus, or recombinant gp120 alone, reduced glutamate uptake by cells and altered cellular gene expression on a population-wide basis [51,71], however such findings are indirect. Using an approach developed by McDonald and colleagues [72], we prepared fluorescently-labeled infectious virions by co-transfection of plasmids encoding intact HIV-1/NL4-3 and a GFP-Vpr fusion protein that is efficiently encapsidated [72] and evaluated this HIV-GFP binding to HFA by fluorescence microscopy. To investigate the specificity of the virus-cell binding, astrocytes were exposed to HIV-GFP in the presence of either unlabeled virus or recombinant soluble CD4 (sCD4) to compete for virion gp120 (Fig. 1). Essentially all HFA bound HIV-1 and this binding was completely blocked by both competitors. To validate our method and reagents, similar studies were carried out with CD4-negative HeLa cells or with MAGI cells, a HeLa derivative engineered to express human CD4 [73] (Fig. 2). HeLa cells failed to bind HIV-1 but MAGI cells bound virus and this inhibition was sensitive to competition by sCD4, indicating that the evaluation of GFP-Vpr-labeled virus binding by microscopy is reliable. These experiments were repeated three times with similar results. We also quantified binding on a population-wide basis by measuring cell-associated p24 1 h after HIV-1 exposure to cells at 4°C (Fig. 3). The extent of binding is expressed relative to the amount of p24 bound to HFA. MAGI cells bound roughly 40% p24 bound to HFA, while CD4-negative 293T or HeLa cells bound less than 15% as much p24 as did HFA. We also exposed HFA to antibodies to the chemokine receptors CCR3, CCR5, or CXCR4, which have been variably detected on HFA [74,75], to test whether any of these receptors are involved in HFA binding of HIV-1. There was a modest increase of virus binding after cells were pretreated with anti-CXCR4; the other antibodies had no effect. Taken together our studies demonstrate that HIV-1 labeled through GFP-Vpr interacts specifically with human cells and that the vast majority of HFA are competent to bind HIV-1. Competitors for gp120 block HIV-1 binding to HFA, confirming the specificity of the interaction of HIV-1 with HFA, however our studies do not identify the cellular receptor.


Human immunodeficiency virus type 1 efficiently binds to human fetal astrocytes and induces neuroinflammatory responses independent of infection.

Li J, Bentsman G, Potash MJ, Volsky DJ - BMC Neurosci (2007)

Extent and specificity of HIV-1 binding to HFA. Primary HFA were exposed for 1 h at 4°C to NL4-3 carrying GFP-Vpr alone (first row), in the presence of excess unlabeled NL4-3 (second row) or in the presence of soluble CD4 (third row). Cells were stained with anti-GFAP and a Texas Red-labeled secondary antibody to label astrocytes and with DAPI to visualize nuclei. Images of red and green fluorescence were superimposed (third column).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1884168&req=5

Figure 1: Extent and specificity of HIV-1 binding to HFA. Primary HFA were exposed for 1 h at 4°C to NL4-3 carrying GFP-Vpr alone (first row), in the presence of excess unlabeled NL4-3 (second row) or in the presence of soluble CD4 (third row). Cells were stained with anti-GFAP and a Texas Red-labeled secondary antibody to label astrocytes and with DAPI to visualize nuclei. Images of red and green fluorescence were superimposed (third column).
Mentions: In previous studies we inferred that HIV-1 interacts with the majority of HFA because exposure to intact or UV-inactivated virus, or recombinant gp120 alone, reduced glutamate uptake by cells and altered cellular gene expression on a population-wide basis [51,71], however such findings are indirect. Using an approach developed by McDonald and colleagues [72], we prepared fluorescently-labeled infectious virions by co-transfection of plasmids encoding intact HIV-1/NL4-3 and a GFP-Vpr fusion protein that is efficiently encapsidated [72] and evaluated this HIV-GFP binding to HFA by fluorescence microscopy. To investigate the specificity of the virus-cell binding, astrocytes were exposed to HIV-GFP in the presence of either unlabeled virus or recombinant soluble CD4 (sCD4) to compete for virion gp120 (Fig. 1). Essentially all HFA bound HIV-1 and this binding was completely blocked by both competitors. To validate our method and reagents, similar studies were carried out with CD4-negative HeLa cells or with MAGI cells, a HeLa derivative engineered to express human CD4 [73] (Fig. 2). HeLa cells failed to bind HIV-1 but MAGI cells bound virus and this inhibition was sensitive to competition by sCD4, indicating that the evaluation of GFP-Vpr-labeled virus binding by microscopy is reliable. These experiments were repeated three times with similar results. We also quantified binding on a population-wide basis by measuring cell-associated p24 1 h after HIV-1 exposure to cells at 4°C (Fig. 3). The extent of binding is expressed relative to the amount of p24 bound to HFA. MAGI cells bound roughly 40% p24 bound to HFA, while CD4-negative 293T or HeLa cells bound less than 15% as much p24 as did HFA. We also exposed HFA to antibodies to the chemokine receptors CCR3, CCR5, or CXCR4, which have been variably detected on HFA [74,75], to test whether any of these receptors are involved in HFA binding of HIV-1. There was a modest increase of virus binding after cells were pretreated with anti-CXCR4; the other antibodies had no effect. Taken together our studies demonstrate that HIV-1 labeled through GFP-Vpr interacts specifically with human cells and that the vast majority of HFA are competent to bind HIV-1. Competitors for gp120 block HIV-1 binding to HFA, confirming the specificity of the interaction of HIV-1 with HFA, however our studies do not identify the cellular receptor.

Bottom Line: Despite extensive binding, only about 1% of HFA were detectably infected by HIV-RevGFP or HIV-NefGFP, but this proportion increased to the majority of HFA when the viruses were pseudotyped with vesicular stomatitis virus envelope glycoprotein G, confirming that HFA impose a restriction upon HIV-1 entry.Our results indicate that HIV-1 binding to human astrocytes, although extensive, is not generally followed by virus entry and replication.Astrocytes respond to HIV-1 binding by rapidly increased cytokine production suggesting a role of this virus-brain cell interaction in HIV-1 neuropathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Virology Division, St. Luke's-Roosevelt Hospital Center and Columbia University, New York, NY 10019, USA. JiLi@coh.org <JiLi@coh.org>

ABSTRACT

Background: HIV-1 infects human astrocytes in vitro and in vivo but the frequency of infected cells is low and its biological significance is unknown. In studies in vitro, recombinant gp120 alone can induce profound effects on astrocyte biology, suggesting that HIV-1 interaction with astrocytes and its functional consequences extend beyond the limited levels of infection in these cells. Here we determined the relative efficiencies of HIV-1 binding and infection in human fetal astrocytes (HFA), mainly at the single cell level, using HIV-1 tagged with green fluorescence protein (GFP)-Vpr fusion proteins, termed HIV-GFP, to detect virus binding and HIV-1 expressing Rev and NefGFP fusion proteins to detect productive infection.

Results: Essentially all HFA in a population bound HIV-GFP specifically and independently of CCR5 and CXCR4. The dynamics of this binding at 37 degrees C resembled binding of an HIV fusion mutant to CD4-positive cells, indicating that most of HIV-GFP arrested infection of HFA at the stage of virus-cell fusion. Despite extensive binding, only about 1% of HFA were detectably infected by HIV-RevGFP or HIV-NefGFP, but this proportion increased to the majority of HFA when the viruses were pseudotyped with vesicular stomatitis virus envelope glycoprotein G, confirming that HFA impose a restriction upon HIV-1 entry. Exposure of HFA to HIV-1 through its native proteins rapidly induced synthesis of interleukin-6 and interleukin-8 with increased mRNA detected within 3 h and increased protein detected within 18 h of exposure.

Conclusion: Our results indicate that HIV-1 binding to human astrocytes, although extensive, is not generally followed by virus entry and replication. Astrocytes respond to HIV-1 binding by rapidly increased cytokine production suggesting a role of this virus-brain cell interaction in HIV-1 neuropathogenesis.

Show MeSH
Related in: MedlinePlus