Recent data suggest that Nef-mediated downmodulation of TCR-CD3 may protect SIVsmm-infected sooty mangabeys (SMs) against the loss of CD4+ T cells. However, the mechanisms underlying this protective effect remain unclear. To further assess the role of Nef in nonpathogenic SIV infection, we cloned nef alleles from 11 SIVsmm-infected SMs with high (>500) and 15 animals with low (<500) CD4+ T-cells/µl in bulk into proviral HIV-1 IRES/eGFP constructs and analyzed their effects on the phenotype, activation, and apoptosis of primary T cells. We found that not only efficient Nef-mediated downmodulation of TCR-CD3 but also of MHC-I correlated with preserved CD4+ T cell counts, as well as with high numbers of Ki67+CD4+ and CD8+CD28+ T cells and reduced CD95 expression by CD4+ T cells. Moreover, effective MHC-I downregulation correlated with low proportions of effector and high percentages of naïve and memory CD8+ T cells. We found that T cells infected with viruses expressing Nef alleles from the CD4low SM group expressed significantly higher levels of the CD69, interleukin (IL)-2 and programmed death (PD)-1 receptors than those expressing Nefs from the CD4high group. SIVsmm Nef alleles that were less active in downmodulating TCR-CD3 were also less potent in suppressing the activation of virally infected T cells and subsequent cell death. However, only nef alleles from a single animal with very low CD4+ T cell counts rendered T cells hyper-responsive to activation, similar to those of HIV-1. Our data suggest that Nef may protect the natural hosts of SIV against the loss of CD4+ T cells by at least two mechanisms: (i) downmodulation of TCR-CD3 to prevent activation-induced cell death and to suppress the induction of PD-1 that may impair T cell function and survival, and (ii) downmodulation of MHC-I to reduce CTL lysis of virally infected CD4+ T cells and/or bystander CD8+ T cell activation.

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The human immunodeficiency virus type 1 (HIV-1) Vpu accessory protein is a transmembrane protein that down regulates CD4 expression and promotes the release of new virions. We screened a human leukocytespecific yeast two-hybrid expression library to discover novel Vpu-interacting cellular proteins. The major histocompatibility complex class II (MHC II)

Isolated adult rat cardiac myocytes were subjected to anoxia and substrate deprivation for 15, 30, 60, 90, and 120 minutes and reoxygenation for 120 seconds. The supernatant and cell extract were analyzed for hydroxyl radicals (.OH) with high-performance liquid chromatography using salicylate as a trapping agent. The production of intracellular H2O2 as a possible precursor of .OH was also documented using the fluorescent probe dichlorofluorescein diacetate. The release of the cytosolic enzyme lactate dehydrogenase (LDH) and malondialdehyde (MDA) formation were used as cell injury markers. Trypan blue and horseradish peroxidase stains were used as markers for altered membrane permeability. Maximum formation of .OH was observed in myocytes subjected to 15 minutes of anoxia/reoxygenation (2.83 +/- 0.27 nmol/mg protein), at which time no injury was observed at light and ultramicroscopic levels. On the other hand, there was no correlation between the amount of .OH production and different parameters of cell injury in myocytes subjected to anoxia/reoxygenation longer than 15 minutes. Myocytes developed extensive blebbing, loss of cell membrane permeability, and ultrastructural damage. The enzyme leakage was minimal at 15 minutes (0.094 +/- 0.021 units/mg protein) and increased fivefold after 120 minutes (0.428 +/- 0.069 units/mg protein). Similarly, MDA increased from 0.78 +/- 0.14 nmol/mg protein at 15 minutes to 1.65 +/- 0.35 nmol/mg protein at 120 minutes. Incubation with 1 mM deferoxamine reduced the .OH production at all anoxic intervals, most significantly at 15 minutes, but did not decrease LDH and MDA release or provide ultrastructural preservation. However, preincubation with 2.5 microM diphenylphenylenediamine markedly reduced both LDH and MDA release and offered prominent ultrastructural protection. These results suggest that 1) myocytes were able to generate .OH endogenously; 2) maximum .OH was produced at 15 minutes after anoxic reoxygenation without compromising cell viability; 3) prolongation of the anoxic period exacerbated cell damage without parallel increase in .OH generation; 4) there was no significant production of .OH after 15 minutes of anoxia/reoxygenation with or without treatment of deferoxamine, suggesting that prolonged anoxia/reoxygenation does not induce additional .OH formation and thus mediate cell injury; and 5) it is likely that the damage to myocytes in this system was still mediated by free radicals other than .OH, as indicated by the protection by diphenylphenylenediamine against the cellular injury.