We previously reported that extraordinarily large numbers of CD4+ T lymphocytes are infected during SIV and SHIV acute infections. At day 7 PI, greater than 30% of CD4+ memory T cells systemically are infected with SIV. At the peak of an X4-tropic SHIV infection on day 10 PI, 30 to 90% of the circulating nave CD4+ T lymphocytes are productively infected. Immunophenotyping by flow cytometry and combined in situ hybridization/ immunohistochemistry revealed that the vast majority of the virus-producing CD4+ T lymphocytes collected from SHIV infected animals were not expressing activation markers. Other studies have shown that on day 12 following SIV inoculation, a majority of the viral RNA positive cells in lymph nodes and colon biopsies were not activated. The observation that the vast majority of virus producing CD4+ T cells during acute infection exhibited a resting phenotype has been extended to answer two additional questions: 1) would virus production by resting CD4+ T lymphocytes, following their isolation from acutely infected animals, continue ex vivo and 2) what was the integration status of viral DNA in non-activated virus producing CD4+ T cells in vivo? The prevailing dogma has long been that HIV is unable to integrate its viral DNA in the genome of resting CD4+ T cells. To answer the first question, negatively selected resting CD4+ T lymphocytes were purified from SIV (day 7 PI) and SHIV (day 10 PI) infected rhesus monkeys and cultivated in the presence of autologous serum and in the absence of IL-2 for 4 days. Low, but detectable amounts of progeny virus, capable of establish spreading infections in cultured macaque PBMC continued to be produced ex vivo. A modified Alu-LTR PCR approach, using pairs of outward-facing PCR primers binding to conserved regions of human and rhesus macaque Alu DNA sequences, in conjunction with a cloned cell line containing a single copy of integrated SIV DNA, were employed to analyze genomic DNA prepared from sorted and endpoint diluted nave and memory CD4+ T cells from acutely SIV and SHIV infected animals. Using this approach, high frequencies of integrated SHIV DNA in nave (12.7 to 50.8%) and memory (6.3 to 12.7%) and SIV DNA in memory (6.3%) CD4+ T cells in blood were detected during the first 10 days of the infection. The very low levels of progeny virions released from the ex vivo cultures of resting CD4+ T cells recovered from acutely SIV and SHIV infected monkeys would, at first glance, appear to be inconsistent with the high levels of plasma viremia (10e7 RNA copies/ml) contemporaneously measured in these animals. A plausible explanation of this apparent paradox is that: 1) the high frequencies of circulating, non-activated CD4+ T lymphocytes containing integrated viral DNA is reflective of the extraordinarily large number of similar productively infected cells in lymphoid tissues and/or effector sites systemically;2) the latter population produces small amounts of virus per cell;and 3) the net effect of the slow release of small quantities of progeny virions by an enormous fraction of the total CD4+ T cell population would result in high SIV or SHIV virus loads.
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