Genetic recombination is believed to assist HIV-1 diversification and escape from host immunity and antiviral therapies, yet this process remains largely unexamined within the natural target-cell populations. We developed a method for measuring HIV-1 recombination directly that employs reporter viruses bearing functional enhanced yellow fluorescent protein (YFP) and enhanced cyan fluorescent protein (CFP) genes in which recombination produces a modified GFP gene and GFP fluorescence in the infected cells. These reporter viruses allow simultaneous quantification of the dynamics of HIV-1 infection, coinfection, and recombination in cell culture and in animal models by flow-cytometric analysis. Multiround infection assays revealed that productive cellular coinfection was subject to little functional inhibition. As a result, generation of recombinants proceeded according to the square of the infection rate during HIV-1 replication in T lymphocytes and within human thymic grafts in severe combined immunodeficient (SCID)-hu (Thy/Liv) mice. These results suggest that increases in viral load may confer a compounding risk of virus escape by means of recombinational diversification. A single round of replication in T lymphocytes in culture generated an average of nine recombination events per virus, and infection of macrophages led to ≈30 crossover events, making HIV-1 up to an order of magnitude more recombinogenic than recognized previously and demonstrating that the infected cell exerts a profound influence on the frequency of recombination

Immunogenic, broadly reactive epitopes of the HIV-1 envelope glycoprotein could serve as important targets of the adaptive humoral immune response in natural infection and, potentially, as components of an acquired immune deficiency syndrome vaccine. However, variability in exposed epitopes and a combination of highly effective envelope-cloaking strategies have made the identification of such epitopes problematic. Here, we show that the chemokine coreceptor binding site of HIV-1 from clade A, B, C, D, F, G, and H and circulating recombinant form (CRF)01, CRF02, and CRF11, elicits high titers of CD4-induced (CD4i) antibody during natural human infection and that these antibodies bind and neutralize viruses as divergent as HIV-2 in the presence of soluble CD4 (sCD4). 178 out of 189 (94%) HIV-1–infected patients had CD4i antibodies that neutralized sCD4-pretreated HIV-2 in titers (50% inhibitory concentration) as high as 1:143,000. CD4i monoclonal antibodies elicited by HIV-1 infection also neutralized HIV-2 pretreated with sCD4, and polyclonal antibodies from HIV-1–infected humans competed specifically with such monoclonal antibodies for binding. In vivo, variants of HIV-1 with spontaneously exposed coreceptor binding surfaces were detected in human plasma; these viruses were neutralized directly by CD4i antibodies. Despite remarkable evolutionary diversity among primate lentiviruses, functional constraints on receptor binding create opportunities for broad humoral immune recognition, which in turn serves to constrain the viral quasispecies.

The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replicationcompetent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.

In human immunodeficiency virus (HIV)-positive individuals, the vast majority of infected peripheral blood cells and lymph node cells may be latently or nonproductively infected. The vpr open reading frame of HIV-1 encodes a 15-kDa virion-associated protein, Vpr. The vpr gene has been shown to increase virus replication in T cells and monocyte/macrophages in vitro. We have previously reported that vpr expression in various tumor lines leads to growth inhibition and differentiation, indicating that Vpr may function as a regulator of cellular permissiveness to HIV replication. Here we show that Vpr protein is present in significant amounts in the serum of AIDS patients. Purified serum Vpr activated virus expression from five latently infected cell lines, U1, OM.10.1, ACH-2, J1.1, and LL58. Serum Vpr also activated virus expression from resting peripheral blood mononuclear cells of HIV-infected individuals. Together, these findings implicate serum Vpr in the activation of HIV replication in vivo and in the control of latency. Anti-Vpr antibodies inhibited Vpr activity, suggesting that humoral immunity modulates Vpr activity in vivo. These results have broad implications for the virus life cycle and for the prospective control of HIV replication and pathogenesis.