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.

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.

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.

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.

Fusogenic properties of reconstituted hybrid vesicles containing Sendai and influenza envelope glycoproteins: fluorescence dequenching and fluorescence microscopy studies.

Co-reconstitution of influenza and Sendai virus phospholipids and glycoproteins resulted in the formation of membrane vesicles containing the envelope glycoproteins from both viruses within the same membrane. Reconstituted influenza-Sendai hybrids (RISH) were able to lyse human erythrocytes and fuse with their membranes or with living cultured cells at pH 5.0 as well as at pH 7.4, thus exhibiting the fusogenic properties of both viruses. This was also inferred from experiments showing that the fusogenic activity of RISH was inhibited by anti-influenza as well as by anti-Sendai virus antibodies. Fusion of FISH and of reconstituted influenza (RIVE) or reconstituted Sendai virus envelopes (RSVE) with recipient membranes was determined by the use of fluorescently labeled envelopes and fluorescence dequenching methods. Observations with the fluorescence microscope were used to study localization of fused reconstituted envelopes within living cells. Incubation of RISH and RSVE with living cells at pH 7.4 resulted in the appearance of fluorescence rings around the cell plasma membranes and of intracellular distinct fluorescent spots indicating fusion with cell plasma membranes and with membranes of endocytic vesicles, respectively. The fluorescence microscopy observations clearly showed that RIVE failed to fuse, at pH 7.4, with cultured cell plasma membranes, but fused with membranes of endocytic vesicles.

Implanted IgE-Fc epsilon R complexes elicit IgE-mediated activation of RBL-2H cells.

The high-affinity receptor for IgE (Fc epsilon R) is the cellular trigger of the antigen-induced activation of mast cells and basophils. To examine the functional integrity of Fc epsilon R, we have adopted a protein implantation procedure whereby the purified receptor complex was coreconstituted with Sendai virus envelopes. The latter promoted fusion of the hybrid vesicles with recipient cells such as rat basophilic leukemia, RBL-2H3, thus serving as a vehicle for the receptor. The implanted Fc epsilon R was complexed with 125I-labeled mouse IgE (anti-DNP) to permit receptor quantification as well as specific triggering by DNP20BSA. Implantation in the presence of unlabeled rat IgE, which blocked the native receptors on the recipient RBL-2H3 cells, resulted in incorporation of up to 15 ng of receptor-bound IgE/10(6) cells. This was roughly equivalent in amount to 10-20% of the native receptors on such cells. The exocytosis which was triggered in the recipient cells by reagents that specifically recognized the implanted IgE reached between 15 and 50% of the maximal response. Various treatments that interfered with the activities of the viral envelopes reduced both receptor incorporation (3-5-fold) and cell degranulation (3-10-fold). These included separation of the receptor from the reconstituted envelopes, addition of serum to the incubation mixture (to inhibit vesicle-cell binding), and trypsinization of the virus (to inhibit vesicle-cell fusion). Poly(ethylene glycol) 8000 (4%) enhanced both the incorporation of the receptor and its functional responses. These treatments distinguished between real incorporation of IgE-Fc epsilon R complexes and other mechanisms of 125I-IgE association with the recipient cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative measurement of fusion between human immunodeficiency virus and cultured cells using membrane fluorescence dequenching.

Human immunodeficiency virus (HIV) was purified by sucrose gradient centrifugation and labeled with octadecylrhodamine B-chloride (R-18) under conditions resulting in 90% quenching of the fluorescence label. Incubation of R-18-labeled HIV (R-18/HIV) with CD4-positive CEM and HUT-102 cells, but not with CD4-negative MLA-144 cells, resulted in fluorescence dequenching (DQ, increase in fluorescence) of 20-25%. Similar level of DQ was observed upon incubation of CEM cells with R-18-labeled Sendai virus. DQ was observed when R-18/HIV was incubated with CD4+ cells at 37 degrees C, but not at 4 degrees C. Most of the increase in fluorescence occurred within 5 min of incubation at 37 degrees C and was independent of medium pH over the range of pH 5-8. Preincubation of cells with the lysosomotropic agent NH4Cl had no inhibitory effect on DQ. Complete inhibition was observed when target cells were fixed with glutaraldehyde prior to R-18/HIV addition. Our results demonstrate application of membrane fluorescence dequenching method to a quantitative measurement of fusion between HIV and target cell membranes. As determined by DQ, HIV penetrates into target cells by a rapid, pH-independent, receptor-mediated and specific process of fusion between viral envelope and cell plasma membrane, quite similar to that observed with Sendai virus.

Reconstitution of fusogenic Sendai virus envelopes by the use of the detergent chaps.

Sendai virus envelopes have been a useful tool in studying the mechanism of membrane-membrane fusion and have served as a vehicle for introducing foreign molecules (e.g., membrane proteins) into recipient cells. Reconstituted Sendai virus envelopes are routinely obtained following solubilization of virus particles with Triton X-100. This detergent has a low critical micellar concentration which precludes it from being the best detergent of choice in reconstitution studies. Nevertheless, it has remained in use since other detergents such as sodium deoxycholate and sodium cholate rendered the resultant vesicles inactive. Triton X-100 may be suboptimal for studies of some proteins that need be coreconstituted with the viral envelopes. Thus, alternative advantageous detergents, which retain the envelope fusogenic activity, have been sought. In this study we show that the synthetic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) effectively solubilizes the Sendai virions, and that the vesicles formed by simple reconstitution protocols appear structurally and biochemically similar to those obtained with Triton X-100. The resultant vesicles retain functional integrity as assessed in both fusion and hemolysis assays. This protocol seems to be useful in sendai envelope-mediated reimplantation of Fc epsilon receptors into the plasma membranes of rat basophilic leukemia cells.

Poly(aspartic acid)-dependent fusion of liposomes bearing the quaternary ammonium detergent [[[(1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl] dimethylbenzylammonium hydroxide.

Addition of the quaternary ammonium detergent [[[(1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl] dimethylbenzylammonium hydroxide (DEBDA[OH]) and the fluorescent probes N-(7-nitro-2-1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-NBD-PE and N-Rh-PE, respectively) to liposomes composed of phosphatidylcholine (PC) and cholesterol (chol) resulted in the formation of fluorescently labeled liposomes bearing DEBDA[OH]. Incubation of the anionic polymer poly(aspartic acid) (PASP) with such liposomes resulted in strong agglutination, indicating an association between the negatively charged PASP and the positively charged liposome-associated DEBDA[OH]. Addition of PASP to a mixture of fluorescently labeled and nonlabeled liposomes, both carrying DEBDA[OH], resulted in a significant increase in the extent of fluorescence, namely, fluorescence dequenching. The degree of the fluorescence dequenching was dependent upon the ratio between the nonfluorescent and the fluorescent liposomes, upon the temperature of incubation, and upon the amount of DEBDA[OH] which was associated with the liposomes. Electron microscopic observations revealed that large liposomes were formed upon incubation of liposomes bearing DEBDA[OH] with PASP. The results of the present work strongly indicate that the fluorescence dequenching observed is due to a process of PASP-induced liposome-liposome fusion.

Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts.

Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient appearance of the luciferase enzyme. In the present work we have used these systems to study the effect of DNA topology on the expression of the above recombinant genes. Linear forms of the above plasmids exhibited much higher activity in supporting gene expression than their corresponding super-coiled structures. CAT activity in protoplasts transfected with the linear forms of pUC8CaMVCAT and pNOSCAT was up to ten-fold higher than that observed in protoplasts transfected by the supercoiled template of these plasmids. This effect was observed in protoplasts derived from two different lines of Petunia hybrida and from a Nicotiana tabacum cell line. Transfection with the relaxed form of pUC8CaMVCAT resulted in very low expression of the CAT gene.Northern blot analysis revealed that the amount of poly(A)(+) RNA extracted from protoplasts transformed with the linear forms of the DNA was about 10-fold higher than that found in protoplasts transformed with supercoiled DNA.Southern blot analysis revealed that about the same amounts of supercoiled and linear DNA molecules were present in nuclei of transfected protoplasts. No significant quantitative differences have been observed between the degradation rates of the various DNA templates used.

Fusion of membrane vesicles bearing only the influenza hemagglutinin with erythrocytes, living cultured cells, and liposomes.

Membrane vesicles, bearing only the influenza viral hemagglutinin glycoprotein, were reconstituted following solubilization of intact virions with Triton X-100. The viral hemagglutinin glycoprotein was separated from the neuraminidase glycoprotein by agarose sulfanilic acid column. The hemagglutinin glycoprotein obtained was homogenous in gel electrophoresis and devoid of any neuraminidase activity. A quantitative determination revealed that the hemolytic activity of the hemagglutinin vesicles was comparable to that of intact virions. Incubation of fluorescently labeled hemagglutinin vesicles with human erythrocyte ghosts (HEG) or with liposomes composed of phosphatidylcholine/cholesterol or phosphatidylcholine/cholesterol/gangliosides, at pH 5.0 but not at pH 7.4, resulted in fluorescence dequenching. Very little, if any, fluorescence dequenching was observed upon incubation of fluorescently labeled HA vesicles with neuraminidase or glutaraldehyde-treated HEG or with liposomes composed only of phosphatidylcholine. Hemagglutinin vesicles were rendered non-hemolytic by treatment with NH2OH or glutaraldehyde or by incubation at 85 degrees C or low pH. No fluorescence dequenching was observed following incubation of non-hemolytic hemagglutinin vesicles with HEG or liposomes. These results clearly suggest that the fluorescence dequenching observed is due to fusion between the hemagglutinin vesicles and the recipient membranes. Incubation of hemagglutinin vesicles with living cultured cells, i.e. mouse lymphoma S-49 cells, at pH 5.0 as well as at pH 7.4, also resulted in fluorescence dequenching. The fluorescence dequenching observed at pH 7.4 was inhibited by lysosomotropic agents (methylamine and ammonium chloride) as well as by EDTA and NaN3, indicating that it is due to fusion of hemagglutinin vesicles taken into the cells by endocytosis.