The fd phage and a peptide derived from its p8 coat protein interact with the HIV-1 Tat-NLS and inhibit its biological functions.

Filamentous fd bacteriophages are used to construct phage-display peptide libraries, which have been instrumental in selecting peptides that interact with specific domains within target molecules. Here we demonstrate that the fd bacteriophage itself, as well as NTP8 - a synthetic peptide derived from it and bearing amino acids 1-20 of the phage p8 protein - interact with the nuclear localization signal (NLS) of the HIV-1 Tat protein. Accordingly, fd bacteriophage and the NTP8 peptide inhibit binding mediated by the Tat-NLS to the nuclear-import receptor importin beta and Tat-NLS-mediated translocation into cell nuclei. The NTP8 peptide, at 100 microM concentration, also caused about 50% inhibition of HIV-1 propagation in cultured cells. The fd bacteriophage prevents heparan sulfate proteoglycans-mediated uptake of extracellular Tat by target cells and consequently transactivation of a chloramphenicol acetyltransferase (CAT) reporter gene. A BSA-NTP8 conjugate inhibits Tat-NLS-mediated binding to heparin immobilized on a BIAcore surface. BLAST analysis of the NTP8 amino-acid sequence revealed similarity to sequences in several human proteins, including ADA2 and CD53.

Antibody fragments selected by phage display against the nuclear localization signal of the HIV-1 Vpr protein inhibit nuclear import in permeabilized and intact cultured cells.

The HIV-1 Vpr protein harbors a nuclear localization signal in its N-terminal domain. A peptide bearing this domain and which is designated VprN has been used as a target to screen a phage display single chain Fv (scFv) library. Here we report the isolation of anti-VprN scFv fragments from this library. The purified scFv fragments were able to bind the VprN peptide in an ELISA-based system and to inhibit VprN-mediated nuclear import in permeabilized as well as in intact microinjected cells. Furthermore, the anti-VprN scFv fragments recognized the full-length recombinant Vpr protein and inhibited its nuclear import. The same scFv fragments did not inhibit nuclear import mediated by the nuclear localization signal of the SV40 large T-antigen demonstrating a specific effect. The use of the described inhibitory anti-VprN scFv fragments to study nuclear import of viral karyophilic proteins and their therapeutic potential is discussed.

Systematic solid-phase synthesis of linear pseudooligolysines containing multiple adjacent CH(2)NH amide bond surrogates: potential agents for gene delivery.

Solid-phase methodology was used to synthesize a series of fully reduced linear oligolysines (pseudooligolysines, abbreviated herein as PLs) containing up to five adjacent CH2NH peptide bond isosteres. The reduced peptide bonds were introduced by the reductive alkylation reaction between Fmoc-Lys-(Boc)-al and a free alpha-amine moiety on the pseudopeptidyl resin, using sodium cyanoborohydride in an acidified mixture of NMP/CH3OH (1 : 1 v/v). The oligomeric molecules, which can be regarded as polyethylene imine and spermine analogs, possess multiple positive charges under physiological conditions and form tight complexes with plasmid DNA. These characteristics and the increased resistance to hydrolysis by trypsin make these molecules potential candidates for future use as DNA carriers in gene delivery.

Development of a functional backbone cyclic mimetic of the HIV-1 Tat arginine-rich motif.

We have used the backbone cyclic proteinomimetics approach to develop peptides that functionally mimic the arginine-rich motif (ARM) of the HIV-1 Tat protein. This consensus sequence serves both as a nuclear localization signal (NLS) and as an RNA binding domain. Based on the NMR structure of Tat, we have designed and synthesized a backbone cyclic ARM mimetic peptide library. The peptides were screened for their ability to mediate nuclear import of the corresponding BSA conjugates in permeabilized cells. One peptide, designated "Tat11," displayed active NLS properties. Nuclear import of Tat11-BSA was found to proceed by the same distinct pathway used by the Tat-NLS and not by the common importin alpha pathway, which is used by the SV40-NLS. Most of the Tat-derived backbone cyclic peptides display selective inhibitory activity as demonstrated by the inhibition of the nuclear import mediated by the Tat-NLS and not by the SV40-NLS. The Tat-ARM-derived peptides, including Tat-11, also inhibited binding of the HIV-1 Rev-ARM to its corresponding RNA element (Rev response element) with inhibition constants of 5 nm. Here we have shown for the first time (a) a functional mimetic of a protein sequence, which activates a nuclear import receptor and (b) a mimetic of a protein sequence with a dual functionality. Tat11 is a lead compound which can potentially inhibit the HIV-1 life cycle by a dual mechanism: inhibition of nuclear import and of RNA binding.

Identification of a nuclear transport inhibitory signal (NTIS) in the basic domain of HIV-1 Vif protein.

The HIV-1 auxiliary protein Vif contains a basic domain within its sequence. This basic region,90RKKR93, is similar to the prototypic nuclear localization signal (NLS). However, Vif is not a nuclear protein and does not function in the nucleus. Here we have studied the karyophilic properties of this basic region. We have synthesized peptides corresponding to this positively charged NLS-like region and observed that these peptides inhibited nuclear transport via the importin pathway in vitro with IC50values in the micromolar range. Inhibition was observed only with peptides derived from the positively charged region, but not from other regions of the Vif protein, showing sequence specificity. On the other hand, the Vif inhibitory peptide Vif88-98 did not confer karyophilic properties when conjugated to BSA. The inactive Vif conjugate and the active SV40-NLS-BSA conjugate both contained a similar number of peptides conjugated to each BSA molecule, as was determined by amino acid analysis of the peptide-BSA conjugates. Thus, the lack of nuclear import of the Vif peptide-BSA conjugate cannot be attributed to insufficient number of conjugated peptide molecules per BSA molecule. Our results suggest that the HIV-1 Vif protein carries an NLS-like sequence that inhibits, but does not mediate, nuclear import via the importin pathway. We have termed such signals as nuclear transport inhibitory signals (NTIS). The possible role of NTIS in controlling nuclear uptake, and specifically during virus infection, is discussed herein. Our results raise the possibility that NLS-like sequences of certain low molecular weight viral proteins may serve as regulators of nucleocytoplasmic trafficking and not neccessarily as mediators of nuclear import.

A peptide derived from the N-terminal region of HIV-1 Vpr promotes nuclear import in permeabilized cells: elucidation of the NLS region of the Vpr.

Viral protein r (Vpr), a HIV-1 auxiliary protein which mediates nuclear import of the viral preintegration complex (PIC), contains two regions, N- and C-terminal, which have been proposed to function as a nuclear localization signal (NLS). We have synthesized peptides corresponding to both regions (designated as VprN and VprC), conjugated them to bovine serum albumin (BSA), and tested their ability to mediate nuclear import in permeabilized cells. Only VprN, and not VprC, functioned as an active NLS and promoted translocation of the conjugate into nuclei. Nuclear import of the conjugate was found to be energy and temperature dependent and was inhibited by wheat germ agglutinin (WGA). However, it did not require the addition of cytosolic factors and was not inhibited by the prototypic SV40 large T-antigen NLS peptide. Our results show that Vpr harbours a non-conventional negatively charged NLS and therefore suggest that Vpr may use a distinct nuclear import pathway.

Backbone cyclic peptide, which mimics the nuclear localization signal of human immunodeficiency virus type 1 matrix protein, inhibits nuclear import and virus production in nondividing cells.

Here, we describe an application of the backbone cyclic (BC) proteinomimetic approach to the design and the synthesis of a BC peptide which functionally mimics the nuclear localization signal (NLS) region of the human immunodeficiency virus type 1 matrix protein (HIV-1 MA). On the basis of the NMR structure of HIV-1 MA, a library of BC peptides was designed and screened for the ability to inhibit nuclear import of NLS-BSA in digitonin-permeabilized HeLa and Colo-205 cultured cells. The screening yielded a lead compound (IC50 = 3 microM) which was used for the design of a second library. This library led to the discovery of a highly potent BC peptide, designated BCvir, with an IC50 value of 35 nM. This inhibitory potency is compared to a value of 12 microM exhibited by the linear parent HIV-1 MA NLS peptide. BCvir also reduced HIV-1 production by 75% in infected nondividing cultured human T-cells and was relatively resistant to tryptic digestion. These properties make BCvir a potential candidate for the development of a novel class of antiviral drugs which will be based on blocking nuclear import of viral genomes.

Translocation of NLS-BSA conjugates into nuclei of permeabilized mammalian cells can be supported by protoplast extract. An experimental system for studying plant cytosolic factors involved in nuclear import.

An heterologous experimental system, which allows the study of the yet unknown cytosolic factors involved in nuclear import of nuclear localization signal (NLS)-containing proteins in plants, has been established. The ability of plant cell extract to substitute mammalian cytosol and to promote translocation of NLS-containing proteins into nuclei of permeabilized HeLa cells was demonstrated. The results described in the present work show that nuclear import of fluorescently labeled BSA conjugates bearing the NLS sequence of SV40 large T antigen could be supported by petunia cell cytoplasmic extract. This heterologous system shows the characteristic features of the homologous mammalian system, namely, is ATP dependent and is inhibited by WGA, GTPgammaS as well as by non-fluorescent NLS-BSA conjugates. The system described here offers an experimental method to study and characterise cytosolic factors which are required for nuclear import in plants.

Interaction of reconstituted Sendai viral envelopes with sperm cells: reconstituted Sendai virus envelope-induced fusion-mediated introduction of foreign material into bull sperm cells.

Reconstituted Sendai virus envelopes (RSVE), i.e. membrane vesicles bearing the viral envelope glycoproteins and phospholipids, are able to fuse with bull sperm cells. This was inferred from the increase in the degree of fluorescence dequenching (DQ) obtained following incubation of fluorescently labeled (R18 labeled) RSVE with bull sperm cells and from electron microscopy studies of RSVE-sperm interaction. Only a low degree of DQ was observed, under the same conditions, with non-fusogenic fluorescently labeled RSVE. This, and electron microscopy results, show that binding and membrane fusion events occur between RSVE and sperm cells. In addition, DQ was observed following incubation of RSVE that had been pre-loaded with the self-quenched fluorochrome Calcein, with bull sperm cells, indicating fusion-mediated injection of the dye from the RSVE space into the sperm cells.

Fusion of enveloped viruses with sperm cells: interaction of Sendai, influenza, and Semliki Forest viruses with bull spermatozoa.

Incubation of fluorescently labeled Sendai, influenza, as well as Semliki Forest viruses with bull sperm cells resulted in fluorescence dequenching. Fluorescence dequenching was observed with Sendai virus at pH 7.4 while with influenza and Semliki Forest viruses at pH 5.0, a pH value which is required for triggering their fusogenic activity. Control experiments performed with nonfusogenic Sendai and influenza viruses, or with bull sperm cells from which the viral receptors have been removed by treatment with neuraminidase, showed little fluorescence dequenching. These results clearly indicate that animal enveloped viruses are able to interact and to fuse with bull sperm cells. The possibility that following virus-sperm fusion spermatozoa can serve as a carrier of the virus genome and introduce it into recipient eggs during fertilization is discussed.

Transient gene expression of foreign genes in preheated protoplasts: stimulation of expression of transfected genes lacking heat shock elements.

Transfection of preheated petunia protoplasts with several biologically active DNA constructs resulted in a significantly higher gene expression than that observed in transfected unheated protoplasts. It was observed with supercoiled, linearized and single-stranded DNA structures that stimulation of transient gene expression in preheated protoplasts was neither dependent on the reporter gene nor on the regulatory elements used. Heat treatment at 42 degrees C also increased expression in protoplasts transfected with a plasmid bearing the tobacco mosaic virus (TMV) translational enhancer, omega. Northern blot analysis revealed that heat treatment of protoplasts before the transfection event greatly increased the amount of the newly synthesized transcripts. Preheating of protoplasts did not affect the transfection efficiency, namely the number of transfected cells in the population, nor the amount of DNA in transfected nuclei, as was inferred from histochemical staining and Southern blot analysis, respectively. The possible mechanism by which heat treatment stimulates transient gene expression of genes lacking obvious heat shock elements is offered. The relevance of the present findings to transient gene expression in plants in general and to viral gene expression in particular is discussed.

Fusion of influenza virus particles with liposomes: requirement for cholesterol and virus receptors to allow fusion with and lysis of neutral but not of negatively charged liposomes.

Influenza virus particles are able to fuse with liposomes composed of negatively charged or neutral phospholipids, as shown by using fluorochrome-labelled virions and fluorescence dequenching methods. Fusion with liposomes composed of only phosphatidylcholine (PC) was dependent on the presence of cholesterol (Chol), whereas fusion with liposomes containing negatively charged phospholipids, such as phosphatidylserine (PS), or of PC and phosphatidylethanolamine (PE) occurred in the absence of Chol. Fusion of influenza virions with PC:Chol liposomes was observed at pH 5.0, but not at pH 7.4, whereas a low degree of fusion with negatively charged liposomes or those containing PE was observed at pH 7.4. In addition, non-fusogenic influenza virions or HA0 influenza virions fused with PS- or PE-containing liposomes, especially at pH 5.0. Influenza virus particles were also able to induce the release of the fluorochrome calcein from negatively charged calcein-loaded liposomes at pH 5.0, as well as at pH 7.4, but failed to do so with PC:Chol liposomes. Lysis of PC:Chol by influenza virions was dependent on the presence of virus receptors, namely gangliosides (sialoglycolipids), and was observed only at pH 5.0. The results show that fusion of influenza virions with negatively charged or PE-containing liposomes does not reflect the biological activity of the virus needed for penetration and infection of living cells. On the other hand, fusion with PC:Chol liposomes is probably due to the activity of the viral fusion protein, the haemagglutinin glycoprotein.

An inducer of NKCF (NK cytotoxic factor) release: localization on target-cell membrane and initial characterization.

Natural killer (NK) cells are probably involved in the elimination of virus-infected cells and of certain tumor cells. NK cell-mediated cytotoxicity (NK-CMC) was extensively studied and was found to consist of several steps. Following recognition and conjugation between the effector and the target cell, the latter one induces release of NK cytotoxic factor (NKCF) from the effector cells. The NKCF binds to the target cell which is subsequently killed. None of the molecules involved in these steps was completely characterized. In the present study it is demonstrated that isolated membranes of target cells can effectively induce the release of NKCF. Furthermore, the activity of such isolated membranes was found to be modulated by interferon (IFN) treatment of the cells prior to membrane isolation. It was therefore concluded that an NKCF-inducing structure (NKIS) is present on plasma membranes and is distinct from the NK-recognition structure. Similarly, the sensitivity to NK-CMC could be transferred from sensitive cells to IFN-gamma-treated (NK-resistant) cells by membrane fusion with the aid of Sendai virus envelope glycoproteins. It is proposed that transfer of NKIS is responsible for the acquired sensitivity to NK-CMC. In addition, it is shown that NKIS activity was recovered following membrane solubilization and reconstitution. Its level on cell surface was modulated by treatment of cells with tunicamycin, thus indicating that NKIS was probably a cell surface glycoprotein.

Resistance to NK cell-mediated cytotoxicity (in K-562 cells) does not correlate with class I MHC antigen levels.

Natural Killer (NK) cells probably function as an early line of defense against virus-infected cells and tumor cells. In all cases, the killing by NK cell-mediated cytotoxicity (NK-CMC) is not MHC-restricted and the factors which determine the sensitivity to NK-CMC have not yet been identified. A positive correlation between resistance to NK-CMC and the level of class I MHC antigen (MHC I) expression on target cells has been reported in many studies, and in some cases a functional linkage between the two has been claimed. Several other studies have shown that there is no such correlation. By employing several experimental systems, we demonstrate here a lack of correlation between the level of MHC I and the sensitivity of K-562 cells to NK-CMC. Transfer of MHC I to MHC I-negative cells via vesicles had no effect on their resistance to NK-CMC. In addition, a decrease in resistance to NK-CMC and increase of MHC I levels was observed following target-cell membrane modulation by both application of cholesterol and hydrostatic pressure. Finally, no correlation between sensitivity to NK-CMC and MHC I expression was found in three sublines of K-562 cells. Since NK-CMC is a multistage process, it is concluded that components other than class I MHC antigens have a more prominent role in modulating the sensitivity of target cells to NK-CMC.

Induction of serotonin release from mast cells by lymphocyte activators is dependent upon implantation of lymphocyte plasma membrane components.

Plasma membranes (PM) isolated from mouse splenic lymphocytes were successfully fused to rat basophilic leukemia (RBL) cells using intact Sendai virus virions (SV). A two-step procedure was used in which SV were first fused with the PM to create PM + SV vesicles; the vesicles formed were then incubated with the RBL cells. Insertion of lymphocyte PM into the RBL cell's membrane endows a high rate of serotonin secretion upon stimulation of the implanted RBL cells with antimouse Ig antibodies or with concanavalin-A. The results of the present work clearly suggest that activation signals can be delivered via implantation of foreign membrane preparation containing specific receptors, thus rendering the target cells susceptible to stimulation by specific reagents.

Reconstituted viral envelopes--'Trojan horses' for drug delivery and gene therapy?

Reconstituted viral envelopes (RVEs) are formed by solubilizing intact virus in detergent and reassembling the envelope on removal of detergent. RVEs can be formed in the presence of agents that become encapsulated and can then be utilized in vitro and in vivo for drug delivery, cell destruction, transfer of membrane components, and as vectors for genetic engineering. The problems with biotechnological applications of RVEs and possible strategies for overcoming them are discussed in this article.

Expression of plant genes in transfected mammalian cells: accumulation of recombinant preLHCIIb proteins within cytoplasmic inclusion bodies.

Transfection of the monkey COS-7 cells with an expression vector bearing the Lemma gibba LHCIIb AB30 or AB19 genes led to the synthesis of the LHCIIb polypeptide precursors (preLHCIIb). This was inferred mainly from Western blot analysis which has revealed the appearance of a single immunoprecipitation band following the use of three different preparations of anti-LHCIIb antibodies. Synthesis of the precursor polypeptides, not the mature processed LHCIIb protein, was evident from the molecular weight of the newly synthesized protein, inferred from its position in the gel. Expression of the AB30 and AB19 genes was also evident from the appearance of specific transcripts only in transfected cells. Immunofluorescence observations revealed the appearance of distinct fluorescent spots in about 1-2% of the transfected cells. The same was observed following immunogold staining and electron microscopy studies, which revealed a specific association of gold particles with amorphous structures only in transfected cells. The preLHCIIb synthesized by transfected COS-7 cells was insoluble in a variety of detergents and could be solubilized only by 8 M urea or 0.1 N NaOH. These properties are characteristic of proteins accumulating within inclusion bodies of prokaryotes.

Effect of CpG methylation on gene expression in transfected plant protoplasts.

Activity of the cat gene driven by the cauliflower mosaic virus 35S promoter has been assayed by transfecting petunia protoplasts with the pUC8CaMVCAT plasmid. In vitro methylation of this plasmid with M.HpaII (methylates C in CCGG sites) and M.HhaI (methylates GCGC sites) did not affect bacterial chloramphenicol acetyltransferase (CAT) activity. It should be noted, however, that no HpaII or HhaI sites are present in the promoter sequence. In contrast, in vitro methylation of the plasmid with the spiroplasma methylase M.SssI, which methylates all CpG sites, resulted in complete inhibition of CAT activity. The promoter sequence contains 16 CpG sites and 13 CpNpG sites that are known to be methylation sites in plant DNA. In the light of this fact, and considering the results of the experiments presented here, we conclude that methylation at all CpG sites leaving CpNpG sites unmethylated is sufficient to block gene activity in a plant cell. Methylation of CpNpG sites in plant cells may, therefore, play a role other than gene silencing.

Fusion-mediated microinjection of liposome-enclosed DNA into cultured cells with the aid of influenza virus glycoproteins.

Influenza viruses were able to mediate fusion of DNA-loaded liposomes with living cultured cells such as monkey COS-7 cells. This was inferred from the appearance of CAT activity in recipient cells incubated with the combination of influenza viruses and liposomes loaded with the plasmid pSV2CAT. Influenza virions were found to be as efficient as intact Sendai virions in mediating microinjection of foreign DNA into living cells. Also, reconstituted envelopes bearing either influenza glycoproteins or the combination of Sendai and influenza glycoproteins were highly efficient in promoting fusion of loaded liposomes with recipient cells. Introduction of DNA into cultured cells required the presence of an active influenza fusion protein; namely, an active HA glycoprotein. Very little or no CAT activity was observed in cells incubated with loaded liposomes and unfusogenic influenza viruses. The virus-induced fusion event probably occurs within intracellular organelles such as endosomes following receptor-mediated endocytosis of virus-liposome complexes. This is due to the fact that the viral fusion glycoprotein is activated only at acidic pH values such as those which characterize the intraendosomal environment. Results of the present work demonstrate for the first time microinjection of foreign DNA via fusion with membranes of intracellular organelles. The potential of the present system to serve as a biological carrier for in vivo use is discussed.

Externally added DNA molecules support initiation of transcription in isolated nuclei from petunia.

Nuclei isolated from protoplasts transfected with the pUC8CaMVCAT and pDO432 plasmids were able to support, in run off experiments, the synthesis of specific transcripts as was evident from analysis by dot blot hybridization. Also the addition of the above plasmids to nuclei, prepared from non-transfected protoplasts, supported the synthesis of specific transcripts. Dot blot analysis showed that most of the transcripts obtained were complementary to the relevant gene sequences. alpha-Amanitin, at concentrations which are known to block the activity of RNA polymerase II, significantly inhibited the synthesis of specific transcripts by the isolated nuclei. The transcription activity was found to be predominantly associated with the nuclear fraction while the transcription products (RNA molecules) appeared in the supernatant obtained following sedimentation of the nuclei.