Structural and functional similarities between HIV-1 reverse transcriptase and the Escherichia coli RNA polymerase beta' subunit.

Four monoclonal antibodies (MAbs) recognizing HIV-1 reverse transcriptase (RT) were shown here to cross-react with the beta' subunit of Escherichia coli RNA polymerase (RNAP). The anti-RT MAbs bind to a peptide comprising residues 294-305 of the RT amino acid sequence. Computer analyses revealed sequence similarity between this peptide and two regions of the RNAP beta' subunit. MAb-binding studies using RT mutants suggested that the epitope is located to amino acids 652-663 of the beta' sequence. One of the MAbs which inhibited the polymerase activity of RT also mediated a dose dependent inhibition of the RNAP activity.

A monoclonal antibody defines a novel HIV type 1 Tat domain involved in trans-cellular trans-activation.

In the present study, a CAT assay, a beta-galactosidase assay, and immunofluorescence analysis have been used to study the cellular uptake of the HIV-1 Tat protein. An anti-Tat MAb binding to an epitope comprising both the basic domain and the RGD sequence inhibits trans-activation by exogenous Tat. Two different full-length recombinant Tat proteins were used in these studies. The inhibitory MAb, however, recognized only one of the recombinant Tat proteins. Immunofluorescence analysis demonstrated that only the Tat protein recognized by the inhibitory anti-Tat MAb was taken up by COS and HeLa cells. This indicates that there are conformational differences between the two Tat proteins and that a correct folding of the epitope recognized by the anti-Tat MAb is required for cellular uptake. The recombinant Tat taken up by the cells was distributed between the nucleoli, the nucleoplasm, and along the nuclear membrane. Interactions between Tat and serum components were shown in vitro and also inhibition of trans-cellular trans-activation by fetal calf serum in tissue culture was demonstrated. The specific inhibition of the cellular uptake of Tat by an anti-Tat monoclonal antibody and the blocking of uptake by serum components implies specific binding of Tat to the cell membrane.

Monoclonal antibodies against human immunodeficiency virus type 1 integrase: epitope mapping and differential effects on integrase activities in vitro.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the integration of viral DNA into the host chromosome, an essential step in retroviral replication. As a tool to study the structure and function of this enzyme, monoclonal antibodies (MAbs) against HIV-1 IN were produced. Epitope mapping demonstrated that the 17 MAbs obtained could be divided into seven different groups, and the selection of MAbs representing these groups were tested for their effect on in vitro activities of IN. Four groups of MAbs recognized epitopes within the region of amino acids (aa) 1 to 16, 17 to 38, or 42 to 55 in and around the conserved HHCC motif near the N terminus of IN. MAbs binding to these epitopes inhibited end processing and DNA joining and either stimulated or had little effect on disintegration and reintegration activities of IN. Two MAbs binding to epitopes within the region of aa 56 to 102 in the central core or aa 186 to 250 in the C-terminal half of the protein showed only minor effects on the in vitro activities of IN. Three Mabs which recognized on epitope within the region of aa262 to 271 of HIV-1 IN cross-reacted with HIV-2 IN. MAbs binding to this epitope clearly inhibited end processing and DNA joining and stimulated or had little effect on disintegration. In contrast to the N-terminal-specific MAbs, these C-terminal-specific MAbs abolished reintegration activity of IN.

Characterization of the DNA-binding activity of HIV-1 integrase using a filter binding assay.

Based on the selective binding of proteins and DNA to distinct filter materials a double-layered dot blot radio assay was developed to evaluate the binding of DNA to HIV-1 integrase. In this assay the DNA-binding was found to be independent of Mn2+ concentration, inhibited by concentrations of Mg2+ above 5 mM, abolished by zinc chelation and inhibited by monoclonal antibodies reacting with either the N-terminal or C-terminal regions of integrase. Atomic absorption spectroscopy revealed the molar ratio between integrase and zinc to be close to 1. It is concluded that both the N-terminal and the C-terminal regions of integrase are involved in DNA-binding and that the reported double-layered dot blot radio assay is well suited for further characterization of the integrase.

DNA end-joining in extracts from human cells.

DNA end-joining is a central feature of several DNA recombination processes. A DNA end-joining activity present in extracts prepared from cells of the human SupT1 lymphocyte cell line was characterised. Joining of blunt ends and ends having complementary single-strand extensions (SSEs) were precise with no insertion or deletion of substrate base pairs. DNA sequencing analysis showed that molecules having non complementary ends of the same polarity, or molecules having one blunt end and one end with a SSE, were joined without loss of nucleotide sequences in the double-stranded region of the substrate molecule. The joining patterns observed have several features that are consistent with DNA end-joining activities previously observed in vitro in extracts from Xenopus eggs and in vivo in mammalian cells and yeast.

Systemic and mucosal antibody responses to group B streptococci following immunization of the colonic-rectal mucosa.

The cervico-vaginal mucosa is poorly designed for inducing a mucosal immune response, but it can effect such a response evoked at other mucosal sites. This study was undertaken to determine whether colonic-rectal immunization with group B streptococci (GBS) might induce a local cervico-vaginal immune response. Mice were immunized with either fragmented GBS rectally, whole GBS rectally, or whole GBS subcutaneously. Cholera toxin (CT) was used as an adjuvant for the rectal immunizations. Following colonic-rectal immunization with whole GBS, the mean anti-GBS IgA antibody level in vaginal secretions was 735 kU/ml, with individual values reaching 3480 kU/ml. Corresponding levels of IgA antibodies never exceeded 10 kU/ml in serum and intestinal secretions, or 90 kU/g in feces. In vaginal secretions IgA antibodies to GBS also constituted a much larger fraction of total IgA than in serum, intestinal secretions and feces. Immunizations with fragmented GBS produced much lower IgA responses. Anti-GBS IgA response at the inductive site in the colon-rectum was not significant, as opposed to a strong anti-CT IgA response. Except in serum, the anti-GBS IgG responses to colonic-rectal immunizations were generally low, or absent. The results may provide a basis for the development of mucosal vaccines against GBS-infection.

Absence of HTLV-1 related sequences in MS from high prevalence areas in western Norway.

In Western Norway, long-term follow up epidemiological studies have revealed significant increases in the incidence and prevalence rates of multiple sclerosis (MS) in stable populations, indicating the impact of exogenous factors. In this study 183 MS patients and 102 controls from high prevalence areas in Western Norway were investigated for human T-lymphotropic virus type I (HTLV-1) related sequences by polymerase chain reaction. Using primers targeting the gag, pol and env genes in the HTLV-1 provirus genome, no amplification products were detected in the 183 MS patients or 102 controls. The results strongly suggest that neither HTLV-1 nor a closely related retrovirus participate in the aetiology of MS.

Nuclear and mitochondrial forms of human uracil-DNA glycosylase are encoded by the same gene.

Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of M(r) = 33,800 contains an N-terminal sequence of 77 amino acids not present in the presumed mature form of M(r) = 25,800. This led to the hypothesis that the N-terminal sequence might be involved in intracellular targeting. To examine this hypothesis, we analysed UDG from nuclei, mitochondria and cytosol by western blotting and high resolution gel filtration. An antibody that recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they are products of the same gene. The nuclear and mitochondrial form had an apparent M(r) = 27,500 and the cytosolic form an apparent M(r) = 38,000 by western blotting. Gel filtration gave essentially similar estimates. An antibody with specificity towards the presequence recognised the cytosolic form of M(r) = 38,000 only, indicating that the difference in size is due to the presequence. Immunofluorescence studies of HeLa cells clearly demonstrated that the major part of the UDG activity was localised in the nuclei. Transfection experiments with plasmids carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochondria, whereas UNG lacking the presequence was translocated to the nuclei. We conclude that the same gene encodes nuclear and mitochondrial uracil-DNA glycosylase and that the signals for mitochondrial translocation resides in the presequence, whereas signals for nuclear import are within the mature protein.

Complex-type N-linked oligosaccharides of gp120 from human immunodeficiency virus type 1 contain sulfated N-acetylglucosamine.

The major envelope glycoproteins gp120 and gp41 of human immunodeficiency virus type 1, the causative agent for human AIDS, contain numerous N-linked oligosaccharides. We report here our discovery that N-acetylglucosamine residues within the complex-type N-linked oligosaccharides of both gp120 and its precursor, gp160, are sulfated. When human Molt-3 cells persistently infected with human T-cell leukemia virus IIIB were metabolically radiolabeled with 35SO4, gp160, gp120, and to some extent gp41 were radiolabeled. The 35SO4-labeled oligosaccharides were quantitatively released by N-glycanase treatment and were bound by immobilized Ricinus communis agglutinin I, a lectin that binds to terminal beta-galactosyl residues. The kinetics of release of sulfate upon acid hydrolysis from 35SO4-labeled gp120 indicate that sulfation occurs in a primary sulfate ester linkage. Methylation analysis of total glycopeptides from Molt-3 cells metabolically radiolabeled with [3H]glucosamine demonstrates that sulfation occurs at the C-6 position of N-acetylglucosamine. Fragmentation of the gp120-derived 35SO4-labeled glycopeptides by treatment with hydrazine and nitrous acid and subsequent reduction generated galactosyl-anhydromannitol-6-35SO4, which is the expected reaction product from GlcNAc-6-sulfate within a sulfated lactosamine moiety. Charge analysis of the [3H]galactose- and [3H]glucosamine-labeled glycopeptides from gp120 and gp160 indicates that approximately 14% of the complex-type N-linked oligosaccharides are sulfated.

Characterization of HIV-1 reverse transcriptase with antibodies indicates conformational differences between the RNAse H domains of p 66 and p 15.

Antibody binding to the p 66 and p 15 RNase H regions of HIV-1 reverse transcriptase was compared using a polyclonal rabbit immune serum raised against a synthetic peptide from the RNase H region of reverse transcriptase (aa 511-527) and six monoclonal antibodies binding to discontinuous epitopes in the RNase H region of p 66. The antigens used in Western blot analysis included recombinantly expressed homodimeric p 66 digested with the HIV-1 protease for generation of the p 51 and p 15 polypeptides and two different length RNase H domains expressed as Trp E fusion proteins (aa 410-560 and aa 441-560). The polyclonal rabbit antibody binding to a continuous epitope recognized both the Trp E-fusion proteins and also the polypeptides p 66 and p 15 generated by processing of homodimeric p 66 with the viral protease. Two additional cleavage products with estimated molecular weights of 9 and 11 kDa were also detected. The anti-RNase H MAbs binding to discontinuous epitopes recognized only the RNase H domain of the p 66 polypeptide and the Trp E-RNase H fusion protein when this was expressed together with the C-terminal part of the polymerase domain. The results indicate conformational differences between the RNase H domain of the p 66 subunit and the RNase H p 15 polypeptide.

Purification to homogeneity and characterization of a redoxyendonuclease from calf thymus.

A redoxyendonuclease from calf thymus was purified to apparent homogeneity. The redoxyendonuclease recognized and induced cleavage of DNA damaged by ultraviolet light. The enzyme preparation produced a single band of a relative molecular mass of approximately 34 kDa upon SDS/PAGE. The apurinic/apyrimidinic endonuclease and the DNA glycosylase activities remained associated in the apparently homogeneous preparation of the enzyme. The redoxyendonuclease activity displayed a broad pH optimum between pH 5.0-8.5 and exhibited no requirement for divalent cations. By application of FPLC columns Mono-S, Mono-Q and Mono-P, the isoelectric point (pI) of the enzyme was found to be approximately 8.0. Using the DNA sequencing procedure of Maxam and Gilbert [Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560] the purified enzyme was found to incise ultraviolet-light-irradiated DNA at pyrimidine sites as observed previously with a more crude form of the enzyme. While the most frequently cleavaged sites for the crude preparation were at cytosine residues, the apparently homogeneous enzyme preparation frequently induced cleavage sites at both cytosine and guanine residues. Predominant incision induced by the apparently homogeneous preparation was observed at guanine residues when a particular DNA sequence was used as substrate. Furthermore, the 16 N-terminal amino acid residues of the purified enzyme were identified. The sequence did not show any significant similarity to other known proteins.

Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and RNase H activities.

Lysates from E. coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice. Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated. Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells. Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT. Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT. The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT. In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide. MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity. Of these, three MAbs also inhibited the RNase H activity. Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity.

gp160 of HIV-I synthesized by persistently infected Molt-3 cells is terminally glycosylated: evidence that cleavage of gp160 occurs subsequent to oligosaccharide processing.

The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent Mr 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV IIIB). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetyl-galactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [35S]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a half-time of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment.

Transcellular transactivation by the human immunodeficiency virus type 1 tat protein.

The human immunodeficiency virus type 1 (HIV-1) transactivator (tat) protein produced in one cell activated HIV-1 promoter-directed gene expression in a second cell, provided the cells were in direct contact with one another. This observation suggests that the tat protein produced in HIV-1-infected cells has a physiological effect on neighboring cells.

Human uracil-DNA glycosylase complements E. coli ung mutants.

We have previously isolated a cDNA encoding a human uracil-DNA glycosylase which is closely related to the bacterial and yeast enzymes. In vitro expression of this cDNA produced a protein with an apparent molecular weight of 34 K in agreement with the size predicted from the sequence data. The in vitro expressed protein exhibited uracil-DNA glycosylase activity. The close resemblance between the human and the bacterial enzyme raised the possibility that the human enzyme may be able to complement E. coli ung mutants. In order to test this hypothesis, the human uracil-DNA glycosylase cDNA was established in a bacterial expression vector. Expression of the human enzyme as a LacZ alpha-humUNG fusion protein was then studied in E. coli ung mutants. E. coli cells lacking uracil-DNA glycosylase activity exhibit a weak mutator phenotype and they are permissive for growth of phages with uracil-containing DNA. Here we show that the expression of human uracil-DNA glycosylase in E. coli can restore the wild type phenotype of ung mutants. These results demonstrate that the evolutionary conservation of the uracil-DNA glycosylase structure is also reflected in the conservation of the mechanism for removal of uracil from DNA.

Action of spermidine, N1-acetylspermidine, and N8-acetylspermidine at apurinic sites in DNA.

The cleavage efficiency of spermidine and its acetyl derivatives (N1-acetylspermidine and N8-acetylspermidine) at apurinic sites in DNA were examined by PAGE-urea analysis. The three polyamines induced different rates of cleavage when compared at 1 mM concentrations. The order of effectiveness were: spermidine greater than N8-acetylspermidine greater than N1-acetylspermidine. Thus a decrease in efficiency was observed when the first order amino-groups of spermidine were blocked. The N-8amino-group of spermidine was less effective in inducing cleavage at AP-sites than the N1-amino-group. Among several proposed models of polyamine-DNA interactions, our results can best be explained by the model postulated by Liquori et al.

Chromosomal assignment of human uracil-DNA glycosylase to chromosome 12.

Using Southern blot analysis of DNA from a panel of rodent-human somatic cell hybrids with known karyotypes, we have assigned the human uracil-DNA glycosylase gene to chromosome 12.

A short review of biotechnological methods of relevance to modern vaccine development.

The development of new methods and techniques in biotechnology over the last two decades has opened the way for fresh strategies to develop new and better vaccines. The relevant techniques have been developed through important discoveries in immunology, gene technology and polymer chemistry. The purpose of this short review is to discuss these techniques in the light of vaccine development, without going into details regarding each specific disease and vaccine.