Novel reporter T-cell line highly susceptible to both CCR5- and CXCR4-using human immunodeficiency virus type 1 and its application to drug susceptibility tests

Abstract

CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) is a major viral population that is transmitted by sexual intercourse and that replicates in infected individuals during the asymptomatic stage of HIV-1 infection, suggesting that agents effective against R5 HIV-1 can be expected to prevent viral transmission and delay disease progression. However, R5 HIV-1 is unable to replicate in human T-cell lines, which is an apparent obstacle to efficient and reliable susceptibility tests of compounds for their activities against R5 HIV-1. To establish a simple and rapid assay system for the monitoring of R5 HIV-1 replication and drug susceptibility, we have established a novel reporter T-cell line, MOCHA (which represents MOLT-4 cells stably expressing CCR5 and carrying the HIV-1 long terminal repeat-driven secretory alkaline phosphatase). Cells of this cell line express CD4, CXCR4, and CCR5 on their surfaces and secrete human placental alkaline phosphatase into the culture supernatants during HIV-1 infection. MOCHA cells proved to be highly permissive for the replication of R5 HIV-1 as well as CXCR4-using (X4) HIV-1, and the alkaline phosphatase activity increased in parallel with increasing HIV-1 p24 antigen levels in the culture supernatants. When HIV-1 reverse transcriptase inhibitors, protease inhibitors, and entry inhibitors, including the CCR5 antagonist TAK-779 and the CXCR4 antagonist AMD3100, were examined for their inhibitory effects on R5 and X4 HIV-1 replication in MOCHA cells, the antiviral activities of these compounds were found to be almost identical to those previously reported in peripheral blood mononuclear cells. Thus, MOCHA cells are an extremely useful tool for detection of R5 and X4 HIV-1 replication and drug susceptibility tests.

FIG. 1.

Schematic presentation of the LTR-SEAP plasmid and MOCHA cells. (A) The LTR (positions −138 to +82 in reference to the transcriptional start site at position +1) from HIV-1_IIIB was cloned into the cloning site of the pSEAP2 basic reporter plasmid, as described in Materials and Methods. (B) MOCHA cells expressing CD4, CXCR4, and CCR5 molecules are permissive for R5 HIV-1 as well as X4 HIV-1. After HIV-1 infection, the viral activator Tat induces HIV-1 LTR-regulated SEAP reporter expression, which is detectable in the culture supernatants. The LTR contains two NF-κB and three Sp1 binding sites as well as the TATA box and the transactivation responsive element (TAR). SV40, simian virus 40.

FIG. 2.

Surface expression of CCR5, CXCR4, and CD4 in MOLT-4, MOLT-4/CCR5, and MOCHA cells. The cells were stained with anti-CD4, anti-CCR5, and anti-CXCR4 MAbs (solid line) or isotype-matched control MAbs (dotted line) and analyzed with a fluorescence-activated cell sorter, as described in Materials and Methods.

FIG. 3.

HIV-1 replication in MOLT-4/CCR5 and MOCHA cells. MOLT-4/CCR5 (closed diamonds) and MOCHA (open diamonds) cells were infected with HIV-1_IIIB (A) or HIV-1_Ba-L (B). The cells were cultured for 8 days after virus infection, and the p24 antigen levels in the culture supernatants were determined on days 5 to 8. The data represent the means ± standard deviations for triplicate wells.

FIG. 4.

Comparison of SEAP activities and p24 antigen levels for HIV-1-infected MOCHA cells. The cells were infected with various amounts of HIV-1_IIIB (A) or HIV-1_Ba-L (B). SEAP activities (closed diamonds) and p24 antigen levels (open diamonds) in the culture supernatants were determined on day 10 after virus infection. Detection limits were 10 pg/ml for p24 antigen and 10 RLUs for SEAP activity. The data represent the means ± standard deviations for triplicate wells.

FIG. 5.

Inhibitory effects of coreceptor antagonists on HIV-1 replication in MOCHA cells. The cells were infected with HIV-1_IIIB (A) or HIV-1_Ba-L (B) in the presence of various concentrations of TAK-779 (closed diamonds) and AMD3100 (open diamonds). The SEAP activities in the culture supernatants were determined on day 10 after virus infection. The data represent the means ± standard deviations for triplicate wells.