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CXCR4/fusin is not a species-specific barrier in murine cells for HIV-1 entry.

Tachibana K, Nakajima T, Sato A, Igarashi K, Shida H, Iizasa H, Yoshida N, Yoshie O, Kishimoto T, Nagasawa T - J. Exp. Med. (1997)

Bottom Line: Recently, CXCR4 was shown to function as a coreceptor for T cell line-tropic HIV-1 entry.Here we demonstrated that cells expressing murine CXCR4 and human CD4 fused with cells expressing the env proteins derived from T cell line-tropic HIV-1 and were infected with T cell line-tropic HIV-1 strains.We conclude that murine CXCR4 is not a species specific barrier to the entry of T cell line-tropic HIV-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Research Institute, Osaka Medical Center for Maternal and Child Health, Osaka 590-02, Japan.

ABSTRACT
Since some murine cells expressing human CD4 fail to internalize HIV-1, another block was thought to be located at the level of viral entry in addition to CD4. Recently, CXCR4 was shown to function as a coreceptor for T cell line-tropic HIV-1 entry. Here we demonstrated that cells expressing murine CXCR4 and human CD4 fused with cells expressing the env proteins derived from T cell line-tropic HIV-1 and were infected with T cell line-tropic HIV-1 strains. In contrast, the same cells were not infected with chimeric clones constructed by substitution of monocyte- or macrophage-tropic strain-derived env region or V3 region into T cell line-tropic HIV-1, indicating V3 loop of envelope protein is required for murine CXCR4mediated HIV-1 entry. We conclude that murine CXCR4 is not a species specific barrier to the entry of T cell line-tropic HIV-1.

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Murine CXCR4 supported membrane fusion mediated by T cell line-tropic HIV-1 env protein. (A) RT-PCR analysis of murine CXCR4  mRNA expression on NIH3T3 cells and DW34 cells. (B) Intracellular Ca2+ response of NIH3T3 cells and CHO cells transfected with murine CXCR4  in response to murine PBSF/SDF-1. Rise in intracellular Ca2+ concentration is represented by the increase in relative fluorescence. (C) Quantitation of fusion by β-galactosidase activity assay. NIH3T3 target cells were infected with a recombinant vaccinia virus that expresses human CD4, T7 polymerase and  ω subunit of β-gal. Then the cells were transfected with murine CXCR4 or human CXCR4 or CCR5. HeLaS3 effector cells were infected with a recombinant vaccinia virus that expresses α subunit of β-gal and env proteins derived from HIV-1 strains NL 432 or SF162. Cells were allowed to fuse and  assayed for β-gal activity.
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Figure 1: Murine CXCR4 supported membrane fusion mediated by T cell line-tropic HIV-1 env protein. (A) RT-PCR analysis of murine CXCR4 mRNA expression on NIH3T3 cells and DW34 cells. (B) Intracellular Ca2+ response of NIH3T3 cells and CHO cells transfected with murine CXCR4 in response to murine PBSF/SDF-1. Rise in intracellular Ca2+ concentration is represented by the increase in relative fluorescence. (C) Quantitation of fusion by β-galactosidase activity assay. NIH3T3 target cells were infected with a recombinant vaccinia virus that expresses human CD4, T7 polymerase and ω subunit of β-gal. Then the cells were transfected with murine CXCR4 or human CXCR4 or CCR5. HeLaS3 effector cells were infected with a recombinant vaccinia virus that expresses α subunit of β-gal and env proteins derived from HIV-1 strains NL 432 or SF162. Cells were allowed to fuse and assayed for β-gal activity.

Mentions: First, to determine if murine CXCR4 allows HIV-1 env-mediated membrane fusion, we used the assay system in which fusion between effector env-expressing cells and target coreceptor- and CD4-expressing cells leads to activation of a reporter enzyme. In this assay, effector HeLaS3 cells were infected with a recombinant vaccinia virus that expresses α subunit of β-gal and HIV-1 env protein. We used NIH3T3 cells as the target cells since no CXCR4 mRNA was seen and 1,000 nM PBSF/SDF-1, a ligand for CXCR4 did not induce an increase in intracellular free Ca2+ in NIH3T3 cells (Fig. 1, A and B). The target NIH3T3 cells were infected with a recombinant vaccinia virus that expresses ω-subunit of β-gal, T7 polymerase and human CD4. Then NIH3T3 cells were transfected with plasmids containing human CXCR4 or CCR5 or murine CXCR4. Effector and target cells were mixed together and incubated. If fusion occurred, the cytoplasmic contents of the fused cells lead to activation of β-gal. As shown in Fig. 1, HeLaS3 cells expressing the env proteins derived from the T-tropic HIV-1 strain NL432 readily fused with NIH 3T3 cells expressing human CXCR4 and human CD4, but not with cells expressing human CCR5 and human CD4. Surprisingly, they also fused with cells expressing murine CXCR4 and human CD4. HeLaS3 cells expressing the env proteins derived from the M-tropic strain SF162 fused with cells expressing human CCR5 and human CD4, but not with cells expressing murine or human CXCR4 in conjunction with human CD4.


CXCR4/fusin is not a species-specific barrier in murine cells for HIV-1 entry.

Tachibana K, Nakajima T, Sato A, Igarashi K, Shida H, Iizasa H, Yoshida N, Yoshie O, Kishimoto T, Nagasawa T - J. Exp. Med. (1997)

Murine CXCR4 supported membrane fusion mediated by T cell line-tropic HIV-1 env protein. (A) RT-PCR analysis of murine CXCR4  mRNA expression on NIH3T3 cells and DW34 cells. (B) Intracellular Ca2+ response of NIH3T3 cells and CHO cells transfected with murine CXCR4  in response to murine PBSF/SDF-1. Rise in intracellular Ca2+ concentration is represented by the increase in relative fluorescence. (C) Quantitation of fusion by β-galactosidase activity assay. NIH3T3 target cells were infected with a recombinant vaccinia virus that expresses human CD4, T7 polymerase and  ω subunit of β-gal. Then the cells were transfected with murine CXCR4 or human CXCR4 or CCR5. HeLaS3 effector cells were infected with a recombinant vaccinia virus that expresses α subunit of β-gal and env proteins derived from HIV-1 strains NL 432 or SF162. Cells were allowed to fuse and  assayed for β-gal activity.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196327&req=5

Figure 1: Murine CXCR4 supported membrane fusion mediated by T cell line-tropic HIV-1 env protein. (A) RT-PCR analysis of murine CXCR4 mRNA expression on NIH3T3 cells and DW34 cells. (B) Intracellular Ca2+ response of NIH3T3 cells and CHO cells transfected with murine CXCR4 in response to murine PBSF/SDF-1. Rise in intracellular Ca2+ concentration is represented by the increase in relative fluorescence. (C) Quantitation of fusion by β-galactosidase activity assay. NIH3T3 target cells were infected with a recombinant vaccinia virus that expresses human CD4, T7 polymerase and ω subunit of β-gal. Then the cells were transfected with murine CXCR4 or human CXCR4 or CCR5. HeLaS3 effector cells were infected with a recombinant vaccinia virus that expresses α subunit of β-gal and env proteins derived from HIV-1 strains NL 432 or SF162. Cells were allowed to fuse and assayed for β-gal activity.
Mentions: First, to determine if murine CXCR4 allows HIV-1 env-mediated membrane fusion, we used the assay system in which fusion between effector env-expressing cells and target coreceptor- and CD4-expressing cells leads to activation of a reporter enzyme. In this assay, effector HeLaS3 cells were infected with a recombinant vaccinia virus that expresses α subunit of β-gal and HIV-1 env protein. We used NIH3T3 cells as the target cells since no CXCR4 mRNA was seen and 1,000 nM PBSF/SDF-1, a ligand for CXCR4 did not induce an increase in intracellular free Ca2+ in NIH3T3 cells (Fig. 1, A and B). The target NIH3T3 cells were infected with a recombinant vaccinia virus that expresses ω-subunit of β-gal, T7 polymerase and human CD4. Then NIH3T3 cells were transfected with plasmids containing human CXCR4 or CCR5 or murine CXCR4. Effector and target cells were mixed together and incubated. If fusion occurred, the cytoplasmic contents of the fused cells lead to activation of β-gal. As shown in Fig. 1, HeLaS3 cells expressing the env proteins derived from the T-tropic HIV-1 strain NL432 readily fused with NIH 3T3 cells expressing human CXCR4 and human CD4, but not with cells expressing human CCR5 and human CD4. Surprisingly, they also fused with cells expressing murine CXCR4 and human CD4. HeLaS3 cells expressing the env proteins derived from the M-tropic strain SF162 fused with cells expressing human CCR5 and human CD4, but not with cells expressing murine or human CXCR4 in conjunction with human CD4.

Bottom Line: Recently, CXCR4 was shown to function as a coreceptor for T cell line-tropic HIV-1 entry.Here we demonstrated that cells expressing murine CXCR4 and human CD4 fused with cells expressing the env proteins derived from T cell line-tropic HIV-1 and were infected with T cell line-tropic HIV-1 strains.We conclude that murine CXCR4 is not a species specific barrier to the entry of T cell line-tropic HIV-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Research Institute, Osaka Medical Center for Maternal and Child Health, Osaka 590-02, Japan.

ABSTRACT
Since some murine cells expressing human CD4 fail to internalize HIV-1, another block was thought to be located at the level of viral entry in addition to CD4. Recently, CXCR4 was shown to function as a coreceptor for T cell line-tropic HIV-1 entry. Here we demonstrated that cells expressing murine CXCR4 and human CD4 fused with cells expressing the env proteins derived from T cell line-tropic HIV-1 and were infected with T cell line-tropic HIV-1 strains. In contrast, the same cells were not infected with chimeric clones constructed by substitution of monocyte- or macrophage-tropic strain-derived env region or V3 region into T cell line-tropic HIV-1, indicating V3 loop of envelope protein is required for murine CXCR4mediated HIV-1 entry. We conclude that murine CXCR4 is not a species specific barrier to the entry of T cell line-tropic HIV-1.

Show MeSH
Related in: MedlinePlus