Isolation of human immunodeficiency virus-type 1 (HIV-1) clones with biological and molecular properties of the primary isolate.
We developed a new biological cloning system for HIV-1 isolates using the U87.CD4 cell lines that express different chemokine receptors. We demonstrate that our method is sensitive and specific because the clones isolated had the same coreceptor usage and genotype as viruses of the primary isolate. We evaluated our cloning system by isolating 27 biological clones from two primary HIV-1 R3R5X4 isolates. Three HIV-1 phenotypes (R3R5X4, R3R5 and R5) were identified in isolate 29 and two (R3R5X4 or R5X4) in isolate 31. Each phenotype was distinguished by a unique genotype. Sequencing of 20 molecular clones from each isolate did not reveal additional genotypes. One of the three genotypes identified from isolate 29 was not found by molecular cloning of the original isolate, suggesting high specificity and sensitivity of the biological cloning system in isolating minor virus populations. Our results suggest that the new cloning approach can be used as an alternative to the existing method for isolating biological clones in PBMC.
Mild
Mattias
M
Department of Laboratory Medicine, Division of Medical Microbiology/Virology, Lund University, Sölvegatan 23, 223 62 Lund, Sweden. mattias.mild@med.lu.se
Björndal
Asa
A
Medstrand
Patrik
P
Fenyö
Eva Maria
EM
D000595:Amino Acid Sequence; D002460:Cell Line; D005838:Genotype; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D054397:Receptors, CCR3; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism; D054380:Receptors, CXCR5; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism; D018121:Receptors, Cytokine / Q000235:genetics / Q000378:metabolism
C515350:CCR3 protein, human; C095085:CXCR5 protein, human; D054397:Receptors, CCR3; D019713:Receptors, CCR5; D054380:Receptors, CXCR5; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine