Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of 'covert' virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

Rac1 is a member of the Rho family of small molecular mass GTPases that act as molecular switches to control actin-based cell morphology as well as cell growth and differentiation. Rac1 Cdc42Hs/Rac -activated kinase (PAK) phosphorylates p67 phox amino acid residues adjacent to the Rac1/2-binding site, and this phosphorylation is stimulated by deletion of the C-terminal SH3 domain or the polyproline-rich motif. These data suggest a role for cryptic Rac-binding and PAK phosphorylation sites of p67 phox in control of the NADPH oxidase.Members of the Rho family, Rac1, Cdc42Hs, and RhoA, play essential roles in growth factor-mediated changes in cell morphology associated with the formation of actin microfilaments and "focal complexes" (1-4). They also act downstream of Ras in distinct parts of the transformation process (5-8) and activate Jun N-terminal protein kinase (9, 10) and entry into the G 1 phase of the cell cycle (11). Rho family interacting proteins that may play a role as effectors in these signaling pathways include kinases (12-15), GTPase-activating proteins (e.g. phox are phosphorylated during oxidase activation (27-30), and the isolation and identification of the kinases involved are essential to gain a better understanding of the mechanism by which complex formation and oxidase activity are controlled in vivo. PAK has been shown to be activated by fMet-Leu-Phe in neutrophils and to phosphorylate p47 phox in vitro (31). Hitherto, kinases that can phosphorylate p67 phox have not been identified.We have been using the NADPH oxidase as a model protein complex to investigate the mechanism by which Rho family GTPases activate cellular pathways. In this study, the Rac1-p67 phox interaction has been investigated in more detail. Using binding assays, we show that the Rac1-binding site of p67 phox is cryptic, located between amino acids 170 and 199, and that the N-terminal fragment (amino acids 1-192) can be used as an inhibitor of the Rac signaling pathway. The binding sites in p67phox for Rac1 and p40 phox are distinct. Recombinant PAK purified from Escherichia coli can phosphorylate p67 phox ; the phosphorylation site(s) are cryptic and located adjacent to the Rac1-binding site. Deletion of either the polyproline-rich sequence (aa 1 226 -236) or the C-terminal SH3 domain (aa 460 -526) led to increases in Rac1 binding and PAK phosphorylation, suggesting that there is an intramolecular interaction between these two domains of p67 phox that gives rise to the crypticity. Taken together, these data suggest that unfolding of p67 phox , via disruption of a potential intramolecular "SH3 domain-polyproline" interaction, may play a role in control of the NADPH oxidase. MATERIALS AND METHODSCell Culture and Microinjection-Swiss 3T3 fibroblast cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum and antibiotic/antimycotic (Life Technologies, Inc.) at 37°C and 5% CO 2 . Swiss 3T3 cells were serum-starved for 24 -48 h before being microinjected with p67 phox protein (0.1-1 mg/ml) and obser...

Thirty-one histologically abnormal brains from patients with AIDS were studied in order to establish the relationship between multinucleated giant cells, viral protein expression, the various forms of human immunodeficiency virus type 1 (HIV-1) DNA, and clinical evidence of dementia. Unintegrated HIV-1 DNA of 2 to 8 kb was found in 22 of the 31 brains. Multinucleated giant cells without any other pathology were found in 14 cases; unintegrated 1-long terminal repeat (1-LTR) circular forms of HIV-1 DNA and strongly positive immunohistochemistry for gp41 and p24 were found in most of these brains. Most of these patients had a clinical diagnosis of HIV-1-associated dementia and cerebral atrophy. In all the other brains studied, 1-LTR circles were absent and immunohistochemistry for gp41 and p24 was usually negative. Very few of these patients had a clinical diagnosis of dementia. Sequence comparison of the LTR region from integrated HIV-1 DNA with that from unintegrated 1-LTR circular forms of HIV-1 DNA in 12 cases showed no significant differences. A further comparison of these brain-derived LTR sequences with LTR sequences derived directly from lymphoid tissue also showed strong sequence conservation. The V3 loop of the virus from the brain was sequenced in 6 cases and had a non-syncytium inducing-macrophage-tropic genotype. Our results show that (i) although unintegrated HIV-1 DNA was present in most brains from patients with AIDS, molecular evidence of high levels of viral replication was associated with the presence of multinucleated giant cells and dementia, and that (ii) the HIV-1 LTR is not a determinant of neurotropism. These observations suggest that replication of HIV-1 and not just the presence of HIV-1 DNA within giant cells makes the important contribution to central nervous system damage.

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