Deletion of naive CD8 T cells requires persistent antigen and is not programmed by an initial signal from the tolerogenic APC

Activation of naive CD8 T cells in vivo requires the recognition of cognate peptide-MHC complexes on APCs. Depending upon the activation status of the APC, such recognition will promote either a vigorous immune response or T cell tolerance and deletion. Recent studies suggest that the initial signals provided by APCs are sufficient to program the proliferation of naive CD8 T cells and their differentiation into effector cells. In this study, we sought to determine whether an initial encounter with tolerogenic APCs was sufficient to program deletion of naive CD8 T cells. Surprisingly, we find that regardless of whether naive CD8 T cells were stimulated by activated or quiescent APCs, transfer of the activated T cells into an Ag-free host was sufficient to ensure survival. Thus, although the extent of clonal expansion and development of effector function is determined by the activation status of the stimulatory APC, peripheral clonal deletion requires persistent Ag and is not determined by the initial stimulatory event.     

Microarray profiling of antibody responses against simian-human immunodeficiency virus: postchallenge convergence of reactivities independent of host histocompatibility type and vaccine regimen

We developed antigen microarrays to profile the breadth, strength, and kinetics of epitope-specific antiviral antibody responses in vaccine trials with a simian-human immunodeficiency virus (SHIV) model for human immunodeficiency virus (HIV) infection. These arrays contained 430 distinct proteins and overlapping peptides spanning the SHIV proteome. In macaques vaccinated with three different DNA and/or recombinant modified vaccinia virus Ankara (rMVA) vaccines encoding Gag-Pol or Gag-Pol-Env, these arrays distinguished vaccinated from challenged macaques, identified three novel viral epitopes, and predicted survival. Following viral challenge, anti-SHIV antibody responses ultimately converged to target eight immunodominant B-cell regions in Env regardless of vaccine regimen, host histocompatibility type, and divergent T-cell specificities. After challenge, responses to nonimmunodominant epitopes were transient, while responses to dominant epitopes were gained. These data suggest that the functional diversity of anti-SHIV B-cell responses is highly limited in the presence of persisting antigen.     

Role of nonstructural proteins 3A and 3B in host range and pathogenicity of foot-and-mouth disease virus

The genome of foot-and-mouth disease virus (FMDV) differs from that of other picornaviruses in that it encodes a larger 3A protein (>50% longer than poliovirus 3A), as well as three copies of protein 3B (also known as VPg). Previous studies have shown that a deletion of amino acids 93 to 102 of the 153-codon 3A protein is associated with an inability of a Taiwanese strain of FMDV (O/TAW/97) to cause disease in bovines. Recently, an Asian virus with a second 3A deletion (amino acids 133 to 143) has also been detected (N. J. Knowles et al., J. Virol. 75:1551-1556, 2001). Genetically engineered viruses harboring the amino acids 93 to 102 or 133 to 143 grew well in porcine cells but replicated poorly in bovine cells, whereas a genetically engineered derivative of the O/TAW/97 virus expressing a full-length 3A (strain A12) grew well in both cell types. Interestingly, a virus with a deletion spanning amino acid 93 to 144 also grew well in porcine cells and caused disease in swine. Further, genetically engineered viruses containing only a single copy of VPg were readily recovered with the full-length 3A, the deleted 3A (amino acids 93 to 102), or the "super" deleted forms of 3A (missing amino acids 93 to 144). All of the single-VPg viruses were attenuated in porcine cells and replicated poorly in bovine cells. The single-VPg viruses produced a mild disease in swine, indicating that the VPg copy number is an important determinant of host range and virulence. The association of VPg copy number with increased virulence in vivo may help to explain why all naturally occurring FMDVs have retained three copies of VPg.     

Reversible oxidative modification as a mechanism for regulating retroviral protease dimerization and activation

Human immunodeficiency virus protease activity can be regulated by reversible oxidation of a sulfur-containing amino acid at the dimer interface. We show here that oxidation of this amino acid in human immunodeficiency virus type 1 protease prevents dimer formation. Moreover, we show that human T-cell leukemia virus type 1 protease can be similarly regulated through reversible glutathionylation of its two conserved cysteine residues. Based on the known three-dimensional structures and multiple sequence alignments of retroviral proteases, it is predicted that the majority of retroviral proteases have sulfur-containing amino acids at the dimer interface. The regulation of protease activity by the modification of a sulfur-containing amino acid at the dimer interface may be a conserved mechanism among the majority of retroviruses.     

Regulatory role of arginine 204 in the catalytic activity of rat alloantigens ART2a and ART2b

ART2a (RT6.1) and ART2b (RT6.2) are NAD glycohydrolases (NADases) that are linked to T lymphocytes by glycosylphosphatidylinositol anchors. Although both mature proteins possess three conserved regions (I, II, III) that form the NAD-binding site and differ by only ten amino acids, only ART2b is auto-ADP-ribosylated and only ART2a is glycosylated. To investigate the structural basis for these differences, wild-type and mutant ART2a and ART2b were expressed in rat mammary adenocarcinoma (NMU) cells and released with phosphatidylinositol-specific phospholipase C. All mutants were immunoreactive NADases. Arginine 204 (Arg204), NH2-terminal to essential glutamate 209 in Region III, is found in ART2b, but not ART2a. Replacement of Arg204 in ART2b with lysine, tyrosine, or glutamate abolished auto-ADP-ribosylation. Unlike wild-type ART2a, ART2a(Y204R) was auto-ADP-ribosylated. The tryptophan mutant ART2b(R204W) was auto-ADP-ribosylated and exhibited enhanced NADase activity. Incubation with NAD and auto-ADP-ribosylation decreased the NADase activities of wild-type ART2b and ART2b (R204W), whereas activity of ART2b(R204K), which is not auto-modified, was unchanged by NAD. Facilitation of auto-ADP-ribosylation by tryptophan 204 suggests that the hydrophobic amino acid mimics an ADP-ribosylated arginine. Thus, Arg204 in ART2b serves as a regulatory switch whose presence is required for additional auto-ADP-ribosylation and regulation of catalytic activity.     

The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.     

Allosteric alpha 1-adrenoreceptor antagonism by the conopeptide rho-TIA

A peptide contained in the venom of the predatory marine snail Conus tulipa, rho-TIA, has previously been shown to possess alpha1-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, rho-TIA inhibited alpha1-adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic alpha2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, rho-TIA displayed slightly greater potency at the alpha 1B than at the alpha 1A or alpha 1D subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the alpha 1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by rho-TIA. N-terminally truncated analogs of rho-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of rho-TIA (Arg4). An alanine walk of rho-TIA confirmed the importance of Arg4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of rho-TIA. The unique allosteric antagonism of rho-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive alpha1-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.     

Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase

The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.     

Functional activity of the complement regulator encoded by Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is closely associated with Kaposi's sarcoma and certain B-cell lymphomas. The fourth open reading frame of the KSHV genome encodes a protein (KSHV complement control protein (KCP, previously termed ORF4)) predicted to have complement-regulating activity. Here, we show that soluble KCP strongly enhanced the decay of classical C3-convertase but not the alternative pathway C3-convertase, when compared with the host complement regulators: factor H, C4b-binding protein, and decay-accelerating factor. The equilibrium affinity constant (KD) of KCP for C3b and C4b was determined by surface plasmon resonance analysis to range between 0.47-10 microM and 0.025-6.1 microM, respectively, depending on NaCl concentration and cation presence. Soluble and cell-associated KCP acted as a cofactor for factor I (FI)-mediated cleavage of both C4b and C3b and induced the cleavage products C4d and iC3b, respectively. In the presence of KCP, FI further cleaved iC3b to C3d, which has never been described before as complement receptor 1 only mediates the production of C3dg by FI. KCP would enhance virus pathogenesis through evading complement attack, opsonization, and anaphylaxis but may also aid in targeting KSHV to one of its host reservoirs since C3d is a ligand for complement receptor 2 on B-cells.