HIV infection is associated with the progressive loss of CD4(+) T cells through their destruction or decreased production. A central, yet unresolved issue of HIV disease is the mechanism for this loss, and in particular whether HIV-specific CD4(+) T cells are preferentially affected. Here we show that HIV-specific memory CD4(+) T cells in infected individuals contain more HIV viral DNA than other memory CD4(+) T cells, at all stages of HIV disease. Additionally, following viral rebound during interruption of antiretroviral therapy, the frequency of HIV viral DNA in the HIV-specific pool of memory CD4(+) T cells increases to a greater extent than in memory CD4(+) T cells of other specificities. These findings show that HIV-specific CD4(+) T cells are preferentially infected by HIV in vivo. This provides a potential mechanism to explain the loss of HIV-specific CD4(+) T-cell responses, and consequently the loss of immunological control of HIV replication. Furthermore, the phenomenon of HIV specifically infecting the very cells that respond to it adds a cautionary note to the practice of structured therapy interruption.

Immune reconstitution is a critical component of recovery after treatment of human immunodeficiency virus (HIV) infection, cancer chemotherapy, and hematopoietic stem cell transplantation. The ability to enhance T-cell production would benefit such treatment. We examined the effects of exogenous interleukin-7 (IL-7) on apoptosis, proliferation, and the generation of T-cell receptor rearrangement excision circles (TRECs) in human thymus. Quantitative polymerase chain reaction demonstrated that the highest level of TRECs (14 692 copies/10 000 cells) was present in the CD1a ؉ CD3 ؊ CD4 ؉ CD8 ؉ stage in native thymus, suggesting that TREC generation occurred following the cellular division in this subpopulation. In a thymic organ culture system, exogenous IL-7 increased the TREC frequency in fetal as well as infant thymus, indicating increased T-cell receptor (TCR) rearrangement. Although this increase could be due to the effect of IL-7 to increase thymocyte proliferation and decrease apoptosis of immature CD3 ؊ cells, the in vivo experiments using NOD/LtSz-scid mice given transplants of human fetal thymus and liver suggested that IL-7 can also directly enhance TREC generation. Our results provide compelling evidence that IL-7 has a direct effect on increasing TCR-␣␤ rearrangement and indicate the potential use of IL-7 for enhancing de novo naïve T-cell generation in immunocompromised patients. ( IntroductionIt has been reported that T-cell numbers are maintained in adults predominantly through the expansion of postthymic, memory T cells, whereas in infants, T cells are predominantly maintained through the production of new naïve T cells by the thymus. 1 However, others and we have recently demonstrated that the adult thymus is still capable of thymopoiesis and can contribute to T-cell reconstitution in adults. 2,3 Several methods have been used to measure thymopoietic capacity. Thymic size as measured by radiographic imaging 1 and volumetric computed tomography measurements 4,5 have been correlated with numbers of CD4 ϩ CD45RA ϩ naïve T cells, and the number of phenotypically naïve T cells after transplantation has been shown to correlate with antigen-specific function. 6 However, there are concerns about limitations of estimating thymic function based on naïve T-cell phenotype alone. T cells expressing a naïve phenotype are not necessarily accurate surrogate markers of thymic function. Following thymic emigration, CD45RA ϩ naïve T cells can have a long quiescent life span, 7 may proliferate in an antigen-independent manner, 8 or may rapidly convert to CD45RO ϩ memory/effector phenotype T cells. 9 Furthermore, naïve T-cell markers may be acquired by memory T cells (especially CD8 ϩ T cells), 9,10 further compounding the difficulty in accurately enumerating naïve T cells. 11,12 To measure thymic function more directly in humans, we recently described an assay that quantifies an episomal DNA by-product of the T-cell receptor (TCR) rearrangement process. 2 These TCR rearrangement excision circles (TRECs) contain the ...

A recombinant Mycobacterium bovis bacillus Calmette-Guerin (BCG) vector-based vaccine that secretes the V3 principal neutralizing epitope of human immunodeficiency virus (HIV) could induce immune response to the epitope and prevent the viral infection. By using the Japanese consensus sequence of HIV-1, we successfully constructed chimeric protein secretion vectors by selecting an appropriate insertion site ofa carrier protein and established the principal neutralizing determinant (PND)-peptide secretion system in BCG. The recombinant BCG (rBCG)-inoculated guinea pigs were initially screened by delayed-type hypersensitivity (DTH) skin reactions to the PND peptide, followed by passive transfer of the DTH by the systemic route. Further, immunization of mice with the rBCG resulted in induction of cytotoxic T lymphocytes. The guinea pig immune antisera showed elevated titers to the PND peptide and neutralized HIVMN, and administration of serum IgG from the vaccinated guinea pigs was effective in completely blocking the HIV infection in thymus/liver transplanted severe combined immunodeficiency (SCID)/hu or SCID/PBL mice. In addition, the immune serum IgG was shown to neutralize primary field isolates of HIV that match the neutralizing sequence motif by a peripheral blood mononuclear cell-based virus neutralization assay. The data support the idea that the antigensecreting rBCG system can be used as a tool for development of HIV vaccines.

Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15. The Ala mutation of penultimate phospho-Ser (S359A) that abolishes 14-3-3 binding resulted in substantially reduced O-glycosylation and the cell surface expression of GPR15. The surface membrane protein CD8 fused with the C-terminal tail of GPR15 S359A mutant was re-localized in the endoplasmic reticulum (ER). In the context of S359A mutation, the additional mutations in the upstream stretch of basic residues (RXR motif) restored O-glycosylation and the cell surface expression. The RXR motif was responsible for the interaction with coatomer protein I (COPI), which was inversely correlated with the 14-3-3 binding and cell surface expression. These results suggest that 14-3-3 binding promotes cell surface expression of GPR15 by releasing the receptor from ER retrieval/retention pathway that is mediated by the interaction of RXR motif and COPI. Moreover, 14-3-3 binding substantially increased the stability of GPR15 protein. Thus 14-3-3 proteins play multiple roles in biogenesis and trafficking of an HIV co-receptor GPR15 to control its cell surface density in response to the phosphorylation signal.

A rapid procedure for isolating subfragment one (SF1) from myosin was found. SF1 can be isolated specifically from proteolytic digests of myosin in the presence of a millimolar concentration of magnesium chloride. Under such ionic conditions all of the rod portion and undigested myosin is selectively precipitated. A nucleotide trapping experiment indicated how important quick preparation of SF1 is for maintaining the active site structure. This method can also be utilized in the preparation of heavy meromyosin.

The active site of skeletal myosin has been photoaffinity labeled (=50%) by the ADP analog N-(4-azido-2-nitrophenyl)-2-aminoethyl triphosphate (NANDP) following the cobalt phenanthroline active site trapping procedure of Wells and Yount [Wells, J. A. & Yount, R. G. (1979) Proc. Natl. Acad. Sci. USA 76,[4966][4967][4968][4969][4970]. Extensive proteolytic digestion of [3H]NANDP-labeled myosin subfragment one yielded two major peptides, Pi and P2, which were purified by reversed-phase high-performance liquid chromatography. These peptides represented 50% of all labeled amino acids and contained 1 mol of the unusual amino acid e-N-trimethyllysine. Analysis of P2 by Edman techniques gave a sequence Val-AsnPro-Tyr-Lys(Me3)-X-Leu-Pro-Val-Tyr, which corresponds to an identical sequence for residues 125-134 determined by Tong and Elzinga [Tong, S. W. & Elzinga, M. (1983) J. Biol. Chem. 258, 13100-13110] for a segment of rabbit skeletal myosin heavy chain in which X is Trp-130. P1 was identical to P2 except it contained an additional three amino acids, Asn-ProGln, at the COOH-terminal end. Amino acid composition, sequence data, spectral measurements, and location of radioactive label in both P1 and P2 all indicate Trp-130 is the major site of labeling by NANDP. The adjacent e-N-trimethyllysine may provide part of the binding site for the triphosphate portion of ATP.Myosin is widely accepted as the essential energy-transducing enzyme and major structural protein in the contractile apparatus of muscle cells. In spite of our detailed knowledge about the kinetic properties of myosin's ATPase activity (1, 2), the chemical structure of the active site is still unknown. The principal aim of this study was to identify the amino acid residues at the ATPase site. Our experimental strategy involved the nearly stoichiometric trapping of a photoaffinity analog of ADP, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (NANDP), at the active site by chemical crosslinking of two reactive thiols (SH1 and SH2) of myosin subfragment one (SF1). As has been previously reported (3), NANDP and its triphosphate analog behave remarkably similarly to ADP and ATP in their interaction with myosin and muscle fibers and in their ability to be stably trapped by thiol crosslinking agents. This crosslinking reaction (4) decreases the off-rate of ADP (and NANDP) by a factor of 104 (t½ at 0°C is >5 days) and allows the NANDP SF1 complex to be purified by conventional ammonium sulfate precipitation and gel filtration. Thus, subsequent photolysis of the NANDP* SF1 complex assures reaction will occur only with active site amino acids.This report shows that >50% of the trapped NANDP becomes covalently attached to the heavy chain of myosin and that of this labeling 50% of the NANDP has reacted with peptides containing one of the two tryptophan residues in the 23-kDa NH2-terminal tryptic peptide from SF1. The amino acid sequence of the two major NANDP peptides isolated identifies the predominant modified residue as Trp-130 in the sequence of Tong and ...

A facile and high-yield synthesis of a new ATP analogue, 2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate (NANTP), is described. NANTP and ATP are hydrolyzed by skeletal myosin subfragment 1 (SF1) at comparable rates in the presence of Ca2+, Mg2+, or NH4+-EDTA. NANTP is also cleaved but less readily by mitochondrial F1-ATPase and by (Na+ + K+)-ATPase from dog brain and hog kidney. F-Actin markedly activates NANTP cleavage by SF1 in the presence of Mg2+, suggesting that the diphosphate product NANDP is slow to be released from the enzyme. [alpha-32P]NANDP binds to a single site on SF1 (KA = 1 X 10(6) M-1) with an affinity identical with that of ADP. The absorption maximum of NANDP was shifted from 474 to 467 nm upon binding to SF1, suggesting that the purine binding site has a dielectric constant of about 45. NANDP was trapped in nearly stoichiometric amounts at the active site by cross-linking SH1 and SH2 with N,N'-p-phenylenedimaleimide (pPDM) or by chelation with cobalt (III) phenanthroline [Wells, J., & Yount, R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966]. The trapped [beta-32P]NANDP X SF1 complex, like the comparable ADP X SF1 complex, was stable for days at 0 degree C and could be purified free of extraneous analogue by ammonium sulfate precipitation and gel filtration. Photolysis of the purified complex gave greater than 50% covalent incorporation of the trapped NANDP into the 95-kilodalton (kDa) heavy chain of SF1. Limited trypsinization and analysis by gel electrophoresis showed that greater than 95% of the bound label was associated with the 25-kDa NH2-terminal peptide. Without trapping, NANDP labeling of SF1 was nonspecific and was not prevented by addition of a large excess of ATP. This new approach of trapping photoaffinity analogues by cross-linking agents before photolysis may prove to be of general usefulness in increasing the specificity and extent of labeling of enzymes that undergo substrate-induced conformation changes.

Fast skeletal myosins were isolated from carp acclimated to 10 and 30 degrees C, and their structural and enzymatic properties were compared. Myosins in 0.5 M KCl were subjected to limited proteolysis by using various proteases including alpha-chymotrypsin, trypsin, and papain, and different SDS-PAGE patterns were seen for the 10- and 30 degrees C-acclimated myosins in all cases. Myosin subfragment-1 (S1) prepared from the 10 degrees C-acclimated myosin by alpha-chymotryptic digestion in 0.12 M NaCl showed higher acto-S1 Mg(2+)-ATPase activity and lower thermostability than S1 from the warm-acclimated myosin. The peptide maps and ATP-induced spectral changes of tryptophan fluorescence also showed an obvious difference between the two types of S1. Temperature acclimation further caused changes in the rod region of myosin, since the apparent sizes of light meromyosin were different from each other for the two types of myosin. Myosin from carp acclimated to 20 degrees C showed intermediate properties between those of the 10- and 30 degrees C-acclimated myosins. Myosin isoforms might be expressed in a temperature-dependent manner to compensate for the effect of seasonal environmental temperature variation on swimming ability.