SummaryConsiderable controversy and uncertainty have surrounded the biological function of the Human Immunodeficiency Virus (HIV)-I nef gene product. Initial studies suggested that this early, nonstructural viral protein functioned as a negative regulatory factor; thus, it was proposed to play a role in establishing or maintaining viral latency. In contrast, studies in Simian Immunodeficiency Virus (SIV)mac-infected rhesus monkeys have suggested that Nef is not a negative factor but rather plays a central role in promoting high-level viral replication and is required for viral pathogenesis in vivo. We sought to define a tissue culture system that would approximate the in vivo setting for virus infection in order to assess the role of HIV-1 Nef in viral replication. We show that infection of mitogen-activated peripheral blood mononuclear cells (PBMC) with Nef + HIV results in enhanced replication as evidenced by earlier gag p24 expression when compared with infections performed with nefmutant viruses. Moreover, when unstimulated freshly isolated PBMC are infected with Nef + and Nef-viruses and then subsequently activated with mitogen, the Nef-induced difference in viral replication kinetics is even more pronounced, with the Nef-viruses requiring much more time in culture for appreciable growth. A positive effect of Nef on viral replication was also observed in primary macrophages infected with a recombinant of YU-2, a patient-derived molecular clone with macrophage tropism. These positive effects of Nef on viral replication are dependent on the initial multiplicity of infection (MOI), in that infections of unstimulated PBMC at low MOI are most dependent upon intact neffor subsequent viral growth. We now provide evidence that the Nef § HIV is more infectious than Nef-HIV from both a tissue culture infectious dose analysis, and a single-cell HIV infection assay. In the latter case, we demonstrate that infection with equivalent doses of HIV based on virion-associated gag p24 yields five-to sixfold more infected cells if Nef + viral stocks were used. Furthermore, we find that the differential infectivity is not dependent on CD4 down-regulation as Nef + virus produced from transfected COS cells lacking CD4 is also more infectious. However, normalization of PBMC infections to equivalent infectivity between that of the Nef + and Nef-viruses continues to reveal delayed viral replication in the absence of Nef, suggesting that secondary viral spread in PBMC is also enhanced in Nef + infections. We demonstrate this directly by showing a 13-15-fold increase in infectivity of PBMCderived Nef + HIV. In summary, these findings demonstrate a consistent positive role for the HIV-1 nef gene in promoting viral infection and replication, and suggest that the basis for this phenotype is the increased infectivity of HIV produced from cells expressing nef. These data suggest that HIV-1 Nef, as previously shown for SIV Nef, may play an important role in establishing a fulminant form of viral infection in vivo.

GLQ223 is a highly purified, formulated preparation of trichosanthin, a 26-kDa plant-derived ribosome-inactivating protein with potent inhibitory activity against human immunodeficiency virus (HIV) in vitro. The compound produced concentration-dependent inhibition of HIV replication in acutely infected cultures of T-lymphoblastoid cells (VB cell line). Treatment with GLQ223 selectively reduced levels of detectable viral proteins compared to total cellular protein synthesis and produced a selective decrease in levels of viral RNA relative to total cellular RNA in acutely infected cells. Substantial inhibition of viral replication was observed at concentrations of GLQ223 that showed little inhibition of parallel uninfected cultures. Selective anti-HIV activity was also observed in cultures of primary monocyte/macrophages chronically infected with HIV in vitro. When freshly drawn blood samples from HIV-infected patients were treated with a single 3-hr exposure to GLQ223. HIV replication was blocked for at least 5 days in subsequently cultured monocyte/macrophages, without further treatment. The anti-HIV activity of GLQ223 in both acutely and chronically infected cells and its activity in cells of both lymphoid and mononuclear phagocytic lineage make it an interesting candidate as a potential therapeutic agent in HIV infection and AIDS.

Baboons, rhesus monkeys, and chimpanzees were injected with the human immunodeficiency virus (HIV) and monitored for up to 4 years. Various immunosuppressive regimens were used during this time in attempts to induce development of the acquired immune deficiency syndrome (AIDS). No infectious virus was recovered or anti-HIV antibodies detected in the baboons and rhesus monkeys. Virus has been recovered from lymphocyte cultures of all five of the chimpanzees at intermittent periods following inoculation. The chimpanzees developed anti-HIV antibodies from 1 to 5 months after virus inoculation and had circulating antibodies that neutralized HIV. All the infected animals were capable of in vitro lymphocyte blastogenic responses to recombinant envelope and core HIV antigens. Despite immunosuppressive therapies and evidence of some immunologic abnormalities, none of the five chimpanzees has yet developed AIDS or a related disorder.

GLQ223 is a formulated version of tricosanthin, a single-chain ribosome-inactivating protein that was shown in earlier studies to inhibit human immunodeficiency virus (HIV) replication in T-lymphoblastoid cells and to decrease HIV p24 levels in HIV-infected monocyte-derived macrophages as measured by flow cytometry. The current studies were performed to test the selectivity of the observed inhibitory effects on HIV replication in chronically infected macrophages infected in vitro. Peripheral blood-derived monocyte/macrophages were infected in vitro and cultivated in suspension for at least two weeks prior to GLQ223 treatment. Anti-HIV effects were quantitated by measurement of cytoplasmic HIV p24, by both enzyme-linked immunosorbent assay (ELISA) and flow cytometry and HIV RNA levels were measured by slot blot analysis. Incorporation of [3H]leucine into trichloroacetic acid- (TCA) precipitable protein was also evaluated as an index of nonspecific inhibitory effects mediated by the compound in infected and uninfected cultures. Five days after a single 3-h treatment with GLQ223 there was a concentration-dependent decrease in all measurable HIV parameters within infected cultures. The anti-HIV effects persisted at least 28 days without evidence for increasing HIV expression. GLQ223 treatment of parallel uninfected macrophage cultures showed no significant inhibition of tritiated leucine uptake. These experiments demonstrate that a single pulsed exposure with GLQ223 of macrophages infected with HIV in vitro caused a sustained, concentration-dependent decrease in both HIV p24 antigen levels as well as HIV RNA without causing measurable toxicity in uninfected cultures.

The soluble human transferrin receptor (TfR) found in blood is the result of a proteolytic cleavage occurring in the ectodomain of the receptor close to the transmembrane domain at Arg-100. We have discovered another cleavage site between Gly-91 and Val-92 even closer to the transmembrane domain. Cleavage at Gly-91 differs markedly from the normal cleavage site. It occurs when the entire cytoplasmic portion or the proximal 31 amino acids of the transmembrane domain are deleted. A soluble disulfide-bonded dimer of the TfR is released into the medium in contrast to the cleavage at Arg-100 where a dimer lacking intersubunit disulfide bonds is released. Whereas the cleavage at Arg-100 is generated by cycling through the endosomal system, pulse-chase experiments indicate that cleavage at Gly-91 occurs predominantly during the biosynthesis of the receptor. Pulse-chase analysis of the biosynthesis of mutant TfRs that lack the membrane-proximal cytoplasmic domain show that they exit the endoglycosidase H-sensitive compartment at a slower rate than the wild type TfR. These results suggest that the cytoplasmic domain influences the trafficking of the TfR either by influencing the folding of the ectodomain or by providing a positive signal for its transport through the biosynthetic pathway.