Establishment of a new system for determination of coreceptor usages of HIV based on the human glioma NP-2 cell line.

CD4 and one of the G-protein-coupled receptors (GPCRs) on the cell surface function as a receptor and a coreceptor, respectively, in infection of cells with human and simian immunodeficiency viruses (HIV/SIV). To determine which GPCRs can be coreceptors for HIV (HIV-1 and HIV-2) or SIV infection, several cell lines, including human osteosarcoma HOS-T4 cells and human glioma U87/CD4 cells, have been used. However, these cells often show susceptibilities to some HIV or SIV strains before transduction of GPCRs. The results of this study showed that a CD4-transduced human glioma cell line, NP-2/CD4, a human erythroleukemia cell line, K562/CD4, and a human ovarian cancer cell line, TYK/CD4, were completely resistant to the HIV-1 and HIV-2 strains tested. After transduction of several GPCRs into NP-2/CD4, K562/CD4, or TYK/CD4 cells, NP-2/CD4 cells but not K562/CD4 or TYK/CD4 cells mostly showed expected susceptibilities to several HIV strains. Therefore, an NP-2 cell system would be useful to determine the coreceptor usage of HIV isolates, to find a new coreceptor for HIV/SIV, and to analyze the early stages of HIV/SIV infection.

Soda Y ，Shimizu N ，Jinno A ，Liu HY ，Kanbe K ，Kitamura T ，Hoshino H ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

CD4-Dependent and CD4-independent utilization of coreceptors by human immunodeficiency viruses type 2 and simian immunodeficiency viruses.

More than 10 G protein-coupled receptors (GPCRs) have been reported to act as coreceptors for entry of human and simian immunodeficiency viruses (HIV and SIV). We investigated the utilization of six GPCRs as coreceptors by T-cell-line-adapted HIV-2 strains (CBL-20, CBL-21, CBL-23, GH-1, ROD, and SBL6669) and SIV strains (SIVagmTYO-1, SIVmac251, and SIVmndGB-1). NP-2/CD4 cells were transduced with CCR3, CCR5, CCR8, CXCR4, GPR1, or APJ, and examined for susceptibilities to cell-free HIV/SIV. HIV-2 strains were grouped into two types by their coreceptor usage. The first group, CBL-20 and CBL-21, used CXCR4 exclusively; the other four strains used a few or all of the six coreceptors. These strains could further infect CD4-negative NP-2/CXCR4 or NP-2/CCR5 cells in the presence (all strains) or absence (SBL6669 and ROD strains) of soluble CD4. SIVagm and SIVmnd infected NP-2/CD4/GPR1 cells. The coreceptors CCR3, CCR8, GPR1, and APJ did not mediate the CD4-independent infection. Although HIV-2ROD and SIVmnd infected both NP-2/CD4/CXCR4 and NP-2/CD4/CCR5 cells, only CXCR4 and CCR5, respectively, were used in CD4-independent infection. Binding of virions to CD4-negative cells occurred at 4 degrees C. These findings suggest that there may be a correlation between the promiscuous use of coreceptors by HIV-2/SIV strains and their ability to infect CD4-negative cells.

Liu HY ，Soda Y ，Shimizu N ，Haraguchi Y ，Jinno A ，Takeuchi Y ，Hoshino H ... -  《VIROLOGY》

A CXC chemokine receptor, CXCR5/BLR1, is a novel and specific coreceptor for human immunodeficiency virus type 2.

G protein-coupled receptors serve as coreceptors in the infection process of human immunodeficiency virus type-1 (HIV-1), type-2 (HIV-2), and simian immunodeficiency virus (SIV). In this study, we showed that a CXC-CKR, CXCR5/BLR1, is a novel coreceptor for HIV-2, but for neither HIV-1 nor SIV. The expression of CXCR5 was detected by polymerase chain reaction after reverse transcription of cellular mRNA from S+L-HOS/CD4 cells and MT-2 human T cells, and the CXCR5 gene was cloned into an expression vector. S+L-HOS/CD4 cells were susceptible to several HIV-2 strains but not most HIV-1 strains. To examine a coreceptor activity of CXCR5, we used NP-2/CD4, which is a human glioma cell line, NP-2, transduced with the CD4 gene that shows strict resistance to infection with HIV-1, HIV-2, SIVmac, SIVagm, or SIVmnd strain. When CXCR5 was transduced into NP-2/CD4 cells, they became highly susceptible to HIV-2ROD and HIV-2CBL23 strains in a CD4-dependent manner but to not to HIV-1 or SIV strains. Anti-CXCR5 monoclonal antibody and a ligand for CXCR5, BCA-1, inhibited HIV-2 infection to NP-2/CD4/CXCR5 cells. Our findings suggest a possibility that CXCR5/BLR1 serves as a coreceptor for HIV-2 strains in vivo.

Kanbe K ，Shimizu N ，Soda Y ，Takagishi K ，Hoshino H ... -  《VIROLOGY》

Changes in and discrepancies between cell tropisms and coreceptor uses of human immunodeficiency virus type 1 induced by single point mutations at the V3 tip of the env protein.

We examined the effect of amino acid substitutions of the GPGR (glycine-proline-glycine-arginine) tip sequence at the V3 domain of the Env protein of human immunodeficiency virus type 1 (HIV-1) on its cell tropism and coreceptor use. We changed the GPGR sequence of a T-cell line (T)- and macrophage (M)-tropic (R5-R3-X4) HIV-1 strain, GUN-1wt, to GA(alanine)GR (the resulting mutant was designated GUN-1/A), GL(leucine)GR (GUN-1/L), GP(proline)GR (GUN-1/P), GR(arginine)GR (GUN-1/R), GS(serine)GR (GUN-1/S), or GT(threonine)GR (GUN-1/T). GUN-1/A, GUN-1/S, and GUN-1/T mutants infected brain-derived cells such as a CD4-transduced glioma cell line, U87/CD4, and a brain-derived primary cell strain, BT-20/N, as well as T-cell lines in a CD4-dependent manner, although the plating of these mutants onto macrophages was inhibited. GUN-1/L, GUN-1/P, and GUN-1/R mutants showed both T- and M-tropism, but did not plate onto the brain-derived cells. A CCR3, CCR5, CCR8, or CXCR4 gene was introduced into a CD4-positive glioma cell line, NP-2/CD4, which demonstrated complete resistance to various HIV-1 strains. Not only HIV-1 strains, which were infectious to macrophages, such as GUN-1wt, GUN-1v, GUN-1/L, and GUN-1/P, but also an HIV-1 strain, GUN-1v, which was hardly infectious to macrophages, grew well in NP-2/CD4 cells expressing CCR3 or CCR5. However, the M-tropic GUN-1/R mutant could not efficiently use CCR5 nor CCR3. No point mutants, except GUN-1/L, grew well in NP-2/CD4 cells expressing CCR8. These findings indicate that the cell tropism of HIV-1 to macrophages and brain-derived cells and their use of the coreceptors were markedly, though not always concomitantly, affected by the tip sequence of the V3 domain.

Shimizu N ，Haraguchi Y ，Takeuchi Y ，Soda Y ，Kanbe K ，Hoshino H ... -  《VIROLOGY》

A formylpeptide receptor, FPRL1, acts as an efficient coreceptor for primary isolates of human immunodeficiency virus.

Shimizu N ，Tanaka A ，Mori T ，Ohtsuki T ，Hoque A ，Jinno-Oue A ，Apichartpiyakul C ，Kusagawa S ，Takebe Y ，Hoshino H ... -  《Retrovirology》