Research Article: A new system for parallel drug screening against multiple-resistant HIV mutants based on lentiviral self-inactivating (SIN) vectors and multi-colour analyses

Date Published: January 3, 2013

Publisher: BioMed Central

Author(s): Maria M Prokofjeva, Kristoffer Riecken, Pavel V Spirin, Dimitriy V Yanvarév, Arne Düsedau, Bernhard Ellinger, Boris Fehse, Carol Stocking, Vladimir S Prassolov.

http://doi.org/10.1186/1742-6405-10-1

Abstract

Despite progress in the development of combined antiretroviral therapies (cART), HIV infection remains a significant challenge for human health. Current problems of cART include multi-drug-resistant virus variants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is urgently needed.

We developed a straightforward screening approach for simultaneously evaluating the sensitivity of multiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral self-inactivating (SIN) LeGO vector technology.

We demonstrated the successful use of this approach for screening compounds against up to four HIV gag-pol variants (wild-type and three mutants) simultaneously. Importantly, the technique was adapted to Biosafety Level 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol exploited by pharmaceutical companies in a successful proof-of-concept experiment.

The technology developed here facilitates fast screening for anti-HIV activity of individual agents from large compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits screening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular principle of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the methodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs.

Partial Text

Introduction of combined antiretroviral therapy (cART) has dramatically altered the fate of HIV infected patients. In most cases, AIDS has changed from a fatal into a chronic, but manageable disease. Today, more than 25 drugs representing different classes of antiviral agents are available for combined therapy [1].

We provide here proof of principle for a novel technique that facilitates the testing of potential novel anti-HIV drugs simultaneously against different variants of the virus, as exemplary shown for gag-pol mutants. Since any compound with the potential to overcome a given escape mutation will be detected in our assay, it allows the identification of novel drugs targeting different key functions of HIV life cycle. Moreover, even molecules interfering with cellular factors restricting HIV infection would be identified in this assay. Thus drugs targeting all viral and cellular factors involved in virus entry, intracellular trafficking, reverse transcription, nuclear import, and integration can be screened. Furthermore, the protocol can be easily modified to test newly identified HIV mutants.

All molecular cloning was performed using standard techniques. A DpnI-mediated site-directed mutagenesis [12] approach was used to introduce point mutations conferring AZT resistance into the gag-pol gene [9] in the packaging vector pMDLg/pPRE [10]. Standard cell culture methods were used as outlined previously [13]. Pseudotyped lentiviral LeGO vector particles were generated as described [7]. Standard-scale transduction procedures of different target cells followed the spinoculation protocol described earlier [7]. FACS analysis of one to four simultaneous infections was performed on FACS Aria using a 405 nm laser for detection of Cerulean (457/50 filter) and a 488 nm laser for detection of eGFP (510/20 filter), Venus (550/30 filter), and tdTomato (610/20 filter) (BD Biosciences). For large-scale screening in 384-well-plates, 3000 SC1 cells per well were incubated, without centrifugation and without polybrene, in the presence of virus for 48 h. Imaging was performed using a PE Opera confocal LSM after fixation with 3% formaldehyde and nuclei staining (Hoechst33342; 1.5 μM) at 20 fold magnification. Images were analysed on single cell level using Perkin Elmer Acapella 2.0. Single-cell data of each well were averaged and used for further analysis. Two-tailed, unpaired Student’s t-test was used to evaluate significance of data groups.

B. Ellinger is an employee of the European Screening Port. This company is involved in the development and performance of large-scale drug screening assays. All other authors declare no conflict of interests.

MMP, KR, DVY, PVS performed small-scale experiments with lentiviral vectors and antiviral drugs; AD performed FACS analysis. KR and BE adapted the system to large-scale screening. BF, CS and VSP conceived and designed the experiments and drafted the manuscript. All authors read and approved the final manuscript.

 

Source:

http://doi.org/10.1186/1742-6405-10-1

 

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