Target cell populations of human immunodeficiency virus type 1 in peripheral blood lymphocytes with different chemokine receptors at various stages of disease progression

Abstract

We studied the distribution of human immunodeficiency virus type 1 (HIV-1) DNA in CCR5-positive and -negative peripheral blood lymphocyte populations in HIV-1-infected individuals. While HIV-1 DNA in the CCR5-positive population showed no correlation with CD4 count, the increase of total HIV-1 DNA with lower CD4 count was mainly contributed by the increase of HIV-1 DNA in the CCR5-negative population. This might indicate the change in coreceptor usage from CCR5 to CXCR4 in later stages of disease progression. However, some of the samples with a high viral DNA load in the CCR5-negative population did not have any characteristic of the V3 loop sequence that is compatible with CXCR4 usage or the syncytium-inducing (SI) phenotype. We also did not find any known characteristic change predictive of the SI phenotype in V1 and V2 sequences. Our findings showed that there might be a shift in target cell populations during disease progression, and this shift was not necessarily associated with the genetic changes characteristic of CXCR4 usage.

FIG. 1

Verification of the quantitative HIV-DNA PCR. (A) A total of 10⁴ copies of HIV DNA in 8E5 cells were coamplified with 300, 1,500, 3,000, 6,000, or 30,000 copies of the competitor as shown in lanes 2 to 6, respectively. Lane 1 is the marker containing both wild-type (115 bp) and competitor (97 bp) amplicons, and lane 7 is blank. (B) Ratios of adjusted density of the competitor and wild-type bands plotted against the copy number of the competitor.

FIG. 2

Representative CCR5 RNA analysis by RT-PCR showing the purity of the cell preparations. The size of the amplification products is 189 bp. Lanes 1 and 2 show amplification from 200,000 cells of a CCR5-negative cell preparation and 60,000 cells of a CCR5-positive preparation, respectively. CCR5-positive (98% purity) and -negative (96% purity) cells highly purified by FACS were mixed to make cell preparations with various percentages of CCR5-positive cells in lanes 4 to 8. RNA from 100,000 cells was analyzed in each of these control lanes. The numbers below the lane are intensities of the bands calculated using Gel-Pro software.

FIG. 3

Scatter plots showing the correlation between CD4 count and total HIV DNA load (in log scale) in presorted non adherent cells (A), HIV DNA load in a CCR5-negative population (B), and HIV DNA load in a CCR5-positive population (C). Nonadherent cells were separated into CCR5-positive and -negative populations by immunomagnetic separation, and the HIV DNA content was measured by a gag quantitative competitive PCR.

FIG. 4

Scatter plot showing correlation between CD4 count and the ratio of the HIV DNA in a CCR5-positive to the HIV DNA in a CCR5-negative population in log scale (A) and in linear scale (B). The HIV DNA content was measured by a gag competitive PCR in the two subpopulations, which were separated by the immunomagnetic technique.

FIG. 5

V3 loop amino acid sequence alignment. The N-linked glycosylation site and amino acid positions 11 and 25 are marked by asterisks, and the tip of the V3 loop is underlined. The net charge, the CD4 count, and the ratio of HIV DNA in a CCR5-positive population to the HIV DNA in a CCR5-negative population are provided for each sequence. A reference subtype E sequence of TH253 is shown on the top.

FIG. 6

V1V2 loop amino acid sequence alignment. The length of the hypervariable locus in V2 loop, the CD4 count, and the ratio of HIV DNA in CCR5-positive versus HIV DNA in CCR5-negative populations are provided for each sequence. A reference subtype E sequence of TH253 is shown on the top.