Disruption of the interactions between the subunits of herpesvirus DNA polymerases as a novel antiviral strategy.

Most biological processes depend on the co-ordinated formation of protein-protein interactions. Besides their importance for virus replication, several interactions between virus proteins have been proposed as attractive targets for antiviral drug discovery, as the exquisite specificity of such cognate interactions affords the possibility of interfering with them in a highly specific and effective manner. There is a considerable need for new drugs active against herpesviruses, since available agents, most of which target the polymerisation activity of the virus DNA polymerase, are limited by pharmacokinetic issues, toxicity and antiviral resistance. A potential novel target for anti-herpesvirus drugs is the interaction between the two subunits of the virus DNA polymerase. This review focuses on recent developments using peptides and small molecules to inhibit protein-protein interactions between herpesvirus DNA polymerase subunits.

Effect of neutral and acidic phospholipids on mitochondrial ATP synthase secondary structure.

The secondary structure of delipidated and egg phosphatidylcholine or asolectin reconstituted mitochondrial ATP synthase complex from beef heart was investigated by Fourier transform infrared spectroscopy. Upon reconstitution, the infrared spectra of ATP synthase revealed an increase in turns and a concomitant decrease in beta-sheet content which occurred to a larger extent in the presence of asolectin rather than in the presence of egg phosphatidylcholine. These data correlate with kinetic data showing a higher ATPase activity of the asolectin reconstituted enzyme protein than the egg phosphatidylcholine reconstituted or delipidated enzyme complexes.

Analysis of the antigen specific T cell repertoires in HIV infection.

In addition to HIV infection, several acquired immunodeficiencies lead to depletion of CD4 lymphocytes. These include immunosuppression resulting from high dose cancer chemotherapy or induced to control graft rejection, as well as in autoimmune diseases. The consequence of this depletion is an increased susceptibility to opportunistic infections or the inability to control primary infection in the case of HIV infection. In all instances a full or partial immunoreconstitution is desirable. In order to monitor the cellular immune state of a patient, rational information cannot be simply derived from phenotypic quantification of T lymphocytes. Instead loss or recovery of CD4 cells should be monitored by defining the specificity, the function and the clonality of the relevant cell population. Several methods are now available for this type of investigation. Here we describe an approach for the definition of clonal heterogeneity of antigen specific CD4 lymphocytes, a parameter that may help monitor loss or reconstitution in acquired immunodeficiencies. As examples of antigen specific CD4 T cell responses we focused on Pneumocystis carinii and on cytomegalovirus, as prototypic opportunistic pathogens which are responsible for severe infections in AIDS and in other immunosuppressive conditions which arise for instance following transplantation. Specific CD4 T cell lines were generated from normal controls and from seropositives in order to select antigen specific lymphocytes. The cells were subsequently analyzed for clonal diversity according to TCR BV gene family usage and according to TCR CDR3 size heterogeneity (spectratyping).

Efficient extracellular production of hybrid E. coli heat-labile enterotoxin B subunits in a marine Vibrio.

Escherichia coli heat-labile enterotoxin B subunit (EtxB) has been proposed as a potential protein carrier for the delivery of heterologous peptides to target cells, particularly for the oral delivery of epitopes to the mucosal immune system. In this study, two extensions to the C-terminus of EtxB were genetically engineered that correspond to a well-characterized neutralising epitope of glycoprotein D from herpes simplex virus (EtxB-gD) and to the C-terminal nine amino acids from the 38 kDa subunit of HSV-encoded ribonucleotide reductase (EtxB-R2). Here we describe the extracellular secretion of the two hybrid EtxBs from a marine Vibrio harbouring a broad-host range inducible expression vector containing the hybrid genes. Large amounts of intact fusion proteins (15-20 mg per liter of culture) were secreted into the medium upon induction. These hybrid proteins maintained the receptor-binding activity of the native toxin as well as being cross-reactive with anti-EtxB and anti-heterologous peptide monoclonal antibodies.

Identification and characterization of an extracellular protease activity produced by the marine Vibrio sp. 60.

The marine fish pathogen Vibrio sp. 60 has been used as a host for heterologous expression of the Escherichia coli heat-labile enterotoxin B-subunit and derivatives carrying a C-terminal extension. In this study, a chimeric enterotoxin B-subunit with an extension corresponding to the carboxy-terminal nine amino acids -Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-COOH from the small subunit of herpes simplex virus type 1-encoded ribonucleotide reductase, is shown to be proteolytically cleaved in the extracellular medium by a single virus type 1-encoded ribonucleotide reductase, is shown to be proteolytically cleaved in the extracellular medium by a single protease that is secreted by the host strain. Such protease behaves as a typical metalloprotease, being inhibited by EDTA but not by a serine protease inhibitor. Purification and amino acid composition analysis of the two proteolysis products revealed a specific cleavage of the peptide bond between amino acids glycine and alanine of the nine amino acid extension with loss of activity. The above observation is relevant for the biotechnological exploitation of Vibrio sp. 60.

Quantitative analysis of ribonucleoside triphosphates in human lymphoid cells by micellar electrokinetic capillary chromatography.

Micellar electrokinetic capillary chromatography (MECC) was applied to develop an analytical method for quantitation of ribonucleoside triphosphates (rNTPs) in human lymphoid cells obtained from patients with B-chronic lymphocytic leukaemia (B-CLL) and cutaneous lymphomas. The results of this analysis showed a significant depression of intracellular rNTPs in patients with B-CLL, compared with rNTPs of healthy controls. These data are in agreement with other studies in which rNTP separations were performed with traditional high-performance liquid chromatography. MECC has proved to be a useful tool for intracellular rNTPs determination, revealing possible new applications in the study of the metabolic state of human cells. In addition, this method can be useful in monitoring the effect of many drugs (antiviral, antineoplastic) which interfere with nucleotide metabolism.

Quantitative analysis of ribonucleotide triphosphates in cell extracts by high-performance liquid chromatography and micellar electrokinetic capillary chromatography: a comparative study.

Two analytical methods have been evaluated for their ability to separate and quantitate the intracellular pool of free ribonucleotide triphosphates in four different cell lines. Micellar electrokinetic capillary chromatography and high performance liquid chromatography were compared in terms of speed, sensitivity, and efficiency of analysis, with the former being 3 times faster (about 10 vs 30 min) and having an average minimun detectable quantity of 50 fmol vs 30 pmol and approximately 950 000 theorical plates/m vs 4500. Different protocols of extraction of nucleotides from whole cells were also evaluated with the two analytical methods. Micellar electrokinetic capillary chromatography proved to be a powerful tool for the reproducible and reliable quantitation of ribonucleotide triphosphates, allowing the fast processing of extremely small volumes of sample.

Separation methods for acyclovir and related antiviral compounds.

Acyclovir (ACV) is an antiviral drug, which selectively inhibits replication of members of the herpes group of DNA viruses with low cell toxicity. Valaciclovir (VACV), a prodrug of ACV is usually preferred in the oral treatment of viral infections, mainly herpes simplex virus (HSV). Also other analogues such as ganciclovir and penciclovir are discussed here. The former acts against cytomegalovirus (CMV) in general and the latter against CMV retinitis. The action mechanism of these antiviral drugs is presented briefly here, mainly via phosphorylation and inhibition of the viral DNA polymerase. The therapeutic use and the pharmacokinetics are also outlined. The measurement of the concentration of acyclovir and related compounds in biological samples poses a particularly significant challenge because these drugs tend to be structurally similar to endogenous substances. The analysis requires the use of highly selective analytical techniques and chromatography methods are a first choice to determine drug content in pharmaceuticals and to measure them in body fluids. Chromatography can be considered the procedure of choice for the bio-analysis of this class of antiviral compounds, as this methodology is characterised by good specificity and accuracy and it is particularly useful when metabolites need to be monitored. Among chromatographic techniques, the reversed-phase (RP) HPLC is widely used for the analysis. C18 Silica columns from 7.5 to 30 cm in length are used, the separation is carried out mainly at room temperature and less than 10 min is sufficient for the analysis at 1.0-1.5 ml/min of flow-rate. The separation methods require an isocratic system, and various authors have proposed a variety of mobile phases. The detection requires absorbance or fluorescence measurements carried out at 250-254 nm and at lambdaex=260-285 nm, lambdaem=375-380 nm, respectively. The detection limit is about 0.3-10 ng/ml but the most important aspect is related to the sample treatment, mainly when body fluids are under examination. The plasma samples obtained from human blood are pre-treated with an acid or acetonitrile deproteinization and the supernatant after centrifugation is successively extracted before RP-HPLC injection. Capillary Electrophoresis methods are also discussed. This new analytical approach might be the expected evolution, in fact the analyses are improved with regard to time and performance, in particular coated capillary as well as addition of stabilisers have been employed. The time of analysis is shortened arriving at less than half a minute. Furthermore by using an electrochemical detection, and having a calibration linearity in the range of 0.2-20.0 ng/ml, the detection limit is 0.15 microg/ml. The measurements of acyclovir and penciclovir have been presented but in the future other related drugs will probably be available using CE methods.

Specific inhibition of herpes virus replication by receptor-mediated entry of an antiviral peptide linked to Escherichia coli enterotoxin B subunit.

Mimetic peptides capable of selectively disrupting protein-protein interactions represent potential therapeutic agents for inhibition of viral and cellular enzymes. This approach was first suggested by the observation that the peptide YAGAVVNDL, corresponding to the carboxyl-terminal 9 amino acids of the small subunit of ribonucleotide reductase of herpes simplex virus, specifically inhibited the viral enzyme in vitro. Evaluation and use of this peptide as a potential antiviral agent has, however, been thwarted by its failure to inhibit virus replication in vivo, presumably because the peptide is too large to enter eukaryotic cells unaided. Here, we show that the nontoxic B subunit of Escherichia coli heat-labile enterotoxin can be used as a recombinant carrier for the receptor-mediated delivery of YAGAVVNDL into virally infected cells. The resultant fusion protein specifically inhibited herpes simplex virus type 1 replication and ribonucleotide reductase activity in quiescent Vero cells. Preincubation of the fusion protein with soluble GM1 ganglioside abolished this antiviral effect, indicating that receptor-mediated binding to the target cell is necessary for its activity. This provides direct evidence of the usefulness of carrier-mediated delivery to evaluate the intracellular efficacy of a putative antiviral peptide.

The catalytic subunit of herpes simplex virus type 1 DNA polymerase contains a nuclear localization signal in the UL42-binding region.

The herpes simplex virus type 1 DNA polymerase consists of a catalytic subunit (POL or UL30) and a processivity factor (UL42). The POL/UL42 interaction, which occurs through the extreme C-terminus of POL, is essential for HSV-1 replication and thus represents a valid target for drug inhibition. We recently showed (A. Loregian et al. (1999) Proc. Natl. Acad. Sci. USA 96, 5221-5226) that an oligopeptide corresponding to the 27 C-terminal amino acids of POL, when delivered into herpes simplex virus type 1-infected cells by a protein carrier, was able to localize into the nucleus and to inhibit viral replication by disruption of the POL/UL42 interaction. In this report, to further characterize the 27 mer (Pol peptide), we investigated whether its nuclear localization was due to the presence of a nuclear localization signal. By testing the ability of the Pol peptide to localize the beta-galactosidase, a normally cytoplasmic protein, to the nucleus, we confirmed that the Pol peptide contained a functional nuclear localization signal, corresponding to the RRMLHR motif. This sequence proved not only necessary but also sufficient for nuclear localization, because its substitution with a six-alanine stretch prevented nuclear translocation of the beta-galactosidase-Pol peptide fusion. Site-directed mutagenesis experiments on this revealed that both the three basic arginines and the two hydrophobic residues Met and Leu were crucial for nuclear targeting. Finally, functionally equivalent sequences were also found in the C-terminus of the catalytic subunits of human cytomegalovirus (RRLHL) and of equine herpesvirus-1 DNA polymerase (RRILH).

Use of Vibrio spp. for expression of Escherichia coli enterotoxin B subunit fusion proteins: purification and characterization of a chimera containing a C-terminal fragment of DNA polymerase from herpes simplex virus type 1.

The nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a convenient carrier molecule for the attachment and delivery of heterologous peptides into eukaryotic cells. To evaluate the properties of such EtxB-based fusion proteins an efficient method for their production and purification is required. High-level production and purification of native EtxB has been achieved using heterologous expression and secretion in a marine Vibrio (Amin, T., and Hirst, T. R., 1994, Protein Expression Purif. 5, 198-204). However, the use of this method to isolate EtxB fusion proteins has been precluded because of their susceptibility to degradation by extracellular proteases secreted by members of the Vibrionaceae. In this paper a method is described for production of EtxB-pol, comprising the enterotoxin B subunit linked to a 27-residue C-terminal fragment of Pol, the catalytic subunit of DNA polymerase of herpes simplex virus type 1 (HSV-1). Following assessment of the relative efficacy of different Vibrio strains as hosts for EtxB-pol expression, the chimera was produced at the highest level of 3.5 mg/liter by cultures of Vibrio sp.60. Addition of 0.3 mM EDTA to the growth medium blocked proteolysis of the secreted EtxB-pol fusion protein, which was then purified to homogeneity using ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 57%. Purified EtxB-pol reacted with both anti-EtxB and anti-Pol peptide antibodies, and was able to specifically bind UL42, a processivity factor which normally binds to the C-terminal region of HSV-1 Pol. This modified method for expression and purification of EtxB-pol should be of general utility for the preparation of other EtxB-based fusion proteins.

Intranuclear delivery of an antiviral peptide mediated by the B subunit of Escherichia coli heat-labile enterotoxin.

We report an intracellular peptide delivery system capable of targeting specific cellular compartments. In the model system we constructed a chimeric protein consisting of the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) fused to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1. Viral DNA synthesis takes places in the nucleus and requires the interaction with an accessory factor, UL42, encoded by the virus. The peptide, designated Pol, is able to dissociate this interaction. The chimeric protein, EtxB-Pol, retained the functional properties of both EtxB and peptide components and was shown to inhibit viral DNA polymerase activity in vitro via disruption of the polymerase-UL42 complex. When added to virally infected cells, EtxB-Pol had no effect on adenovirus replication but specifically interfered with herpes simplex virus 1 replication. Further studies showed that the antiviral peptide localized in the nucleus, whereas the EtxB component remained associated with vesicular compartments. The results indicate that the chimeric protein entered through endosomal acidic compartments and that the Pol peptide was cleaved from the chimeric protein before being translocated into the nucleus. The system we describe is suitable for delivery of peptides that specifically disrupt protein-protein interactions and may be developed to target specific cellular compartments.

Crystal structure of the B subunit of Escherichia coli heat-labile enterotoxin carrying peptides with anti-herpes simplex virus type 1 activity.

Two chimeric proteins, consisting of the B subunit of Escherichia coli heat-labile enterotoxin with different peptides fused to the COOH-terminal ends, have been crystallized and their three-dimensional structure determined. The two extensions correspond to (a) a nonapeptide representing the COOH-terminal sequence of the small subunit of herpes simplex virus type 1 ribonucleotide reductase and (b) a 27-amino acid long peptide, corresponding to the COOH-terminal end of the catalytic subunit (POL) of DNA polymerase from the same virus. Both proteins crystallize in the P41212 space group with one pentameric molecule per asymmetric unit, corresponding to a solvent content of about 75%. The overall conformation of the B subunit pentamer in the two chimeric proteins, which consists of five identical polypeptide chains, is very similar to that in the native AB complex and conforms strictly to 5-fold symmetry. On the contrary, the peptide extensions are essentially disordered: in the case of the nonapeptide, only 5 and 6 amino acids were, respectively, positioned in two monomers, while in the other three only 2 residues are ordered. The extension is fully confined to the surface of the pentamer opposite to the face that interacts with the membrane and consequently it does not interfere with the ability of the B subunit to interact with membrane receptors. Moreover, the conformational flexibility of the two peptide extensions could be correlated to their propensity for proteolytic processing and consequent release of a biologically active molecule into cultured cells.