Vaccinia Expression System Research Articles

Recombinant Poxviruses: Versatile Tools for Immunological Assays.

The study of antigen processing and presentation is critical to our understanding of the mechanisms that govern immune surveillance. A typical requirement of assays designed to examine antigen processing and presentation is the de novo biosynthesis of a model antigen. Historically, Vaccinia virus, a poxvirus closely related to Cowpox virus, has enjoyed widespread use for this purpose. Recombinant poxvirus-based expression has a number of advantages over other systems. Poxviruses accommodate the insertion of large pieces of recombinant DNA into their genome, and recombination and selection are relatively efficient. Poxviruses readily infect a variety of cell types, and they drive rapid and high levels of antigen expression. Additionally, they can be utilized in a variety of assays to study both MHC class I restricted and MHC class II restricted antigen processing and presentation. Ultimately, the numerous advantages of poxvirus recombinants have made the Vaccinia expression system a mainstay in the study of processing and presentation over the past two decades. In an attempt to address one shortcoming of Vaccinia virus while simultaneously retaining the benefits inherent to poxviruses, our laboratory has begun to engineer recombinant Ectromelia viruses. Ectromelia virus, or mousepox, is a natural pathogen of murine cells and performing experiments in the context of a natural host-pathogen relationship may elucidate unknown factors that influence epitope generation and host response. This chapter will describe several recombinant poxvirus system protocols used to study both MHC class I and class II antigen processing and presentation, as well as provide insight and troubleshooting techniques to improve the reproducibility and fidelity of these experiments.

Human multidrug and toxin extrusion 1 (MATE1/SLC47A1) transporter: functional characterization, interaction with OCT2 (SLC22A2), and single nucleotide polymorphisms

Renal elimination of a number of cationic compounds is thought to be mediated by the organic cation transporter 2 (OCT2, SLC22A2), a drug uptake transporter expressed at the basolateral domain of renal tubular cells. Recently, the key efflux transporter for the secretion organic cations was identified as an electroneutral H(+)/organic cation exchanger termed the multidrug and toxin extrusion (MATE)-type transporter 1 (MATE1, SLC47A1). The key goals of this study were to assess the interplay between the renal cationic transporters OCT2 and MATE1 and the functional assessment of genetic variation in human MATE1. First, the ability of various agents to interact with OCT2- or MATE1-mediated transport was determined using a recombinant vaccinia expression system. We were able to identify several drugs in clinical use with a divergent inhibitory capacity for these transporters. Subsequently, we further assessed the effect of those compounds on the cellular accumulation of shared substrates using OCT2 and MATE1 double-transfected cells. Consistent with data obtained using single transporter transfected cells, compounds that exhibited preferential inhibition of MATE1 such as rapamycin and mitoxantrone induced significant cellular accumulation of cationic substrates. We next assessed the functional relevance of MATE1 genetic polymorphisms. Significant loss of transport activity for metformin and tetraethylammonium was noted for two nonsynonymous single nucleotide polymorphisms (SNPs), c.404T>C (p.159T>M) and c.1012G>A (p.338V>A). The c.404T>C was only seen in Asian subjects with an allele frequency of 1%, and the c.1012G>A SNP was much more common, especially among those of African descent. In conclusion, we show that coordinate function of MATE1 with OCT2 likely contributes to the vectorial renal elimination of organic cationic drugs and that altered activity of MATE1, whether by drugs or polymorphisms, should be considered as an important determinant of renal cationic drug elimination.

Small Molecules VP-14637 and JNJ-2408068 Inhibit Respiratory Syncytial Virus Fusion by Similar Mechanisms

Here we present data on the mechanism of action of VP-14637 and JNJ-2408068 (formerly R-170591), two small-molecule inhibitors of respiratory syncytial virus (RSV). Both inhibitors exhibited potent antiviral activity with 50% effective concentrations (EC50s) of 1.4 and 2.1 nM, respectively. A similar inhibitory effect was observed in a RSV-mediated cell fusion assay (EC50=5.4 and 0.9 nM, respectively). Several drug-resistant RSV variants were selected in vitro in the presence of each compound. All selected viruses exhibited significant cross-resistance to both inhibitors and contained various single amino acid substitutions in two distinct regions of the viral F protein, the heptad repeat 2 (HR2; mutations D486N, E487D, and F488Y), and the intervening domain between HR1 and HR2 (mutation K399I and T400A). Studies using [3H]VP-14637 revealed a specific binding of the compound to RSV-infected cells that was efficiently inhibited by JNJ-2408068 (50% inhibitory concentration=2.9 nM) but not by the HR2-derived peptide T-118. Further analysis using a transient T7 vaccinia expression system indicated that RSV F protein is sufficient for this interaction. F proteins containing either the VP-14637 or JNJ-2408068 resistance mutations exhibited greatly reduced binding of [3H]VP-14637. Molecular modeling analysis suggests that both molecules may bind into a small hydrophobic cavity in the inner core of F protein, interacting simultaneously with both the HR1 and HR2 domains. Altogether, these data indicate that VP-14637 and JNJ-2408068 interfere with RSV fusion through a mechanism involving a similar interaction with the F protein.

Transient expression of wild-type and mutant glucocerebrosidases in hybrid vaccinia expression system.

Gaucher disease, the most prevalent lysosomal storage disease, is characterised by a significant phenotypic variation caused by more than 150 mutations. In order to verify pathogenicity of mutations found in the Czech Gaucher population, the vaccinia expression system was used. The wild-type human beta-glucocerebrosidase cDNA and cDNAs carrying the mutations 72delC, 1326insT, 1263del55, S196P, N370S, L444P, G202E, D409H, T369M, L444P+V460V, and D409H+T369M were expressed in Gaucher fibroblast cell line (L444P/S107L), BSC40, and HeLa G cells. The enzymatic activity and immunological reactivity were analysed. Only beta-glucocerebrosidase-deficient fibroblasts were suitable for expression using plasmid transfection. The expressed beta-glucosidase activity of mutant glucocerebrosidases was in good correlation with the presumed severity of the mutations.

HIV-Nef enhances interleukin-2 production and phosphatidylinositol 3-kinase activity in a human T cell line.

The Nef protein has a major influence on disease pathogenesis in HIV-infected individuals. The objective of the present study was to examine the effects of Nef on T lymphocyte activation and associated signalling events. A recombinant vaccinia expression system was used to express Nef in a human T cell line. Stimulation of these cells with anti-CD28 antibody, and either phorbol 12-myristate 13-acetate (PMA) or anti-CD3, activates signal transduction pathways and results in IL-2 production and IL-2 receptor alpha-chain (CD25) expression. Cellular responses were examined in cells expressing either Nef or an irrelevant control protein. Activation of signalling was assessed by immunoblot analysis, or by in-vitro phosphatidylinositol 3-kinase (PI3K) assays. IL-2 production was measured by enzyme-linked immunosorbent assay, and CD25 cell surface expression was examined using flow cytometry. Infection of cells with recombinant vaccinia expressing HIV-nef resulted in a marked increase in the production of IL-2 when cells were activated. The enhanced IL-2 response was accompanied by an increase in the level of PI3K activity. IL-2 production remained sensitive to inhibition with the PI3K competitive inhibitor Ly294002, and to the fungal macrolide, rapamycin. In contrast, CD25 expression was not affected, and there were no measurable changes to nuclear factor kappaB (NFkappaB) activation pathways. Enhanced IL-2 production in stimulated T cells expressing HIV-Nef is associated with increased activation of PI3K-dependent signalling pathways. The results support a model in which Nef affects HIV disease progression by distorting T cell responses.

Conserved Residues of Human XPG Protein Important for Nuclease Activity and Function in Nucleotide Excision Repair

The human XPG endonuclease cuts on the 3' side of a DNA lesion during nucleotide excision repair. Mutations in XPG can lead to the disorders xeroderma pigmentosum (XP) and Cockayne syndrome. XPG shares sequence similarities in two regions with a family of structure-specific nucleases and exonucleases. To begin defining its catalytic mechanism, we changed highly conserved residues and determined the effects on the endonuclease activity of isolated XPG, its function in open complex formation and dual incision reconstituted with purified proteins, and its ability to restore cellular resistance to UV light. The substitution A792V present in two XP complementation group G (XP-G) individuals reduced but did not abolish endonuclease activity, explaining their mild clinical phenotype. Isolated XPG proteins with Asp-77 or Glu-791 substitutions did not cleave DNA. In the reconstituted repair system, alanine substitutions at these positions permitted open complex formation but were inactive for 3' cleavage, whereas D77E and E791D proteins retained considerable activity. The function of each mutant protein in the reconstituted system was mirrored by its ability to restore UV resistance to XP-G cell lines. Hydrodynamic measurements indicated that XPG exists as a monomer in high salt conditions, but immunoprecipitation of intact and truncated XPG proteins showed that XPG polypeptides can interact with each other, suggesting dimerization as an element of XPG function. The mutation results define critical residues in the catalytic center of XPG and strongly suggest that key features of the strand cleavage mechanism and active site structure are shared by members of the nuclease family.

Assignment of the possible HTLV receptor gene to chromosome 17q21-q23.

We have determined the HTLV (Human T-cell leukemia virus) receptor localization more precisely on the human chromosome 17. Based on the fact that HTLV infection induces syncytium formation of infected cells as a result of interaction between the viral envelope and viral receptor, we performed the sensitive biological assay using recombinant vaccinia expression system. Our results from the induced syncytium pattern of the somatic hybrid cell lines with different deletions indicated that the HTLV receptor gene may reside from q21 to q23 on the long arm of the human chromosome 17.

Downstream signals initiated in mast cells by FcεRI and other receptors

The significant contributions this past year to our understanding of IgE receptor (FcεRI) signaling in mast cells include studies with truncated Syk in a vaccinia expression system and Syk-negative variants of rat basophilic (RBL-2H3) cells. These studies demonstrate an essential role for Syk in initiating signals for secretion and release of arachidonic acid via phospholipase A 2 and mitogen-activated protein kinase. A newly recognized addition to the repertoire of FcεRI-mediated signaling systems is the activation of sphingosine kinase, which contributes to calcium mobilization in mast cells. Advances have been made in our understanding of other receptors that regulate proliferation and differentiation of mast cells, and in our understanding of the ability of mast cells to mount acquired and acute responses to antigenic and bacterial challenge.

Characterization of a Novel Baboon Virus Closely Resembling Human T-Cell Leukemia Virus

We report the isolation of a virus from a baboon imported from Kenya and the analysis of the nucleotide sequence of theenvgene. Comparison of the complete nucleotide sequence of theenvgene of different HTLV-1 strains and the baboon T-cell leukemia virus (designated BTLV) indicated similarities ranging from 92.5 to 97.4%. In contrast, only 89.1% similarity was observed between the BTLVenvsequence and that of simian T-cell leukemia virus (PtM3). The sequences corresponding to the glycosylation sites, endoproteolytic processing site, and major immunological determinants were strictly conserved between BTLV and HTLV-1. To characterize the expressed protein we used a vaccinia expression system, which indicated that a protein of 62 kDa is encoded by the envelope gene. The protein acquired mostly high mannose modifications and was localized predominantly in the endoplasmic reticulum. A fraction of the protein was expressed at the cell surface, where it could induce membrane fusion of target cells. The existence of HTLV-1-like viruses in baboons indicates the potential risk of transmission of such virus from these nonhuman primates to humans, thus highlighting the need for specific screening for such viruses during xenotransplantation.

Identification of an Interferon-γ Receptor α Chain Sequence Required for JAK-1 Binding

We have shown previously that a four-amino acid block residing at positions 266-269 (LPKS) in the intracellular domain of the human interferon-gamma (IFN-gamma) receptor alpha chain is critical for IFN-gamma-dependent tyrosine kinase activation and biologic response induction. Herein we show that this sequence is required for the constitutive attachment of the tyrosine kinase JAK-1. Using a vaccinia expression system, a receptor alpha chain-specific monoclonal antibody coprecipitated JAK-1 from cells coexpressing JAK-1 and either (a) wild type IFN-gamma receptor alpha chain, (b) a receptor alpha chain truncation mutant containing only the first 59 intracellular domain amino acids, or (c) a receptor mutant containing alanine substitutions for the functionally irrelevant residues 272-275. In contrast, JAK-1 was not coprecipitated when coexpressed with a receptor alpha chain mutant containing alanine substitutions for the functionally critical residues 266-269 (LPKS). Mutagenesis of the LPKS sequence revealed that Pro-267 is the only residue obligatorily required for receptor function. In addition, Pro-267 is required for JAK-1 binding. These results thus identify a site in the IFN-gamma receptor alpha chain required for constitutive JAK-1 association and establish that this association is critical for IFN-gamma signal transduction.

Identification and Site-Directed Mutagenesis of the Primary (2A/2B) Cleavage Site of the Hepatitis A Virus Polyprotein: Functional Impact on the Infectivity of HAV RNA Transcripts

The junction between 2A and 2B proteins of the hepatitis A virus (HAV) polyprotein is processed by the virus-encoded 3C protease to liberate the precursor for capsid proteins, but details of this cleavage remain poorly defined. We identified the location of this primary cleavage by a novel approach involving expression of HAV polypeptides in eukaryotic cells via recombinant vaccinia viruses. A substrate polyprotein spanning the putative HAV 2A/2B site was fused at its C-terminus to a poliovirus VP1 reporter sequence. This substrate was cleaved efficiently in trans by protease 3C derived from another recombinant vaccinia virus expressing a 3C precursor protein. N-terminal sequencing of the 2B-poliovirus VP1 fusion product identified the site of cleavage as the Gln836/Ala837 dipeptide, 144 residues upstream of the originally predicted site. Two mutations were introduced at the P1 position of the 2A/2B site: Gln836 → Asn, and Gln836 → Arg. Asn substitution at the P1 residue reduced the efficiency of cleavage in the vaccinia expression system and resulted in a small replication focus phenotype of virus rescued from infectious HAV RNA transcripts. Arg substitution abolished cleavage and was lethal to HAV replication. In addition to identifying the site of the primary HAV polyprotein cleavage, these results shed light on the in vivo specificities of the HAV 3C protease.

Low Affinity Binding of Interleukin-1β and Intracellular Signaling via NF-κB Identify Fit-1 as a Distant Member of the Interleukin-1 Receptor Family

The fit-1 gene gives rise to two different mRNA isoforms, which code for soluble (Fit-1S) and membrane-bound (Fit-1M) proteins related to the type I interleukin (IL)-1 receptor. To investigate IL-1 binding, we have synthesized and purified histidine-tagged polypeptides corresponding to Fit-1S and the extracellular domain of the type I IL-1 receptor using a vaccinia expression system. Fit-1S is shown to interact with IL-1 beta, but not with IL-1 alpha. However, Fit-1S binds IL-1 beta only with low affinity in contrast to the IL-1 receptor, suggesting that IL-1 beta is not a physiological ligand of Fit-1S. Moreover, expression of the membrane-bound protein Fit-1M in transiently transfected Jurkat cells did not result in activation of the transcription factor NF-kappa B following IL-1 beta treatment. However, a chimeric protein consisting of the extracellular domain of the type I IL-1 receptor and of the transmembrane and intracellular regions of Fit-1M stimulated NF-kappa B-dependent transcription as efficiently as the full-length type I IL-1 receptor. These data indicate that Fit-1M is a signaling molecule belonging to the IL-1 receptor family.

TheDfur2Gene ofDrosophila melanogaster:Genetic Organization, Expression During Embryogenesis, and Pro-Protein Processing Activity of Its Translational Product Dfurin2

The gene structure and expression of the Dfur2 gene of Drosophila melanogaster, which encodes the subtilisin-like serine endoprotease Dfurin2, was studied. The Dfur2 gene is very compact in contrast to the related Dfur1 gene, which has an estimated size of over 100 kbp. The 6-kb Dfur2 mRNA is encoded by 16 exons dispersed over a genomic region of about 9 kbp. The exon/intron organization shows conservation of intron positions not only in comparison with Dfur1, but also with the related mammalian genes FUR, PC1/PC3, PC2, and PC4. This conservation supports the hypothesis that all genes belonging to the family of subtilisin-like pro-protein processing enzymes are evolutionary related by descent from a common ancestral gene. In primer extension experiments, Dfur2 transcription initiation sites were identified in the presumed Dfur2 promoter region. This region was found to contain general RNA polymerase II promoter elements like a potential TATA box, a potential CAP signal, and several potential CCAAT boxes. Also, several sequence motifs putatively corresponding to binding sites for Drosophila transcription factors like zeste, bicoid, and engrailed were found to be present. RNA in situ hybridization experiments on Drosophila embryos revealed presumably maternal Dfur2 expression until the syncytial blastoderm (stage 5 of embryogenesis), no expression during gastrulation (stage 9), transient expression in a subset of neurons in the central nervous system of stage 12-13 embryos, and, from stage 13 onwards, expression in the developing tracheal tree. In a vaccinia expression system, the endoprotease Dfurin2 not only cleaved wild-type precursor of von Willebrand factor (pro-vWF) with pro-region cleavage site R-S-K-R decreases, but also, although to a lesser extent, pro-vWF mutants in which the P2 (vWFK-2A) or P4 (vWFR-4A) basic residue with respect to the pro-region cleavage site had been mutated. This cleavage specificity resembles that of human furin. The cleavage of pro-vWF by Dfurin2 shows that the previously reported lack of cleavage of the precursor of the beta A-chain of activin-A by Dfurin2 in this vaccinia expression system is substrate determined.

Phospholipase C-gamma 1 can induce DNA synthesis by a mechanism independent of its lipase activity.

Inositol phospholipid-specific phospholipase C (PLC) is involved in several signaling pathways leading to cellular growth and differentiation. Our previous studies reported the induction of DNA synthesis in quiescent NIH 3T3 cells after microinjection of PLC and the inhibition of serum- or Ras-stimulated DNA synthesis by a mixture of monoclonal antibodies to PLC-gamma 1. In the course of our investigation of anti-PLC-gamma 1 monoclonal antibodies, we found that each antibody exerts different inhibitory effects on the phosphatidylinositol-hydrolyzing activity of PLC-gamma 1 and that the inhibition of enzymatic activity does not correlate with the inhibition of DNA synthesis observed in the microinjection assay. PLC-gamma 1 with defective enzymatic activity was synthesized by substituting phenylalanine for histidine within the PLC-gamma 1 catalytic domain at amino acids 335 and 380, and mutant enzymes were expressed using a vaccinia expression system. The mutant enzymes were purified and microinjected into quiescent NIH 3T3 cells to evaluate their mitogenic activity. A moderate induction of DNA synthesis occurred after injection of mutant PLC-gamma 1. This mitogenic activity was inhibited by an antibody (alpha E 8-4) that does not significantly inhibit PLC-gamma 1 enzyme activity, which indicates that something else has to be inhibited. Furthermore, the partial induction of DNA synthesis observed with mutant PLC-gamma 1 was increased to levels seen with wild-type PLC-gamma 1 by coinjection of mutant PLC-gamma 1 with two second messengers, diacylglycerol and inositol trisphosphate. These results suggest that the mitogenic activity of PLC-gamma 1 does not exclusively result from the enzymatic activity of the lipase and that another activity inherent to the PLC-gamma 1 molecule can also induce DNA synthesis in quiescent cells.

Molecular Cloning and Expression of the Transformation Sensitive Epithelial Marker Stratifin: A Member of a Protein Family that has been Involved in the Protein Kinase C Signalling Pathway

We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126, and 9231 in the master two-dimensional gel database of human keratinocyte proteins) that share peptide sequences with each other, with protein 14-3-3 and with the kinase C inhibitory protein. Immunofluorescence staining of keratinocytes showed that two of these proteins (IEF SSPs 9124 and 9126) localize to the Golgi apparatus, while stratifin is distributed diffusely in the cytoplasm. Significant levels of stratifin, and in smaller amount the sample spot proteins 9124, 9125 and 9126, were detected in the medium of cultured human keratinocytes suggesting that they are partially secreted by these cells.Two-dimensional gel analysis of proteins from cultured human cells and fetal tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium.We have cloned and sequenced cDNAs coding for members of this family. The complete identity of the sequenced peptides from stratifin with the amino acid sequence translated from the stratifin cDNA clone indicated that this cDNA codes for stratifin. The identity of clones 1054, HS1 and AS1 is less clear as, with few exceptions, none of the individual peptide sequences fits the predicted protein sequences. The polypeptides synthesized by clones 1054 and HS1 in the vaccinia expression system, on the other hand, comigrate with proteins 9126 and 9124, suggesting cell-type-specific expression of members of the protein family. Database searches indicated that clone HS1 corresponds to a human T-cell cDNA 14-3-3 clone, while the high level of similarity of clones 1054 and AS1 with the 14-3-3 β and η sequences respectively, suggested that they code for the human equivalent of the two bovine proteins. Microsequence data indicated that IEF SSP 9124 corresponds to the human homolog bovine 14-3-3γ.

Human immunodeficiency virus type 2 envelope glycoprotein: Differential CD4 interactions of soluble gpl20 versus the assembled envelope complex

Utilizing a recombinant vaccinia expression system, we investigated the biological properties and CD4 receptor interactions of the envelope glycoproteins of a noncytopathic human immunodeficiency virus type 2 strain, termed HIV-2/ST, and a highly cytopathic variant derived from it. The efficiency and host cell range of syncytium formation by the recombinant glycoproteins of both viruses were highly restricted compared to those of prototypic strains of HIV (HIV-2/ROD or HIV-1/11113). However, the glycoprotein of cytopathic but not wild-type ST generated numerous large syncytia in the human T-cell line Sup T1 from which it was derived. A single cell line (Molt 4 clone 8) was permissive to fusion by both wild-type and cytopathic ST envelopes, but only the glycoprotein of cytopathic ST could be inhibited with a soluble form of the viral receptor CD4 (sCD4). While these results indicated major differences in the envelope glycoprotein-CD4 receptor interactions of wild-type versus cytopathic ST, direct and competition binding assays utilizing soluble external glycoprotein (SU) and sCD4 surprisingly revealed equivalent low binding affinity for both viruses. From these experiments we conclude that relevant biological properties (e.g., CD4 binding, cytopathic potential, and sCD4 neutralization) of HIV viruses which differ in their pathogenic potential are reflected in the sCD4 interactions of the assembled native envelope complex (as on cell or virion surfaces) but not the soluble SU glycoprotein.

Processing of nonstructural proteins NS4A and NS4B of dengue 2 virus in vitro and in vivo

The production, from polyprotein precursors, of two hydrophobic nonstructural proteins of dengue 2 (DEN2) virus, NS4A and NS4B, was analyzed both in cell-free systems and in infected cells. In DEN2-infected cells, NS4B is first produced as a peptide of apparent size 30 kDa; NS4B is then post-translationally modified, in an unknown way, to produce a polypeptide of apparent size 28 kDa. The rate and extent of NS4B modification was found to be cell-dependent; in BHK cells the half-time for the conversion of the 30-kDa form to the 28-kDa form was 90 min. N-terminal sequence analysis of NS4B suggests that the N-terminus is produced by an enzyme with a specificity similar to that of signalase. Low levels of a putative polyprotein, NS4AB, were also found in mammalian cells, but not mosquito cells, infected with DEN2, suggesting that a small proportion of DEN2 4A/4B cleavage can occur post-translationally or that some nonstructural polyproteins escape normal processing. Cleavage of the 4A/4B bond in infected cells required expression of DEN2 sequences in addition to those in NS4A and NS4B, as NS4AB produced in cells by a vaccinia expression system was not cleaved. NS4AB produced in cells by a vaccinia expression system was modified post-translationally, presumably in the same way as NS4B. We show that upon translation of DEN2 polyproteins in a cell-free system, the N-terminus of NS4A is generated by cleavage by the viral nonstructural proteinase NS3 and that processing of DEN2 polyproteins occurs with a preferred, but nonobligatory order.

The 5′ noncoding region sequence of the Choristoneura biennis entomopoxvirus spheroidin gene functions as an efficient late promoter in the mammalian vaccinia expression system

About 100 nucleotides of DNA sequence at the 5′ noncoding region of the Choristoneura biennis entomopoxvirus spheroidin gene was chemically synthesized and inserted into a vaccinia expression vector, interrupting the vaccinia thymidine kinase gene. When the bacterial β-galactosidase gene was introduced downstream of this sequence and a recombinant vaccinia virus containing these inserts was obtained by homologous recombination, β-galactosidase was shown to be expressed at a high level late in the vaccinia infection cycle. The level of β-galactosidase expression was four- to fivefold higher with this spheroidin-vaccinia recombinant virus than with a similar recombinant in which the β-galactosidase gene was under the control of the vaccinia 7.5-kDa promoter. Primer extension and S1 mapping of the 5′ terminus of the β-galactosidase transcript located the transcription initiation site within the spheroidin DNA sequence, confirming the promoter nature of this DNA sequence in the vaccinia system. Dot blot analysis indicated that the difference in β-galactosidase expression with these two recombinant viruses can be attributed to the difference in their transcript levels. We also demonstrated that full promoter activity encoded in the spheroidin 5′ noncoding sequence was contained within a 38-nucleotide DNA fragment.

The oct-1 homeo domain contacts only part of the octamer sequence and full oct-1 DNA-binding activity requires the POU-specific domain.

The ubiquitous octamer-binding protein oct-1 contains a POU domain required for DNA binding, which can be subdivided into a POU-specific domain and a POU homeo domain. We have overproduced the POU domain and the POU homeo domain in a vaccinia expression system, purified both polypeptides to near homogeneity, and compared their DNA-binding properties. In contrast to the POU domain, the homeo domain protects only part of the octamer sequence in the Ad2 origin against breakdown by DNase I or hydroxyl radicals. Analysis of purine contacts by DMS and DEPC interference assays shows that the Ad2 octamer can be divided into two regions: one that is recognized both by the POU domain and the homeo domain in an identical fashion, and one that is only recognized by the POU domain. This suggests that the POU-specific domain is responsible for the additional contacts located at one side of the octamer. In agreement with this, mutating the first 3 nucleotides (ATG) of the octamer affected binding by the POU domain but not by the homeo domain. The apparent binding affinities to different octamer sites were compared. The homeo domain binds 600-fold less efficiently to the canonical octamer sequence (ATGCAAAT) than the POU domain. The difference is only sevenfold for the Ad2 octamer, whereas both Kd values are almost identical for the HSV ICP4 TAATGARAT motif. Both the POU and homeo domains recognize target sequences for mammalian homeo box proteins. We conclude that the octamer can act as a bipartite recognition sequence for oct-1 and that the POU-specific domain contributes to the binding affinity, as well as to the specificity, by providing additional contacts.

Association of antibodies blocking HIV‐1 gp160‐sCD4 attachment with virus neutralizing activity in human sera

Sera, from HIV-1 and HIV-2 seropositive individuals, were tested for the presence of antibodies able to inhibit the binding (BI) of HIV-IIIB gp 160 (produced in mammalian cells using a vaccinia expression system) to the extracellular portion of the CD4 receptor. A competition enzyme immunoassay (EIA) with soluble CD4 (sCD4) was used. BI antibodies were highly prevalent among HIV-1 seropositives but not in HIV-2 infected individuals. Attempts to localize the binding site for these BI antibodies on the primary sequence of gp 120 by using synthetic peptides encompassing the putative CD4 binding site on gp 120 (aa 397-439) were not successful. This study did not reveal a significant correlation between the presence of BI antibodies and disease evolution. BI antibody titres correlated less well with anti-gp 160 titres (r = 0.51, P less than or equal to 0.011) than with neutralizing antibody (NA) titres using either the isolates HIV-SF2 (SF2) (r = 0.77, P less than or equal to 0.000) and HIV-MN (MN) (r = 0.61, P less than or equal to 0.002) or the isolate HIV-IIIB (HX10) (r = 0.89, P less than or equal to 0.000) of which the gp 160 for the assays was derived. An HIV-IIIB neutralizing serum, elicited in a rabbit by immunization with a 17-mer synthetic peptide derived from the third variable domain (V3) of gp 120, did bind gp 160 without inhibiting the subsequent attachment of sCD4 to gp 160.(ABSTRACT TRUNCATED AT 250 WORDS)