A Tat-grafted anti-nucleic acid antibody acquires nuclear-localization property and a preference for TAR RNA

The 3D8 single chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can hydrolyze nucleic acids and enter the cytosol of cells without reaching the nucleus. The Tat peptide, derived from the basic region of the HIV-1 Tat protein, translocates to cell nuclei and has TAR RNA binding activity. In this study, we generated a Tat-grafted antibody (_H3Tat-3D8) by replacing complementarity-determining region 3 (CDR3) within the VH domain of the 3D8 scFv with a Tat_48–60 peptide (GRKKRRQRRRPPQ). _H3Tat-3D8 retained the DNA-binding and DNA-hydrolyzing activity of the scFv, and translocated to the nuclei of HeLa cells and preferentially recognized TAR RNA. Thus, the properties associated with the Tat peptide were transferred to the antibody via Tat-grafting without loss of the intrinsic DNA-binding and hydrolyzing activities of the 3D8 scFv antibody.     

The NK model of rugged fitness landscapes and its application to maturation of the immune response

Adaptive evolution is, to a large extent, a complex combinatorial optimization process. Such processes can be characterized as "uphill walks on rugged fitness landscapes". Concrete examples of fitness landscapes include the distribution of any specific functional property such as the capacity to catalyze a specific reaction, or bind a specific ligand, in "protein space". In particular, the property might be the affinity of all possible antibody molecules for a specific antigenic determinant. That affinity landscape presumably plays a critical role in maturation of the immune response. In this process, hypermutation and clonal selection act to select antibody V region mutant variants with successively higher affinity for the immunizing antigen. The actual statistical structure of affinity landscapes, although knowable, is currently unknown. Here, we analyze a class of mathematical models we call NK models. We show that these models capture significant features of the maturation of the immune response, which is currently thought to share features with general protein evolution. The NK models have the important property that, as the parameter K increases, the "ruggedness" of the NK landscape varies from a single peaked "Fujiyama" landscape to a multi-peaked "badlands" landscape. Walks to local optima on such landscapes become shorter as K increases. This fact allows us to choose a value of K that corresponds to the experimentally observed number of mutational "steps", 6-8, taken as an antibody sequence matures. If the mature antibody is taken to correspond to a local optimum in the model, tuning the model requires that K be about 40, implying that the functional contribution of each amino acid in the V region is affected by about 40 others. Given this value of K, the model then predicts several features of "antibody space" that are in qualitative agreement with experiment: (1) The fraction of fitter variants of an initial "roughed in" germ line antibody amplified by clonal selection is about 1-2%. (2) Mutations at some sites of the mature antibody hardly affect antibody function at all, but mutations at other sites dramatically decrease function. (3) The same "roughed in" antibody sequence can "walk" to many mature antibody sequences. (4) Many adaptive walks can end on the same local optimum. (5) Comparison of different mature sequences derived from the same initial V region shows evolutionary hot spots and parallel mutations. All these predictions are open to detailed testing by obtaining monoclonal antibodies early in the immune response and carrying out in vitro mutagenesis and adaptive hill climbing with respect to affinity for the immunizing antigen.     

Efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery

One of the most commonly used recombinant antibody formats is the single-chain variable fragment (scFv) that consists of the antibody variable heavy chain connected to the variable light chain by a flexible linker. Since disulfide bonds are often necessary for scFv folding, it can be challenging to express scFvs in the reducing environment of the cytosol. Thus, we sought to develop a method for antigen-independent selection of scFvs that are stable in the reducing cytosol of bacteria. To this end, we applied a recently developed genetic selection for protein folding and solubility based on the quality control feature of the Escherichia coli twin-arginine translocation (Tat) pathway. This selection employs a tripartite sandwich fusion of a protein-of-interest with an N-terminal Tat-specific signal peptide and C-terminal TEM1 β-lactamase, thereby coupling antibiotic resistance with Tat pathway export. Here, we adapted this assay to develop intrabody selection after Tat export (ISELATE), a high-throughput selection strategy for the identification of solubility-enhanced scFv sequences. Using ISELATE for three rounds of laboratory evolution, it was possible to evolve a soluble scFv from an insoluble parental sequence. We show also that ISELATE enables focusing of an scFv library in soluble sequence space before functional screening and thus can be used to increase the likelihood of finding functional intrabodies. Finally, the technique was used to screen a large repertoire of naïve scFvs for clones that conferred significant levels of soluble accumulation. Our results reveal that the Tat quality control mechanism can be harnessed for molecular evolution of scFvs that are soluble in the reducing cytoplasm of E. coli.     

Furin cleavage of the HIV-1 Tat protein

Extracellular human immunodeficiency virus-1 (HIV-1) Tat protein and Tat-derived peptides are biologically active but mechanisms of Tat processing are not known. Within the highly conserved basic region of HIV-1 Tat protein (amino acids, a.a. 48-56), we identified two putative furin cleavage sites and showed that Tat protein was cleaved in vitro at the second site, RQRR\ (a.a. 53-56\). This in vitro cleavage was blocked by a monoclonal antibody that binds near the cleavage site or by the furin inhibitor alpha-1 PDX. Monocytoid cells rich in furin also degraded Tat and this process was slowed by the furin inhibitor or the specific monoclonal antibody. Furin processing did not affect the rates for Tat uptake and nuclear accumulation in HeLa or Jurkat cells, but the transactivation activity was greatly reduced. Furin processing is a likely mechanism for inactivating extracellular HIV-1 Tat protein.     

Endocytosis and targeting of exogenous HIV-1 Tat protein.

The human immunodeficiency virus-1 (HIV-1) Tat protein has previously been shown to transactivate the HIV-1-LTR when added exogenously to HeLa, H9 lymphocytic and U937 promonocytic cells growing in culture. Here we show that Tat enters these cells by adsorptive endocytosis. Tat appears to bind non-specifically to the cell surface, with greater than 10(7) sites per cell. A specific receptor was not detected by protein crosslinking experiments, and uptake was not affected by treating cells with trypsin, heparinase or neuraminidase. Uptake and transactivation could be inhibited by incubation with heparin, dextran sulfate, an anti-Tat monoclonal antibody, or by incubation at 4 degrees C. In contrast, transactivation by Tat was markedly stimulated by the addition of basic peptides, such as Tat 38-58 or protamine. Fluorescence experiments with rhodamine-conjugated Tat show punctate staining on the cell surface and then localization to the cytoplasm and nucleus. The lack of a specific receptor makes it unclear whether Tat uptake is biologically important in HIV infection, however, the efficiency of uptake raises the possibility that Tat may be useful for delivery of protein molecules into cells.     

HIV-1 Tat directly binds to NFkappaB enhancer sequence: role in viral and cellular gene expression

HIV-1 Tat protein reprograms cellular gene expression of infected as well as uninfected cells apart from its primary function of transactivating HIV-1 long terminal repeat (LTR) promoter by binding to a nascent RNA stem–loop structure known as the transactivator response region (TAR). Tat also induces chromatin remodeling of proviral LTR-mediated gene expression by recruiting histone acetyl transferases to the chromatin, which results in histone acetylation. Furthermore several studies have shown convincing evidence that Tat can transactivate HIV-1 gene expression in the absence of TAR, the molecular mechanism of which remains to be elucidated. Here we show a direct interaction of Tat with nuclear factor kappa B (NFκB) enhancer, a global regulatory sequence for many cellular genes both in vitro and in vivo. This interaction not only provides a novel molecular basis to explain TAR-independent transactivation in HIV-1, but also points toward the potential mechanism of Tat- mediated modulation of cellular genes.     

Genetic evidence for a TatC dimer at the core of the Escherichia coli twin arginine (Tat) protein translocase

The twin arginine protein transport (Tat) system transports folded proteins across the cytoplasmic membranes of prokaryotes and the thylakoid membranes of plant chloroplasts. In Escherichia coli, the TatB and TatC components form a multivalent receptor complex that binds Tat substrates. Here, we have used a genetic fusion approach to construct covalent TatC oligomers in order to probe the organisation of TatC. A fused dimer of TatC supported Tat transport activity and was fully stable in vivo. Inactivating point mutations in one or other of the TatC units in the fused TatC dimer did not inactivate TatC function, indicating that only one TatC protomer in the TatC fused dimer needs to be active. Larger covalent fusions of TatC also supported Tat transport activity but were degraded in vivo to release smaller TatC forms. Taken together, these results strongly suggest that TatC forms a functional dimer, and support the idea that there is an even number of TatC protomers in the TatBC complex.     

1H-13C nuclear magnetic resonance assignment and structural characterization of HIV-1 Tat protein

Tat is a viral protein essential for activation of the HIV genes and plays an important role in the HIV-induced immunodeficiency. We chemically synthesized a Tat protein (86 residues) with its six glycines C alpha labelled with 13C. This synthetic protein has the full Tat activity. Heteronuclear nuclear magnetic resonance (NMR) spectra and NOE back-calculation made possible the sequential assignment of the 86 spin systems. Consequently, 915 NMR restraints were identified and 272 of them turned out to be long range ([i-j] > 4), providing structural information on the whole Tat protein. The poor spectral dispersion of Tat NMR spectra does not allow an accurate structure to be determined as for other proteins studied by 2D NMR. Nevertheless, we were able to determine the folding for Tat protein at a 1-mM protein concentration in a 100 mM, pH 4.5 phosphate buffer. The two main Tat functional regions, the basic region and the cysteine-rich region, are well exposed to solvent while a part of the N-terminal region and the C-terminal region constitute the core of Tat Bru. The basic region adopts an extended structure while the cysteine-rich region is made up of two loops. Resolution of this structure was determinant to develop a drug design approach against Tat. The chemical synthesis of the drugs allowed the specific binding and the inhibition of Tat to be verified.     

Antibody to HIV-1 Tat protein, a key molecule in HIV-1 pathogenesis. A brief review

In the last few years, literature reports have unequivocally established that the 86-101 aminoacid Tat protein, essential for an efficient viral replication, can be actively secreted by infected cells. The contribution of extracellular Tat to the progression of viral infection is underlined by the ability of neutralizing anti Tat antibody to reduce the viral load in vitro and possibly also in vivo. Considering that at least some of the effect of Tat protein seem to be the consequence of an autocrine loop and that anti Tat antibody is an efficient inhibitor of viral replication, it is reasonable to suppose that extracellular Tat play a functional role in HIV-1 infection and that HIV antibody may interfere with a possible Tat driven pathogenesis. This review explores the meaning of anti Tat antibody in vitro and in vivo and its importance to shed more light on viral pathogenesis and the recent development of Tat containing vaccine.     

HIV-1 Tat protein-induced VCAM-1 expression in human pulmonary artery endothelial cells and its signaling

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-kappaB, facilitated nuclear translocation of NF-kappaB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-kappaB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-kappaB translocation, which are the characteristics of pulmonary endothelial cell activation.     

Immunopotentiating properties of a multispecific α-anti-idiotype antibody

Multispecificity is not a well-understood property of some antibodies. Different functions have been attributed to multispecific natural antibodies, commonly associated with the neutralization and clearance of antigens. Much less is known about the role of antibodies like these, based on their idiotypic connectivity. B7Y33 is a chimeric IgG1 version of a polyreactive α anti-idiotype antibody that is able to interact with different immunoglobulin and non-immunoglobulin antigens. Here we report the capacity of this antibody to enhance the immunogenicity of several autologous IgMs in adjuvant-free conditions. Our results suggest that the formation of immune complexes seems to be necessary, but not sufficient, to this activity. The potential involvement of the interaction of B7Y33 with the FcγRIIb is discussed.     

Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins

The transactivator protein of human immunodeficiency virus type 1 Tat has the unique property of mediating the delivery of large protein cargoes into the cells when present in the extracellular milieu. Here we show that Tat fusion proteins are internalized by the cells through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. These conclusions are supported by the study of the slow kinetics of the internalization of Tat endosomes, by their resistance to nonionic detergents, the colocalization of internalized Tat with markers of caveolar endocytosis, and the impairment of the internalization process by drugs that disrupt lipid rafts or disturb caveolar trafficking. These results are of interest for all those who exploit Tat as a vehicle for transcellular protein delivery.     

Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.