Real-time PCR assay of individual human immunodeficiency virus type 1 variants in coinfected human lymphoid tissues.
In the course of infection, human immunodeficiency virus type 1 (HIV-1) mutates, diverging into a "swarm" of viral quasispecies, and the predominance of CCR5- or CXCR4-utilizing quasispecies is strongly associated with the pattern of disease progression. Quantification of CCR5- and CXCR4-utilizing viruses in viral swarms is important in the investigation of the mechanisms of this phenomenon. Here, we report on a new real-time PCR-based methodology for the evaluation of replication of individual CCR5- and CXCR4-utilizing variants. The assay is highly reproducible, with a coefficient of variation of <3%, and it accurately estimates the numbers of virus-specific RNA copies even when their difference in the mixture is 2 orders of magnitude. We demonstrate that replications of CCR5- and CXCR4-utilizing variants can be evaluated and distinguished in experimentally coinfected human lymphoid tissue. The assay we developed may facilitate study of the mechanisms of the R5-to-X4 switch in viral swarms in human tissues infected with HIV-1.
Ito
Yoshinori
Y
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Grivel
Jean-Charles
JC
Margolis
Leonid
L
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D014644:Genetic Variation; D016655:HIV Core Protein p24 / Q000032:analysis; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008221:Lymphoid Tissue / Q000821:virology; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D019713:Receptors, CCR5 / Q000502:physiology; D019718:Receptors, CXCR4 / Q000502:physiology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral; D016655:HIV Core Protein p24; D019713:Receptors, CCR5; D019718:Receptors, CXCR4