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Journal of Clinical Microbiology, February 2008, p. 792-795, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.01470-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Dual-Reporter Phenotypic Assay for Human Immunodeficiency Viruses{triangledown}

Keiko Kajiwara, Eiichi Kodama,* Yasuko Sakagami, Takeshi Naito, and Masao Matsuoka

Laboratory of Virus Immunology, Institute for Virus Research, Kyoto University, 53 Kawaramachi, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan

Received 22 July 2007/ Returned for modification 27 September 2007/ Accepted 5 December 2007


   ABSTRACT
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 ABSTRACT
TEXT
 REFERENCES
 
We have established a novel human immunodeficiency virus (HIV) tandem-reporter assay using HIV receptor-transduced NP-2 cells with long terminal repeat-controlled β-galactosidase, inserted internal ribosome entry site, and secretary alkaline phosphatase genes. This assay allows users to detect replication of clinical isolates, indicating its useful application as an HIV phenotypic assay.


   TEXT
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Assays for detecting the emergence of resistant variants, as well as evaluating clinical efficacy, provide useful information regarding chemotherapy for human immunodeficiency virus (HIV) infection. To date, two types of assay systems have been developed and approved, namely, genotypic and phenotypic assays (13, 31). Genotypic assays detect genetic mutations that are associated with drug resistance and lead to rapid and sensitive detection of the emergence of resistant variants (9), although they only provide estimated resistance profiles (16, 30). Lists of significant resistance-associated mutations in reverse transcriptase (RT), protease, and envelope genes are maintained by some organizations and Universities, such as the International AIDS Society-USA (http://iasusa.org/resistance mutations); Los Alamos National Laboratory, Los Alamos, NM (http://resdb.lanl.gov/Resist_DB); and the Stanford University, Stanford, CA (http://hivdb.stanford.edu/index.html). PCR-based genotypic assays are heavily dependent on the primers used. Therefore, some biases must unfortunately be presumed, since most primer-matched sequences are preferentially amplified, resulting in some discordance with phenotypic assays (21, 33) that are time-consuming and require tedious procedures because isolation of replication-competent viruses is required. To date, phenotypic assays for clinical isolates have been mainly performed in experiments with a p24 production assay in phytohemagglutinin-stimulated peripheral blood mononuclear cells (27, 32, 34).

For more rapid and simple phenotypic assays, recombinant viruses containing the region responsible for resistance have been utilized instead of isolated viruses (14, 18, 35). However, since protease resistance mutations are introduced simultaneously with gag mutations (4, 25), cloning of the entire gag and protease coding region is occasionally required. Recently, mutations for 3'-azido-3'-deoxythymidine (AZT) resistance have been identified in the connection and/or RNase H subdomain (5, 7, 26), which no commercially available genotypic and phenotypic assays include for the analysis. Moreover, the mechanism of resistance to a fusion inhibitor, enfuvirtide, is a complex issue, since mutations in not only the gp41 coding region (6, 24) but also the V3 region (29) and the CD4-binding site (2) of gp120 influence the susceptibility, indicating that patient-derived viruses are ideally required for evaluation of drug susceptibility.

Recently, Hachiya et al. established a simple and rapid phenotypic assay using MAGIC5 cells (CCR5-transduced MAGI cells) (11). This system efficiently isolates clinical HIV variants and has proven to be useful for evaluating drug susceptibility (12). However, the expression of transduced receptors on MAGIC5 cells declines during prolonged culture, as described for MAGI cells (17). Therefore, in order to obtain HIV isolates efficiently and perform the assay, relatively fresh cells are required. More recently, we established a tetrazolium-based colorimetric assay for monitoring replications of not only CXCR4 (X4)-tropic but also CCR5 (R5)-tropic HIVs and drug susceptibilities (17). We reported that NCK45 cells stably express HIV receptors on their cell surface and provide reproducible results (17). Since this assay depends on the cytopathic effect induced by HIV, it appears to be insufficient for assessing infections with no or a few cytopathic variants. Furthermore, it requires 7 days of culture to obtain the drug susceptibility. In the present study, we have established a novel single long terminal repeat (LTR)-driven tandem two-reporter system using the internal ribosome entry site (IRES) (15), which enables the evaluation of drug susceptibility within 2 days for various HIVs, including clinical isolates.

To construct an LTR-driven reporter vector, an amplified LTR region (the –138 to +89 region of the transcriptional start site of HIV-1 molecular clone pNL4-3) was inserted into pβgal-Basic (Clontech Laboratories, Inc., Palo Alto, CA) between the NheI and HindIII sites (pLTR-βgal). The 5' region (HindIII to EcoRV) of the β-galactosidase gene was replaced with the responsible β-galactosidase fragment with nuclear localization signal sequence (MPKKKRK) amplified from genomic DNA of MAGI cells (20). Fragments of IRES and secretory alkaline phosphatase (SEAP) were amplified from pIRES2-EGFP and pSEAP2-Basic (Clontech Laboratories, Inc.), respectively. A puromycin-resistance gene (Puror) under the control of the phosphoglycerate kinase promoter as a selection marker was inserted at the SalI site of the vector (pLTR-β-Gal/SEAP-Puror), as shown in Fig. 1A. All fragments were verified by sequencing.


Figure 1
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  		FIG. 1. Establishment of a cell line with β-galactosidase (β-Gal) and SEAP genes driven by an LTR. (A) Schematic diagram of the vector used in the present study, which simultaneously expresses genes for β-Gal and SEAP under the control of the HIV-1 LTR promoter (pLTR-β-Gal/SEAP-Puror). The enhancer region (positions –138 to +89) of the LTR, the nuclear localization signal (NLS) derived from the T-antigen of simian virus 40, the IRES, the phosphoglycerate kinase promoter (PGK), and the polyadenylation signal (pA) are also shown. (B) Microscopic image of X-Gal-stained NCK45-β-Gal/SEAP cells at 48 h after virus inoculation. (C) Correlation between β-Gal and SEAP activities in culture supernatants. NCK45-β-Gal/SEAP cells were infected with HIV at various infectious doses and incubated for 48 h. Culture supernatants were examined for their SEAP activities and expressed as relative right units (RLU). BFU, blue-cell-forming units.

 
The pLTR-β-Gal/SEAP-Puror plasmid was transfected into NCK45 cells (CD4, CXCR4, and CCR5-transduced NP-2 cells derived from a glioma) (17, 36) to detect intracellular Tat expression through the LTR-driven tandem reporter genes. At 48 h after transfection, the cells were cultured in Dulbecco modified Eagle medium (Sigma, St. Louis, MO) supplemented with 5% heat-inactivated fetal calf serum, 0.5 mg of G418 disulfate (Nacalai Tesque, Kyoto, Japan)/ml, 0.2 mg of hygromycin B (Calbiochem, La Jolla, CA)/ml, and 10 µg of puromycin (Sigma)/ml and designated NCK45-β-Gal/SEAP cells. The expression levels of receptors on NCK45 cells confirmed by a flow cytometer (17) were 97, 83, and 99%, while those on H9 cells as a control were 65, 73, and 0.3% for CD4, CXCR4, and CCR5, respectively.

To evaluate anti-HIV agents, NCK45-β-Gal/SEAP cells (5 x 104 cells/ml) in Dulbecco modified Eagle medium-based culture medium supplemented with 5% fetal calf serum, penicillin, and streptomycin were seeded onto 96-well plates. On the following day, the cells were inoculated with sample viruses at 60 blue-cell-forming units (BFU)/well, incubated for 48 h, and then cultured in the presence of various concentrations of drugs. After 48 h of culture, the cells were fixed with 1% formaldehyde and 0.2% glutaraldehyde for 3 min, washed three times with phosphate-buffered saline, and incubated with X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) for 2 h (Fig. 1B). To evaluate the SEAP activities, the culture supernatants were harvested and analyzed by using a Great EscAPe SEAP chemiluminescent detection kit (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer's protocol. Samples were measured by using a Wallac 1450 MicroBeta Jet Luminometer (Perkin-Elmer, Wellesley, MA). For comparison, MAGI-CCR5 cells were analyzed as previously described (17).

The activities of various anti-HIV agents toward NCK45-β-Gal/SEAP cells were compared to those in the MAGI assay. The BFU and SEAP activities were well correlated with the viral input (Fig. 1C; correlation coefficient R2 = 0.928). We tested various anti-HIV agents (Table 1) : DS5000, an adsorption inhibitor; AZT and 2',3'-dideoxycytidine (ddC), RT inhibitors; T-140 (37), a CXCR4 antagonist; and TAK-779, a CCR5 antagonist (1). The antiviral activities of the compounds determined by each reporter in NCK45-β-Gal/SEAP cells were comparable to those obtained using MAGI cells, although some were statistically significant (Table 1). Intracellular nucleoside/nucleotide metabolisms, especially thymidine kinase (10), and expression levels of receptors (19) may alter the 50% effective concentrations of the AZT and CCR5 antagonists, respectively.


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  		TABLE 1. Comparison of anti-HIV activities in MAGI and NCK45-β-Gal/SEAP cells

 
NCK45-β-Gal/SEAP cells also supported the replication of various clinical isolates, as well as laboratory strains. The clinical isolates used in the present study were kindly provided by Y. Maeda (Kumamoto University School of Medicine, Kumamoto, Japan) and S. Oka (AIDS Clinical Center, International Medical Center of Japan, Tokyo, Japan). The drug susceptibilities of not only R5-tropic isolates but also X4-tropic and dualtropic isolates were comparable to those in the MAGI assay (Table 2). IVR405 and IVR406 were highly resistant to AZT, whereas IVR409 and IVR416 showed moderate resistance to AZT, and KMT and IVR417 were susceptible to AZT. These susceptibilities were confirmed by the MAGI assay.


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  		TABLE 2. Drug susceptibility against HIV clinical isolates in MAGI and NCK45-β-Gal/SEAP cells

 
In the present study, we established an IRES-mediated tandem-reporter assay using NCK45 cells for the rapid and simultaneous evaluation of the antiviral activities of compounds. This assay enables the evaluation of various HIV strains and clinical isolates within 3 days, including the cell preparation procedure. Secreted SEAP from HIV-infected NCK45 cells is also useful for monitoring the isolation of viruses without cell destruction and enables the continuous propagation of the isolates during isolation. Since NCK45-β-Gal/SEAP cells are susceptible to various viruses, including clinical strains, depending on the experimental purpose any HIV variants may be used for a reference virus, e.g., an isolate prior to the therapy. The LTR promoter for the dual reporters used in the present study includes four CpG methylation-sensitive sites, although Tat can transactivate the HIV-1 LTR regardless of the methylation state (3, 28). Thus, reporter genes in NCK45 cells are less affected by gene silencing.

Compared to the commercially available phenotypic assays, our established system may be useful especially for inhibitors targeting new molecules, including envelope proteins and integrase. A fusion inhibitor T-20 can suppress variants refractory to most of approved RT and protease inhibitors (22, 23). Both phenotypic and genotypic assays for T-20 are needed to evaluate the clinical outcome of patients with T-20-containing regimens. An approved CCR5 antagonist, maraviroc, may also suppress such refractory variants (8). Needless to say, to assess the drug susceptibility experimentally and/or clinically, a combination of established databases accumulated with various assays appears to be useful and important.

In conclusion, the tandem reporters β-Gal and SEAP can evaluate the exact viral infectivity and proportional LTR activation level by repetitive analyses of culture supernatants, respectively. This IRES-mediated tandem-reporter system may be applicable to other reporter genes, e.g., the luciferase gene. Thus, our assay system may provide simple, rapid and stable results for HIV phenotypic assays.


   ACKNOWLEDGMENTS
 
We thank J. Overbaugh for providing the HeLa-CD4/CCR5-LTR/β-Gal cells through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (Bethesda, MD).

This study was supported in part by a grant for the Promotion of AIDS Research from the Ministry of Health and Welfare of Japan (to M.M. and E.K.); a grant for Research for Health Science Focusing on Drug Innovation from the Japan Health Science Foundation (to E.K.); and a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to E.K.). K.K. and T.N. are supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science, and Technology.


   FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Virus Immunology, Institute for Virus Research, Kyoto University, 53 Kawaramachi, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Phone and fax: 81-75-751-3986. E-mail: ekodama{at}virus.kyoto-u.ac.jp Back

{triangledown} Published ahead of print on 19 December 2007. Back


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Journal of Clinical Microbiology, February 2008, p. 792-795, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.01470-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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