1 Dept. of Microbiology, Fact. of Life Sci., Kumamoto Univ. 2 Dept. of Virology, Noguchi Memorial Inst. for Medical Research, Univ. of Ghana. 3 Grad. Sch. of Sci., Technol. and Innov., Kobe Univ. 4 Collaborative Res. Program, Joint Res. Center for Human Retrovirus Infection, Kumamoto Univ. 5Dept. of Nursing, Kibi International Univ.6 Dept. of Med. Virology, Fact. of Life Sci., Kumamoto Univ.7 Collaboration Unit for Infection, Joint Res. Center for Human Retrovirus Infection, Kumamoto Univ.Assembly of CCR5 with gp120 inhibits the HIV-1 infectivity in a Tcell-specific manner ○Joyce Appiah-Kubi1,2 Yuan Yuzhe3 Takeo Kuwata4 Hiromi Terasawa1 Perpetual Nyame1 Wright Ofotsu Amesimeku1 Md Jakir Hossain1 Shuzo Matsushita4 Tomohiro Sawa1 Yosuke Maeda1,5 Shinji Harada6 Keisuke Yusa3 Kazuaki Monde1,7CCR5 is one of the major coreceptors implicated in susceptibility to HIV-1 infection and disease progression. Functional studies have demonstrated that after initial interaction with the CD4 receptor, the gp120 V3 loop interacts with the N-terminal extracellular domain and the extracellular loop 2 of CCR5. The CD4 receptor in infected cells is strictly down-regulated, however, the behavior of CCR5 post-infection remains unclear. In this study, we examined the expression level and distribution of CCR5 on the cell surface of PM1 and PM1/CCR5 cells after infection with HIV-1JR-FL.The expression level and distribution of CCR5 on the cell surface of PM1 and PM1/CCR5 cells were examined after infection with HIV-1JR-FL by Flow cytometry. Co-localization co-efficiency was determined with confocal microscopy by staining cells with MAbs: anti-gp120 (49G2) and FITC-labeled anti-p24 antibodies, anti-gp120 and anti-CCR5 (T21/8), and anti-CCR5 (2D7). CCR5 late-stage inhibitory mechanisms were clarified by virus Immunoprecipitation and Infectivity, p24 Gag was quantified by a p24 Gag enzyme-linked immunosorbent assay (ELISA).No downregulation of CCR5 was observed after HIV-1 infection as detected by an anti-CCR5 MAb (T21/8) in HIV-1-infected cell lines. Juxtaposed with another anti-CCR5 MAb (2D7) reveals an apparent down-regulation of CCR5 in infected PM1 and PM1/CCR5 cells, suggesting that the 2D7 epitope on CCR5 was impaired or masked by HIV-1 infection, leading to an alternative form of CCR5 that could not be recognized by 2D7. In this outcome, we designated the alternative form of CCR5 as CCR5A recognized by T21/8, and normal CCR5 designated as CCR5N recognized by both T21/8 and 2D7. At the plasma membrane of infected PM1/CCR5 cells, the distribution of CCR5 was highly colocalized with HIV-1JR-FL Env at the Gag-assembled regions. Additionally, CCR5 detected by T21/8 MAb significantly colocalized with HIV-1JR-FL Env compared to 2D7 MAb. We observed that the presence of HIV-1JR-FL Env enhanced the incorporation of CCR5A into viruses generated from PM1 and PM1/CCR5, this mechanism was clarified by comparing CCR5 incorporation into virions in the presence or absence of Env. The efficiency of CCR5A incorporation from CD4+ T cells was 83-fold higher than that from CCR5A in adherent cells, strongly suggesting that the incorporation of CCR5A into viruses from CD4+ T cells is distinct from that of adherent cells. Interestingly, CCR5A incorporated into virions reduced the viral infectivity of HIV-1JR-FL, indicating the inhibitory effect CCR5 has on viral infectivity. Altogether, CCR5 exerts an inhibitory effect at the late stage of HIV-1 replication. The study also provides the characterization of CCR5 from the early to late stages of the HIV-1 life cycle.57P 027
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