Document Detail

Prediction of HIV-1 coreceptor usage (tropism) by sequence analysis using a genotypic approach.
MedLine Citation:
PMID:  22157596     Owner:  NLM     Status:  MEDLINE    
Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 2011. For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3). This region carries enough information to perform a reliable prediction. The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL < 1000 copies/μl roughly increased in the last years (Fig. 3). The main steps for tropism testing (Fig. 4) demonstrated in this video: Collection of a blood sample Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells Amplification of the env region Amplification of the V3 region Sequence reaction of the V3 amplicon Purification of the sequencing samples Sequencing the purified samples Sequence editing Sequencing data interpretation and tropism prediction.
Saleta Sierra; Rolf Kaiser; Nadine Lübke; Alexander Thielen; Eugen Schuelter; Eva Heger; Martin Däumer; Stefan Reuter; Stefan Esser; Gerd Fätkenheuer; Herbert Pfister; Mark Oette; Thomas Lengauer
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Video-Audio Media     Date:  2011-12-01
Journal Detail:
Title:  Journal of visualized experiments : JoVE     Volume:  -     ISSN:  1940-087X     ISO Abbreviation:  J Vis Exp     Publication Date:  2011  
Date Detail:
Created Date:  2011-12-14     Completed Date:  2012-02-10     Revised Date:  2013-12-09    
Medline Journal Info:
Nlm Unique ID:  101313252     Medline TA:  J Vis Exp     Country:  United States    
Other Details:
Languages:  eng     Pagination:  -     Citation Subset:  IM    
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MeSH Terms
DNA, Viral / blood,  chemistry,  genetics,  isolation & purification
Genotyping Techniques / methods*
HIV Envelope Protein gp120 / chemistry,  genetics
HIV Infections / blood,  virology
HIV-1 / genetics,  metabolism,  physiology*
Oligonucleotide Array Sequence Analysis
Peptide Fragments / chemistry,  genetics
Polymerase Chain Reaction / methods
RNA, Viral / blood,  chemistry,  genetics,  isolation & purification
Receptors, CCR5 / metabolism
Receptors, CXCR4 / metabolism
Tropism / genetics,  physiology*
env Gene Products, Human Immunodeficiency Virus / chemistry,  genetics
Reg. No./Substance:
0/DNA, Viral; 0/HIV Envelope Protein gp120; 0/HIV envelope protein gp120 (305-321); 0/Peptide Fragments; 0/RNA, Viral; 0/Receptors, CCR5; 0/Receptors, CXCR4; 0/env Gene Products, Human Immunodeficiency Virus

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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