SCS. CD4⁺ T cells and monocytes from HIV-1–infected patients were sorted by FACS into PCR plates at dilutions selected so that each well would contain an average of much less than one infected cell. The cells in each well were lysed and their DNA distributed over 10 wells. PCR using primers spanning a 1.3-kb fragment of HIV-1 DNA encompassing part of the gag-pol region was performed, and the products were detected by gel electrophoresis and sequenced. The number of viral DNA molecules per infected cell was estimated from the number of positive wells. Viral DNA amplicons were sequenced and aligned to pNL43. The relatedness of viral DNA sequences to one another and to single-genome sequences derived from contemporaneous plasma virus RNA was determined by phylogenetic analysis.

Frequency of infection of CD4⁺ T cells from peripheral blood in early (n = 5) and chronic infection (n = 4) vs. plasma viral RNA level. Frequency of infection for each patient and time point was calculated using two methods. The first method assumed that each observed HIV-1 amplicon represented a unique infected cell, corresponding to an assumption that no multiple infection was present. The second method assumed that any row with multiple proviruses represented only one infected cell, corresponding to an assumption that the maximum possible rate of multiple infection was present. Frequency of infection is shown as total CD4⁺ T cells per HIV DNA molecule. The frequency of infection with 95% confidence interval in early and chronic infection was compared, as well as the correlation between frequency of infection and plasma viral RNA levels within the two groups. The correlation analysis is represented as Spearman r values in the figure.

Phylogenetic analysis of viral sequences from cells and plasma. Maximum likelihood trees of plasma (open circles) and cell-derived sequences (filled circles) from early HIV infection (patient 2) (A), chronic HIV infection (patient 7, time point 1) (B), and patient 9, the longest-infected patient (C). All trees are rooted to pNL43 (open square). Colored triangles denote sequences from different wells in the same row.

Genetic diversity of HIV-1 in CD4⁺ T cells and plasma during early (n = 5) and chronic (n = 4) stages of infection. Diversity was measured as APD and shown as percent differences. Symbols in gray represent values from early infection, and symbols in black represent values from chronic infection. Intrapatient genetic diversity of HIV-1 DNA from CD4⁺ T cells vs. RNA from plasma and the combined interpopulation APD of cells and plasma during early (A) and chronic infection (B). Comparison of HIV-1 diversity in CD4⁺ T cells (C) and plasma (D) collected from patients during early vs. the two different time points for chronic infection. Statistical analysis was conducted using the Mann–Whitney test.