Determination of coreceptor usage of human immunodeficiency virus type 1 from patient plasma samples by using a recombinant phenotypic assay

Abstract

We developed a recombinant virus technique to determine the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from plasma samples, the source expected to represent the most actively replicating virus population in infected subjects. This method is not subject to selective bias associated with virus isolation in culture, a step required for conventional tropism determination procedures. The addition of a simple subcloning step allowed semiquantitative evaluation of virus populations with a different coreceptor (CCR5 or CXCR4) usage specificity present in each plasma sample. This procedure detected mixtures of CCR5- and CXCR4-exclusive virus populations as well as dualtropic viral variants, in variable proportions. Sequence analysis of dualtropic clones indicated that changes in the V3 loop are necessary for the use of CXCR4 as a coreceptor, but the overall context of the V1-V3 region is important to preserve the capacity to use CCR5. This convenient technique can greatly assist the study of virus evolution and compartmentalization in infected individuals.

FIG. 1

Schematic representation of the TRT.

FIG. 2

Multiple alignment of deduced amino acid sequences of the V3 domain from plasma samples from patients 136 (A) and 145 (B) obtained at different time points. Sequences of the clones are aligned against the prevalent clone from the first time point. Letters on the left of each sequence correspond to letters on the phylogenetic tree in Fig. 3. The frequency of the clones with identical amino acid sequences is given in parentheses, and the net positive charge of the domain is given. Dots indicate identity and dashes represent gaps introduced to maximize alignment.

FIG. 3

Unrooted neighbor-joining tree of the V3 nucleotide sequences of virus clones from plasma samples. Branch lengths are drawn to scale. Bar, 1% nucleotide sequence distance. The number at the node indicates the proportion of support in 200 bootstrap replicates. The single-letter code corresponds to letters in Fig. 2 to allow the identification of each sequence on the tree. A number in parentheses following the letter indicates the number of clones with an identical nucleotide sequence.