HIV-1 - Viral tropism for CCR5 and CXCR4 receptors. V3 gene sequencing for
quasispecies CCR5 and CXCR4.
HIV-1 infects cells by their tropism for the CD4 receptor and binding to one of the chemokine receptors CCR5 or CXCR4. There virus quasispecies with affinity for one of the two -monotropics- receptors, either CCR5 (R5 viruses) or the CXCR4 coreceptor (X4 viruses) and double -dualtropics- quasispecies with tropism for both receptors (X4R5 viruses). These quasispecies, besides having the indicated tropism have also been linked to the stages of infection, with the evolution of the infection and the tendency to disease progression.
CCR5 antagonist drugs, already in use (Maraviroc and vicriviroc) try to block cellular infection by HIV-1 quasispecies by using the CCR5. For this reason, it is necessary to know whether a patient has one or another of the variants, R5 or X4, because if the variant is a X4 virus, it is not a candidate for treatment with CCR5 antagonist drugs.
The procedure of reference for typing tropism of major quasispecie infecting a patient is a tropism test using phenotypic assays to determine coreceptor usage (Trofile HIV coreceptor tropism assay, PhenoSense HIV-Entry Assay, Monogram Bioscience, or TRT test test Recombinant -Tropism, Eurofins Viralliance Inc.). These tests are based on using a producing cells pseudovirus, such as 293 or 293T, which have been introduced genes for envelope glycoprotein gp120 obtained from the patient, cloned and transfected into cells together with pseudovirus cloned HIV without genes of the envelope protein for the infecting pseudovirus. Subsequently a target cells with CD4 receptors and one of the two coreceptors CCR5 or CXCR4 (U87-CD4-CCR5 and U87-CD4-CXCR4 in Trofile; U373-CD4-CCR5 and U373-CD4-CXCR4 in TRT test) are infected with the recombinant pseudovirus, to be infected based on the coreceptor present, and thus determine the quasispecie R5 or X4 existing in the patient.
Phenotypic methods (Trofile and TRT) are not available to most laboratories, and on the other hand the time required for implementation prevents obtain an immediate results for clinical use, so they have tried to find genotypic methods that could correlate with phenotypic methods.
The gp120 glycoprotein on which depends coreceptor interaction would be corresponding to V1-V2 and V3, V3 preferably. Therefore, it has been sought a genotypic method could be used to predict the tropism of existing viruses in the patient. The main problem has been to correlate the amino acid sequence derived from the V3 region of the glycoprotein gp120, with the tropism of the virus, because in the tropism not only influences the sequence of existing amino acids in the epitopes of virus interaction with CCR5 or CXCR4 receptor cell, but also the conformational arrangement of that amino acid sequence. It is now recognized that minor changes in the amino acid sequence of the V3 loop of gp120 envelope glycoprotein is sufficient to change the use of CCR5 by CXCR4. At the same time, some changes in the V1-V2 sequence could influence the use of one or other of the coreceptor.
Today it is known that the sequence of the V3 loop of gp120 envelope glycoprotein is highly polymorphic, and even 7.3% of patients who have not been treated with CCR5 antagonists (Maraviroc) have mutations that confer resistance to V3 CCR5 antagonists.
With the knowledge available, studies have investigated the correlation between the results of genotypic and phenotypic tests and have concluded that the genotypic changes that determine the amino acid sequence are not sufficient to achieve good sensitivity and specificity the results, so have established rules that allow fairly sensitivity and specificity to know the variant virus in the patient.
It now appears that the most accepted is to follow the 11/25 rule. This rule proposes that a basic amino acid (K or R -arginina- -lisina-) at position 11 or 25 of the V3 region is associated with CXCR4 tropism, whereas the presence of acidic or neutral amino acids at these positions are associated with the use of CCR5. However, only with this rule can not be fully determined the sensitivity and specificity to confer tropism.
The rule proposes that the net charge greater or equal to +5 at the V3 loop (calculated by subtracting the number of negative charges of the amino acids "D" -aspartate- and "E" -glutamate- number of loads positive (-lisina- K and R -arginina-) is associated with CXCR4 coreceptor usage, while a net charge <+ 5, is associated with CCR5.
With the combination of rule and rule 11/25 and net charge, a 93% sensitivity and a 100% specificity, with a positive predictive value of 100% and a negative predictive value of 96.7 for improving the CXCR4 coreceptor use. For other authors would be 88% sensitivity and 96% specificity.
There are several tools that can help to know fairly accurately phenotype predominant virus in a patient (R5 or X4) from the genotype obtained. In our laboratory we use algorithms PSSM (Position-specificc scoring matrices) and Geno2-pheno.
From the V3 sequence obtained, using bioinformatics tools such determined quite sensitive and specific tropism for the majority quasispecie and if the patient has the quasispecie R5 (CCR5) or X4 (CXCR4).
Tests in IVAMI:
- Amplification and sequencing of the genomic region encoding the V3 region of gp120 envelope glycoprotein and determinaton of ythe majority quasispecie using bioinformatic tools.
- Uncoagulated whole blood obtained with EDTA (5 mL), or plasma separated from blood are obtained with EDTA.
Preservation and shipment of sample:
Refrigerated for less than 2 days (anticoagulated blood or plasma).
Frozen (plasma separated from whole blood).
Cost of the test: