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A small-molecule inhibitor directed against the chemokine receptor CXCR4 prevents its use as an HIV-1 coreceptor.

Doranz BJ, Grovit-Ferbas K, Sharron MP, Mao SH, Goetz MB, Daar ES, Doms RW, O'Brien WA - J. Exp. Med. (1997)

Bottom Line: ALX40-4C inhibited HIV-1 use of the coreceptor CXCR4 by T- and dual-tropic HIV-1 strains, whereas use of CCR5 by M- and dual-tropic strains was not inhibited.ALX40-4C blocked stromal-derived factor (SDF)-1alpha-mediated activation of CXCR4 and binding of the monoclonal antibody 12G5 to cells expressing CXCR4.Overlap of the ALX40-4C binding site with that of 12G5 and SDF implicates direct blocking of Env interactions, rather than downregulation of receptor, as the mechanism of inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104, USA.

ABSTRACT
The chemokine receptor CXCR4 is the major coreceptor used for cellular entry by T cell- tropic human immunodeficiency virus (HIV)-1 strains, whereas CCR5 is used by macrophage (M)-tropic strains. Here we show that a small-molecule inhibitor, ALX40-4C, inhibits HIV-1 envelope (Env)-mediated membrane fusion and viral entry directly at the level of coreceptor use. ALX40-4C inhibited HIV-1 use of the coreceptor CXCR4 by T- and dual-tropic HIV-1 strains, whereas use of CCR5 by M- and dual-tropic strains was not inhibited. Dual-tropic viruses capable of using both CXCR4 and CCR5 were inhibited by ALX40-4C only when cells expressed CXCR4 alone. ALX40-4C blocked stromal-derived factor (SDF)-1alpha-mediated activation of CXCR4 and binding of the monoclonal antibody 12G5 to cells expressing CXCR4. Overlap of the ALX40-4C binding site with that of 12G5 and SDF implicates direct blocking of Env interactions, rather than downregulation of receptor, as the mechanism of inhibition. Thus, ALX40-4C represents a small-molecule inhibitor of HIV-1 infection that acts directly against a chemokine receptor at the level of Env-mediated membrane fusion.

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(A) Inhibition of virus infection on PM1 cells. The PM1 cell  line was infected with single-cycle pseudotyped HIV-1 virions and assayed for luciferase production. (B) Feline CCCS+L-CD4 cells transiently  expressing either CXCR4 or CCR5 were infected with pseudotyped luciferase viruses. Results in both panels represent the average of two identical experiments using 10 μM ALX40-4C and 10 μg/ml Leu3A, and error bars represent the range of the two independent experiments. Relative  light unit (RLU) values are normalized to 100% for direct infection of  permissive cells.
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Figure 1: (A) Inhibition of virus infection on PM1 cells. The PM1 cell line was infected with single-cycle pseudotyped HIV-1 virions and assayed for luciferase production. (B) Feline CCCS+L-CD4 cells transiently expressing either CXCR4 or CCR5 were infected with pseudotyped luciferase viruses. Results in both panels represent the average of two identical experiments using 10 μM ALX40-4C and 10 μg/ml Leu3A, and error bars represent the range of the two independent experiments. Relative light unit (RLU) values are normalized to 100% for direct infection of permissive cells.

Mentions: Reporter viruses pseudotyped with the T-tropic Envs NL4-3 and HXB2 (that use CXCR4 as a coreceptor), the M-tropic Env ADA (that uses CCR5), or the murine leukemia virus (MLV) Env that enters cells independently of CD4 or chemokine receptors, were used to infect PM1 cells, a T cell line that expresses CD4, CXCR4, and CCR5 (6). As shown in Fig. 1 A, ALX40-4C inhibited infection of viruses containing the NL4-3 and HXB2, but not the ADA or MLV Envs. An antibody to CD4 inhibited all HIV-1 strains tested. Inhibition of HXB2 infection in PM1 cells was achieved with an IC50 of ∼0.3 μM (data not shown). Since ALX40-4C did not prevent infection by ADA, we conclude that it did not block CD4 binding or postentry steps of viral replication, suggesting that its inhibitory effects are at the level of virus entry and might be coreceptor specific, consistent with involvement of the V3 loop in ALX40-4C sensitivity (21). To test this, we transiently transfected feline cells that stably express human CD4 with plasmids encoding the coreceptor of interest. HIV-1 NL4-3 only infected cells expressing CXCR4, whereas HIV-1 ADA only infected cells expressing CCR5 (Fig. 1 B). As with PM1 cells, ALX40-4C inhibited the entry of NL4-3, but did not significantly affect entry of ADA.


A small-molecule inhibitor directed against the chemokine receptor CXCR4 prevents its use as an HIV-1 coreceptor.

Doranz BJ, Grovit-Ferbas K, Sharron MP, Mao SH, Goetz MB, Daar ES, Doms RW, O'Brien WA - J. Exp. Med. (1997)

(A) Inhibition of virus infection on PM1 cells. The PM1 cell  line was infected with single-cycle pseudotyped HIV-1 virions and assayed for luciferase production. (B) Feline CCCS+L-CD4 cells transiently  expressing either CXCR4 or CCR5 were infected with pseudotyped luciferase viruses. Results in both panels represent the average of two identical experiments using 10 μM ALX40-4C and 10 μg/ml Leu3A, and error bars represent the range of the two independent experiments. Relative  light unit (RLU) values are normalized to 100% for direct infection of  permissive cells.
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Related In: Results  -  Collection

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Figure 1: (A) Inhibition of virus infection on PM1 cells. The PM1 cell line was infected with single-cycle pseudotyped HIV-1 virions and assayed for luciferase production. (B) Feline CCCS+L-CD4 cells transiently expressing either CXCR4 or CCR5 were infected with pseudotyped luciferase viruses. Results in both panels represent the average of two identical experiments using 10 μM ALX40-4C and 10 μg/ml Leu3A, and error bars represent the range of the two independent experiments. Relative light unit (RLU) values are normalized to 100% for direct infection of permissive cells.
Mentions: Reporter viruses pseudotyped with the T-tropic Envs NL4-3 and HXB2 (that use CXCR4 as a coreceptor), the M-tropic Env ADA (that uses CCR5), or the murine leukemia virus (MLV) Env that enters cells independently of CD4 or chemokine receptors, were used to infect PM1 cells, a T cell line that expresses CD4, CXCR4, and CCR5 (6). As shown in Fig. 1 A, ALX40-4C inhibited infection of viruses containing the NL4-3 and HXB2, but not the ADA or MLV Envs. An antibody to CD4 inhibited all HIV-1 strains tested. Inhibition of HXB2 infection in PM1 cells was achieved with an IC50 of ∼0.3 μM (data not shown). Since ALX40-4C did not prevent infection by ADA, we conclude that it did not block CD4 binding or postentry steps of viral replication, suggesting that its inhibitory effects are at the level of virus entry and might be coreceptor specific, consistent with involvement of the V3 loop in ALX40-4C sensitivity (21). To test this, we transiently transfected feline cells that stably express human CD4 with plasmids encoding the coreceptor of interest. HIV-1 NL4-3 only infected cells expressing CXCR4, whereas HIV-1 ADA only infected cells expressing CCR5 (Fig. 1 B). As with PM1 cells, ALX40-4C inhibited the entry of NL4-3, but did not significantly affect entry of ADA.

Bottom Line: ALX40-4C inhibited HIV-1 use of the coreceptor CXCR4 by T- and dual-tropic HIV-1 strains, whereas use of CCR5 by M- and dual-tropic strains was not inhibited.ALX40-4C blocked stromal-derived factor (SDF)-1alpha-mediated activation of CXCR4 and binding of the monoclonal antibody 12G5 to cells expressing CXCR4.Overlap of the ALX40-4C binding site with that of 12G5 and SDF implicates direct blocking of Env interactions, rather than downregulation of receptor, as the mechanism of inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104, USA.

ABSTRACT
The chemokine receptor CXCR4 is the major coreceptor used for cellular entry by T cell- tropic human immunodeficiency virus (HIV)-1 strains, whereas CCR5 is used by macrophage (M)-tropic strains. Here we show that a small-molecule inhibitor, ALX40-4C, inhibits HIV-1 envelope (Env)-mediated membrane fusion and viral entry directly at the level of coreceptor use. ALX40-4C inhibited HIV-1 use of the coreceptor CXCR4 by T- and dual-tropic HIV-1 strains, whereas use of CCR5 by M- and dual-tropic strains was not inhibited. Dual-tropic viruses capable of using both CXCR4 and CCR5 were inhibited by ALX40-4C only when cells expressed CXCR4 alone. ALX40-4C blocked stromal-derived factor (SDF)-1alpha-mediated activation of CXCR4 and binding of the monoclonal antibody 12G5 to cells expressing CXCR4. Overlap of the ALX40-4C binding site with that of 12G5 and SDF implicates direct blocking of Env interactions, rather than downregulation of receptor, as the mechanism of inhibition. Thus, ALX40-4C represents a small-molecule inhibitor of HIV-1 infection that acts directly against a chemokine receptor at the level of Env-mediated membrane fusion.

Show MeSH
Related in: MedlinePlus