Background:  Determining the relationship among proviruses in cells from peripheral blood and tissue compartments is important for understanding the pathogenesis of HIV-1. Therefore, we developed a single-cell sequencing technique to examine individual HIV DNA molecules from single cells in the peripheral blood (PB) and lymph node tissue (LN).

Methods:  Memory and naïve CD4⁺ T cells from paired PB and LN samples from 5 chronically untreated HIV-infected patients (0.5 to 11 years, 3000 to 290,000 HIV RNA copies/mL) were sorted into PCR plates with each well containing, on average, far less than 1 infected cell. The cells in each well were lysed and their DNA distributed over a row of 10 wells. A 1.3-kb gag-pol fragment was amplified and sequenced using a method validated to detect a single copy of DNA. The number of viral DNA molecules per infected cell was estimated from the number of positive wells in each row. The genetic relatedness of intracellular viral DNA sequences from PB, LN, and contemporaneous plasma RNA was determined by phylogenetic analysis.

Results:  Analysis of CD4⁺ T cells from PB revealed 1 HIV DNA copy per 120 to 3600 memory T cells and 1 DNA copy per 200 to <10,000 naïve T cells. The infection frequency of memory T cells from LN from 4 of the 5 patients was 2- to 17-fold higher than from PB, whereas LN-naïve T cells had a similar frequency of infection (1 HIV DNA copy per 381 to 10,667 cells) as PB-naïve T cells (1 DNA copy per 200 to <10,000 naïve T cells ). The majority (70 to 90%) of infected memory and naïve CD4⁺ T cells from the PB and LN contained a single HIV DNA molecule and the number of wells with more than 1 copy of viral DNA was similar to that predicted by the Poisson distribution. Sequence analysis revealed that intracellular HIV DNA in memory CD4⁺ T cells from PB and LN samples and plasma RNA were phylogenetically indistinguishable in each of the patients and had similar average genetic diversity (average pair-wise distance 1.5%, 1.5%, and 1.4%, respectively).

Conclusions:  Most (70 to 90%) infected memory and naïve CD4⁺ T cells from PB and LN contain only 1 copy of HIV DNA, implying a limited potential for recombination in virus produced by these cells. The genetic similarity of HIV DNA populations in CD4⁺ T cells from PB, LN, and HIV RNA from plasma implies ongoing exchange of virus and/or infected cells between these compartments during untreated chronic HIV infection.