Limits...
The B-oligomer of pertussis toxin deactivates CC chemokine receptor 5 and blocks entry of M-tropic HIV-1 strains.

Alfano M, Schmidtmayerova H, Amella CA, Pushkarsky T, Bukrinsky M - J. Exp. Med. (1999)

Bottom Line: T cells treated with B-oligomer did not initiate signal transduction in response to macrophage inflammatory protein (MIP)-1beta or RANTES (regulated upon activation, normal T cell expressed and secreted); however, cell surface expression of CCR5 and binding of MIP-1beta or HIV-1 to such cells were not impaired.B-oligomer also blocked cocapping of CCR5 and CD4 induced by R5 HIV-1 in primary T cells, but did not affect cocapping of CXCR4 and CD4 after inoculation of the cultures with X4 HIV-1.These results suggest that the B-oligomer of PTX cross-deactivates CCR5 to impair its function as a coreceptor for HIV-1.

View Article: PubMed Central - PubMed

Affiliation: Picower Institute for Medical Research, Manhasset, New York 11030, USA.

ABSTRACT
Infection of target cells by HIV-1 requires initial binding interactions between the viral envelope glycoprotein gp120, the cell surface protein CD4, and one of the members of the seven-transmembrane G protein-coupled chemokine receptor family. Most primary isolates (R5 strains) use chemokine receptor CCR5, but some primary syncytium-inducing, as well as T cell line-adapted, strains (X4 strains) use the CXCR4 receptor. Signaling from both CCR5 and CXCR4 is mediated by pertussis toxin (PTX)-sensitive G(i) proteins and is not required for HIV-1 entry. Here, we show that the PTX holotoxin as well as its binding subunit, B-oligomer, which lacks G(i)-inhibitory activity, blocked entry of R5 but not X4 strains into primary T lymphocytes. Interestingly, B-oligomer inhibited virus production by peripheral blood mononuclear cell cultures infected with either R5 or X4 strains, indicating that it can affect HIV-1 replication at both entry and post-entry levels. T cells treated with B-oligomer did not initiate signal transduction in response to macrophage inflammatory protein (MIP)-1beta or RANTES (regulated upon activation, normal T cell expressed and secreted); however, cell surface expression of CCR5 and binding of MIP-1beta or HIV-1 to such cells were not impaired. The inhibitory effect of B-oligomer on signaling from CCR5 and on entry of R5 HIV-1 strains was reversed by protein kinase C (PKC) inhibitors, indicating that B-oligomer activity is mediated by signaling events that involve PKC. B-oligomer also blocked cocapping of CCR5 and CD4 induced by R5 HIV-1 in primary T cells, but did not affect cocapping of CXCR4 and CD4 after inoculation of the cultures with X4 HIV-1. These results suggest that the B-oligomer of PTX cross-deactivates CCR5 to impair its function as a coreceptor for HIV-1.

Show MeSH

Related in: MedlinePlus

The effect of PTX and B-oligomer on HIV-1 entry. (A) Monocyte-depleted PBMC cultures were treated with PTX (1 nM) or left untreated (control), and inoculated with R5 HIV-192US660 or X4 HIV-1LAI. Viral entry was analyzed by PCR using primers LTR R/U5 specific for the early reverse transcription product, as described previously 34. Results were quantified on a Direct Imager (Packard Instrument Company) and are presented as a percentage of counts in treated versus control samples. The error bars show the deviation from the mean for triplicate samples. Similar results were obtained in two independent experiments with cells from different donors. (B) Cells were treated with the indicated concentrations of B-oligomer or an anti-CD4 mAb Leu3A for 10 min or were left untreated and inoculated with HIV-192US660 (left) or HIV-1LAI (middle). Viral entry was analyzed as in A. In parallel, each sample was amplified with α-tubulin–specific primers to control for the total amount of DNA. Results were quantified on a Packard Direct Imager and are presented on bar graphs as mean counts for each set of duplicate samples, expressed as counts per minute (cpm). The error bars show the deviation from the mean for each duplicate. Uninfected cells (Neg.) served as negative control. Dilutions of 8E5/LAI cells containing one HIV-1 genome per cell 45 were used as PCR standards (right).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195621&req=5

Figure 1: The effect of PTX and B-oligomer on HIV-1 entry. (A) Monocyte-depleted PBMC cultures were treated with PTX (1 nM) or left untreated (control), and inoculated with R5 HIV-192US660 or X4 HIV-1LAI. Viral entry was analyzed by PCR using primers LTR R/U5 specific for the early reverse transcription product, as described previously 34. Results were quantified on a Direct Imager (Packard Instrument Company) and are presented as a percentage of counts in treated versus control samples. The error bars show the deviation from the mean for triplicate samples. Similar results were obtained in two independent experiments with cells from different donors. (B) Cells were treated with the indicated concentrations of B-oligomer or an anti-CD4 mAb Leu3A for 10 min or were left untreated and inoculated with HIV-192US660 (left) or HIV-1LAI (middle). Viral entry was analyzed as in A. In parallel, each sample was amplified with α-tubulin–specific primers to control for the total amount of DNA. Results were quantified on a Packard Direct Imager and are presented on bar graphs as mean counts for each set of duplicate samples, expressed as counts per minute (cpm). The error bars show the deviation from the mean for each duplicate. Uninfected cells (Neg.) served as negative control. Dilutions of 8E5/LAI cells containing one HIV-1 genome per cell 45 were used as PCR standards (right).

Mentions: Several previously published reports 1617181920 demonstrated that activity of chemokine receptors as coreceptors for HIV-1 does not involve signaling via coupled Gi proteins. A somewhat conflicting result has been reported recently 21, demonstrating that entry of X4 HIV-1 strains into primary T cells correlated with actin-dependent cocapping of CD4 and CXCR4 receptors, thus implicating signaling in the process of HIV-1 entry. To better define the role of signaling from chemokine receptors in HIV-1 entry into primary T cells, we used PTX to specifically inactivate Gi-like proteins that transduce signals from both CCR5 and CXCR4 13, and measured HIV-1 entry by PCR, using primers LTR R/U5 specific for early products of reverse transcription 34. The fragment of HIV-1 cDNA amplified by these primers is produced either within the virion or very early after virus–cell fusion, and thus reflects the efficiency of virus entry. Surprisingly, PTX inhibited entry of R5 HIV-1 strains 92US660 (Fig. 1 A) and ADA (not shown), but not of X4 strains LAI (Fig. 1 A) and 92UG21 (not shown). PTX is a complex protein composed of an active (A) and a binding (B) subunit, and certain T cell activities have been shown to be initiated by the B-oligomer of PTX independently of inactivation of Gi-like proteins by the A-protomer 35. We therefore tested whether activity of B-oligomer could account for the observed inhibitory effect of PTX on entry of R5 HIV-1. Similar to results observed with PTX, B-oligomer inhibited entry of R5 HIV-1 strains in a dose-dependent fashion, but not of X4 HIV-1 strains (Fig. 1 B). Analysis of the B-oligomer preparation by SDS-PAGE confirmed the lack of A-protomer (not shown), and Gi-mediated signaling was not impaired in B-oligomer–treated cells (see Fig. 4, right). We thus conclude that PTX blocks entry of R5 HIV-1 strains by a mechanism that is independent of Gi protein inhibition. Interestingly, no inhibitory activity of PTX or B-oligomer was observed on entry of R5 HIV-1 into PM1 cells (not shown), consistent with recently reported results 36. This result underscores the differences between the primary cells and T cell lines.


The B-oligomer of pertussis toxin deactivates CC chemokine receptor 5 and blocks entry of M-tropic HIV-1 strains.

Alfano M, Schmidtmayerova H, Amella CA, Pushkarsky T, Bukrinsky M - J. Exp. Med. (1999)

The effect of PTX and B-oligomer on HIV-1 entry. (A) Monocyte-depleted PBMC cultures were treated with PTX (1 nM) or left untreated (control), and inoculated with R5 HIV-192US660 or X4 HIV-1LAI. Viral entry was analyzed by PCR using primers LTR R/U5 specific for the early reverse transcription product, as described previously 34. Results were quantified on a Direct Imager (Packard Instrument Company) and are presented as a percentage of counts in treated versus control samples. The error bars show the deviation from the mean for triplicate samples. Similar results were obtained in two independent experiments with cells from different donors. (B) Cells were treated with the indicated concentrations of B-oligomer or an anti-CD4 mAb Leu3A for 10 min or were left untreated and inoculated with HIV-192US660 (left) or HIV-1LAI (middle). Viral entry was analyzed as in A. In parallel, each sample was amplified with α-tubulin–specific primers to control for the total amount of DNA. Results were quantified on a Packard Direct Imager and are presented on bar graphs as mean counts for each set of duplicate samples, expressed as counts per minute (cpm). The error bars show the deviation from the mean for each duplicate. Uninfected cells (Neg.) served as negative control. Dilutions of 8E5/LAI cells containing one HIV-1 genome per cell 45 were used as PCR standards (right).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195621&req=5

Figure 1: The effect of PTX and B-oligomer on HIV-1 entry. (A) Monocyte-depleted PBMC cultures were treated with PTX (1 nM) or left untreated (control), and inoculated with R5 HIV-192US660 or X4 HIV-1LAI. Viral entry was analyzed by PCR using primers LTR R/U5 specific for the early reverse transcription product, as described previously 34. Results were quantified on a Direct Imager (Packard Instrument Company) and are presented as a percentage of counts in treated versus control samples. The error bars show the deviation from the mean for triplicate samples. Similar results were obtained in two independent experiments with cells from different donors. (B) Cells were treated with the indicated concentrations of B-oligomer or an anti-CD4 mAb Leu3A for 10 min or were left untreated and inoculated with HIV-192US660 (left) or HIV-1LAI (middle). Viral entry was analyzed as in A. In parallel, each sample was amplified with α-tubulin–specific primers to control for the total amount of DNA. Results were quantified on a Packard Direct Imager and are presented on bar graphs as mean counts for each set of duplicate samples, expressed as counts per minute (cpm). The error bars show the deviation from the mean for each duplicate. Uninfected cells (Neg.) served as negative control. Dilutions of 8E5/LAI cells containing one HIV-1 genome per cell 45 were used as PCR standards (right).
Mentions: Several previously published reports 1617181920 demonstrated that activity of chemokine receptors as coreceptors for HIV-1 does not involve signaling via coupled Gi proteins. A somewhat conflicting result has been reported recently 21, demonstrating that entry of X4 HIV-1 strains into primary T cells correlated with actin-dependent cocapping of CD4 and CXCR4 receptors, thus implicating signaling in the process of HIV-1 entry. To better define the role of signaling from chemokine receptors in HIV-1 entry into primary T cells, we used PTX to specifically inactivate Gi-like proteins that transduce signals from both CCR5 and CXCR4 13, and measured HIV-1 entry by PCR, using primers LTR R/U5 specific for early products of reverse transcription 34. The fragment of HIV-1 cDNA amplified by these primers is produced either within the virion or very early after virus–cell fusion, and thus reflects the efficiency of virus entry. Surprisingly, PTX inhibited entry of R5 HIV-1 strains 92US660 (Fig. 1 A) and ADA (not shown), but not of X4 strains LAI (Fig. 1 A) and 92UG21 (not shown). PTX is a complex protein composed of an active (A) and a binding (B) subunit, and certain T cell activities have been shown to be initiated by the B-oligomer of PTX independently of inactivation of Gi-like proteins by the A-protomer 35. We therefore tested whether activity of B-oligomer could account for the observed inhibitory effect of PTX on entry of R5 HIV-1. Similar to results observed with PTX, B-oligomer inhibited entry of R5 HIV-1 strains in a dose-dependent fashion, but not of X4 HIV-1 strains (Fig. 1 B). Analysis of the B-oligomer preparation by SDS-PAGE confirmed the lack of A-protomer (not shown), and Gi-mediated signaling was not impaired in B-oligomer–treated cells (see Fig. 4, right). We thus conclude that PTX blocks entry of R5 HIV-1 strains by a mechanism that is independent of Gi protein inhibition. Interestingly, no inhibitory activity of PTX or B-oligomer was observed on entry of R5 HIV-1 into PM1 cells (not shown), consistent with recently reported results 36. This result underscores the differences between the primary cells and T cell lines.

Bottom Line: T cells treated with B-oligomer did not initiate signal transduction in response to macrophage inflammatory protein (MIP)-1beta or RANTES (regulated upon activation, normal T cell expressed and secreted); however, cell surface expression of CCR5 and binding of MIP-1beta or HIV-1 to such cells were not impaired.B-oligomer also blocked cocapping of CCR5 and CD4 induced by R5 HIV-1 in primary T cells, but did not affect cocapping of CXCR4 and CD4 after inoculation of the cultures with X4 HIV-1.These results suggest that the B-oligomer of PTX cross-deactivates CCR5 to impair its function as a coreceptor for HIV-1.

View Article: PubMed Central - PubMed

Affiliation: Picower Institute for Medical Research, Manhasset, New York 11030, USA.

ABSTRACT
Infection of target cells by HIV-1 requires initial binding interactions between the viral envelope glycoprotein gp120, the cell surface protein CD4, and one of the members of the seven-transmembrane G protein-coupled chemokine receptor family. Most primary isolates (R5 strains) use chemokine receptor CCR5, but some primary syncytium-inducing, as well as T cell line-adapted, strains (X4 strains) use the CXCR4 receptor. Signaling from both CCR5 and CXCR4 is mediated by pertussis toxin (PTX)-sensitive G(i) proteins and is not required for HIV-1 entry. Here, we show that the PTX holotoxin as well as its binding subunit, B-oligomer, which lacks G(i)-inhibitory activity, blocked entry of R5 but not X4 strains into primary T lymphocytes. Interestingly, B-oligomer inhibited virus production by peripheral blood mononuclear cell cultures infected with either R5 or X4 strains, indicating that it can affect HIV-1 replication at both entry and post-entry levels. T cells treated with B-oligomer did not initiate signal transduction in response to macrophage inflammatory protein (MIP)-1beta or RANTES (regulated upon activation, normal T cell expressed and secreted); however, cell surface expression of CCR5 and binding of MIP-1beta or HIV-1 to such cells were not impaired. The inhibitory effect of B-oligomer on signaling from CCR5 and on entry of R5 HIV-1 strains was reversed by protein kinase C (PKC) inhibitors, indicating that B-oligomer activity is mediated by signaling events that involve PKC. B-oligomer also blocked cocapping of CCR5 and CD4 induced by R5 HIV-1 in primary T cells, but did not affect cocapping of CXCR4 and CD4 after inoculation of the cultures with X4 HIV-1. These results suggest that the B-oligomer of PTX cross-deactivates CCR5 to impair its function as a coreceptor for HIV-1.

Show MeSH
Related in: MedlinePlus