Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus.

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem-loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

Endogenous DNA damage and testicular germ cell tumors.

Testicular germ cell tumors are comprised of two histologic groups, seminomas and non-seminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable levels of net endogenous DNA damage. To test our hypothesis, we conducted a case-case analysis of 51 seminoma and 61 non-seminoma patients using data and specimens from the Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort. A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95% CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modelled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with non-seminoma compared with seminoma (OR(50th percentile) = 3.31, 95% CI: 1.00, 10.98; OR(75th percentile) = 3.71, 95% CI: 1.04, 13.20; p for trend = 0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR(50th percentile) = 2.27, 95% CI: 0.75, 6.87; OR(75th percentile) = 2.40, 95% CI: 0.75, 7.71; p for trend = 0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that net endogenous levels are higher in patients who develop non-seminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

Functions of HIV envelope glycans.

The unusually highly glycosylated state of the major envelope glycoprotein (gp160) of the human immunodeficiency virus has offered a challenge to both glycobiologists and virologists. What is the functional significance of such a mass of glycans and how might they be manipulated to disadvantage virus pathogenesis? Some answers to each of these questions have already been obtained: N-linked glycans are necessary for the creation, but not the maintenance, of a bioactive conformation, and drug-induced alteration of the glycosylation pattern can lead to impaired virus infectivity. As a model for studying glycan function and as a target for antiviral therapy, gp160 represents a unique candidate.

Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans.

The removal or repair of DNA damage has a key role in protecting the genome of the cell from the insults of cancer-causing agents. This was originally demonstrated in individuals with the rare genetic disease xeroderma pigmentosum, the paradigm of cancer genes, and subsequently in the relationship between mismatch repair and colon cancer. Recent reports suggest that individuals with less dramatic reductions in the capacity to repair DNA damage are observed at polymorphic frequency in the population; these individuals have an increased susceptibility to breast, lung, and skin cancer. We report initial results from a study to estimate the extent of DNA sequence variation among individuals in genes encoding proteins of the DNA repair pathways. Nine different amino acid substitution variants have been identified in resequencing of the exons of three nucleotide excision repair genes (ERCC1, XPD, and XPF), a gene involved in double-strand break repair/recombination genes (XRCC3), and a gene functioning in base excision repair and the repair of radiation-induced damage (XRCCI). The frequencies for the nine different variant alleles range from 0.04 to 0.45 in a group of 12 healthy individuals; the average allele frequency is 0.17. The potential that this variation, and especially the six nonconservative amino acid substitutions occurring at residues that are identical in human and mouse, may cause reductions in DNA repair capacity or the fidelity of DNA repair is intriguing; the role of the variants as cancer risk factors or susceptibility alleles remains to be addressed.

The glycosylation pattern of baculovirus expressed envelope protein E2 affects its ability to prevent infection with bovine viral diarrhoea virus.

We have investigated the role of glycosylation of the envelope glycoprotein E2 of bovine viral diarrhoea virus (BVDV), produced in insect cells, in BVDV infection. When amino acids predicated to code for the C-terminal N-linked glycosylation site were mutated the resulting protein was less efficient than wild type protein at preventing infection of susceptible cells with BVDV. In addition, mutational analysis showed that a further two predicted N-terminal N-linked glycosylation sites of E2 are required for efficient production of recombinant protein.

Monoclonal antibodies to a CD4 peptide derivative which includes the region corresponding to an immunoglobulin CDR3: evidence of the involvement of pre-CDR3-related region in HIV-1 and host cell interaction.

A CD4 peptide of amino acid residues 68-130 [CD4(68-130)], which had the capacities to inhibit HIV-1 replication and HIV-1-induced syncytium formation, was used as an immunogen for the preparation of mAb. The mAbs prepared were classified into at least five types (I-V) in terms of their recognition sites by ELISA using various kinds of smaller CD4 peptides. Among them, the type I mAb no. 35 recognizing amino acid residues 72-84, which lies just before the region corresponding to an immunoglobulin third complementarity-determining region (CDR3), showed the strongest effects in reducing both HIV-1 infection and HIV-1-induced syncytium formation, although a large amount of no. 35 mAb was necessary to reduce such HIV-1 activities compared with those of anti-Leu-3a and OKT4A mAbs which recognize CD4 epitopes near a portion corresponding to an immunoglobulin CDR2. Western blot analysis showed that the reactivities of CD4 molecule in CD4-positive cells or sCD4 molecule with types I-V mAbs were stronger than that with anti-Leu-3a mAb. Flow cytometry showed that no. 35 mAb was faintly reactive with native CD4 molecule on cell surface at the concn showing the inhibitory effects on HIV-1 infection and syncytium formation. In addition, a smaller peptide CD4(66-92), one of the good epitope peptides for no. 35 mAb, also showed strong inhibitory effect on HIV-1 infection as well as a weaker inhibitory effect on syncytium formation. These results suggest that, in addition to the CD4 CDR2-related region, the pre-CDR3-related region is also involved in the early events of the interactions between the host cell and HIV-1.

Expression and purification of p24, the core protein of HIV, using a baculovirus-insect cell expression system.

The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been genetically manipulated to yield a recombinant virus capable of expressing p24, the major core protein of HIV-1, in insect cell culture. The expressed product is a p24 protein flanked by short regions of p17 at the amino terminus and p12 at the carboxy terminus. It has been identified and characterized using monoclonal antibodies on Western blots and by amino-terminal sequence analysis. The presence of p24 in the soluble fraction of infected cells following lysis by detergent or sonication, combined with a high level of expression (in excess of 50 mg/l of culture) facilitates the enrichment of large quantities of recombinant HIV antigen in a simple two-step procedure involving ammonium sulphate fractionation and gel filtration. p24 antigen purified in this way is shown to be an efficient diagnostic reagent.

Complement activation upon binding of mannan-binding protein to HIV envelope glycoproteins.

OBJECTIVE: Retroviruses can activate the complement system in the absence of antibodies, and the purpose of this study was to examine whether the serum collection, mannan-binding protein (MBP), could mediate such complement activation. DESIGN: Virus envelope proteins gp120 and gp110 from HIV-1 and HIV-2 were incubated in microtitre wells coated with anti-gp120 or anti-gp110 antibodies. After further incubation with serum, complement activation was measured as deposition of complement factor C4 and C3 onto the wells. Deposited C4 and C3 were detected with enzyme-labelled antibodies. Normal human serum depleted of endogenous lectins by affinity chromatography was used as the complement source. Serum from C1q-deficient patients was used in some experiments. Complement activation was then assessed with and without prior addition of MBP to the wells. Complement activation was also correlated with the quantity of endogenous MBP in a number of normal sera. RESULTS: Complement activation by HIV envelope glycoproteins was found to be mediated by the binding of MBP to carbohydrates on natural envelope protein produced in virus-infected cells, as well as on glycosylated recombinant envelope proteins produced in insect cells. Non-glycosylated recombinant envelope proteins produced in Escherichia coli did not induce this type of complement activation. CONCLUSIONS: Activation of the classical complement pathway by retrovirus envelope proteins can be initiated by the binding of MBP to carbohydrate side chains of envelope glycoproteins.

Characterization of recombinant gp120 and gp160 from HIV-1: binding to monoclonal antibodies and soluble CD4.

We compared four preparations of recombinant HIV-1 envelope glycoprotein: mammalian (Chinese hamster ovary cells) gp120 (Celltech); baculovirus gp120 from American Biotechnologies Inc. (ABT) and from MicroGeneSys (MGS); and baculovirus gp160 (Institute of Virology, Oxford, UK). Each envelope glycoprotein binds to a neutralizing monoclonal antibody (MAb) directed against the V3 loop, confirming the integrity of this type-specific neutralization epitope. MGS gp120 binds abnormally well to a MAb which recognizes an epitope preferentially exposed on denatured gp120. Consistent with this finding, MGS gp120 binds to soluble CD4 (sCD4) with an affinity 50-100-fold lower than that of Celltech gp120. The affinity of Celltech gp120 from sCD4 is 2.3 nM, indistinguishable from that of gp120 extracted from HIV-1 virions. Baculovirus gp120 (ABT) and gp160 also have a high affinity for sCD4. A significant proportion of anti-gp120 antibodies in HIV-positive human sera recognize epitopes that are dependent on the mammalian glycosylation pattern, and a human HIV-positive serum inhibits the binding of mammalian gp120 to sCD4 five- to 10-fold more potently than it inhibits baculovirus gp120 binding to sCD4.

Differential expression of influenza N protein and neuraminidase antigenic determinants in Escherichia coli.

Two influenza gene products of similar size and codon usage have been expressed in Escherichia coli under control of the phage lambda pR promoter. The influenza N protein (NP) was expressed in its entirety after fusion to a short (12 amino acid) segment of the lambda cro gene product and constituted about 1-2% of total soluble cell protein after induction. By contrast, constructions using the full length neuraminidase (NA) gene failed to give rise to detectable amounts of NA antigen after fusion to either the 12 amino acid Cro peptide or after fusion to bacterial beta-galactosidase (beta gal). Rather, expression of NA antigenic determinants was only achieved after deletion of coding sequences at the 3' end of the beta gal-NA fusion construct such that the encoded protein precipitated within the cell.

Molecular cloning and nucleotide sequence of human alpha 1 acid glycoprotein cDNA.

A cDNA clone has been isolated from a pEX expression library that encodes alpha 1 acid glycoprotein. We present the complete nucleotide sequence encoding this protein and compare the derived amino acid sequence with pre-existing data.

Nonsense mutations in the vpr gene of HIV-1 during in vitro virus passage and in HIV-1 carrier-derived peripheral blood mononuclear cells.

Long-term, persistent infection by HIV-1 is a prerequisite for the development of AIDS. However, little is known of the determinants required for HIV-1 to cause persistence. We have reported previously that persistent infection of a T cell line by a cytopathogenic strain of HIV-1 became increasingly likely with in vitro serial passage of the virus. DNA sequencing of the persistent strains revealed a nonsense mutation in the vpr gene in all isolates tested. Here, we report the development and use of a semi-quantitative PCR method to detect the vpr nonsense mutation within populations of virus. Our results show that vpr mutants also arise in cells during acute infection and increase progressively with serial passage of the virus. In addition, HIV-1-seropositive individuals were examined and found to carry the same vpr nonsense mutation at high frequency in virus-infected PBMC. These data are consistent with a mechanism of HIV-1 persistence in vivo and in vitro in which virus cytopathogenic potential is lost by the build up of nonsense mutations in vpr.

Factors affecting HPRT mutant frequency in T-lymphocytes of smokers and nonsmokers.

The frequency of thioguanine-resistant, hypoxanthine phosphoribosyltransferase-deficient lymphocytes in the peripheral blood of human subjects was used to study the genotoxic effects of smoking. Sixty-two nonsmokers and 58 smokers, aged 19 to 45 years with average ages of 30 and 32 years, respectively, and with no other known exposures, were studied using an in vitro assay of the frequency of mutant lymphocyte clones. Analysis of variance explained 68% of the variation in the mutant frequencies. Mutant frequency was dependent upon lymphocyte cloning efficiency, length of smoking history, age, and interactions between these variables. Four nonsmokers and three smokers had high mutant frequencies that were not explained by these variables. Mutant frequencies were inversely related to lymphocyte cloning efficiencies; the effect was twice as great for smokers as for nonsmokers. The time-dependent effect of smoking dominated, with mutant frequency increasing 10%/year of smoking as compared with an independent 1%/year of age. Smoking had a greater effect on young smokers' lymphocytes. Heterogeneity of mutant frequency among both smokers and nonsmokers and its implications for use of lymphocyte mutation assays as biomarkers are discussed.

Release of [3H]noradrenaline from descending tracts in the cat spinal cord in vivo.

The release of [3H]noradrenaline into the perfused central canal of the cat lumbar-sacral spinal cord was monitored in vivo. Stimulation of descending tracts produced an increase efflux into artificial cerebrospinal fluid containing 10(-6) M phenoxybenzamine which could be dissociated from any concurrent rise in blood pressure. No release was produced by stimulating dorsal roots over the length of cord perfused. It appears, therefore, that noradrenaline can be released from descending nerve terminals, but not from dorsal root afferents in the spinal cord.

Secretion and purification of HCV E1 protein forms as glutathione-S-transferase fusion in the baculovirus insect cell system.

We have expressed the E1 protein of Hepatitis C Virus (HCV) in a new recombinant form by using a baculovirus transfer vector directing the expression of proteins fused to the carboxy-terminus of glutathione-S-transferase (GST). The E1 domain was expressed varying at its carboxy terminus in order to retain (GST-E1) or delete (GST-E1b) the C-terminal hydrophobic region that may be involved in membrane association. Following infection with the recombinant virus, GST-E1b was efficiently secreted into the culture media and could be purified in a single step with the minimum of denaturation by glutathione affinity chromatography. The purified product was specifically immunoprecipitated by HCV positive human sera suggesting the maintenance of an immuno-relevant tertiary structure despite removal of the hydrophobic anchor. By contrast, cells infected with a recombinant baculovirus expressing GST-E1 gave a fusion protein with an appropriate molecular weight but also a series of polypeptides of lower molecular weight consistent with cleavage at the C-terminus of E1. GST-E1 was not secreted into the medium and was associated predominantly with the membrane fraction following cell disruption; the lower molecular weight forms were soluble and secreted.

Parameters influencing measurement of the Gag antigen-specific T-proliferative response to HIV type 1 infection.

We have analyzed factors that might influence the in vitro quantitation of the T-proliferative response to HIV-1 Gag antigens, a common and increasingly used clinical measurement of helper T cell function in the context of HIV-1 infection. We have compared the rate and extent of T cell proliferation in freshly prepared and previously frozen PBMC samples, and have concluded that frozen cells can be used successfully; we have assessed whether the suppression of any HIV-1 replication in the PBMC cultures affects the extent of T cell proliferation; we have studied which forms of the Gag antigens are the most efficient at inducing T cell proliferation. From the latter experiments, we conclude that Gag proteins that include p17, and perhaps also p7, sequences flanking the central p24 capsid protein, are better stimulants than proteins that comprise only p24 sequences.

Expression of HIV-1 gp120 and human soluble CD4 by recombinant baculoviruses and their interaction in vitro.

The soluble domains of the envelope glycoprotein of HIV-1 (gp120) and human CD4 (sCD4) have been individually expressed in insect cells using recombinant baculoviruses. Each product is secreted from infected cells and accumulates in the surrounding media to levels of 1-2 mg/liter of 2 x 10(9) cells. Both molecules have full biological activity, and conditioned media from infected cells have been used to establish a simple assay for gp120-sCD4 interaction that is highly specific and amenable to mass screening. The crystallization of sCD4 purified from this source is reported.

Characterization of in vivo somatic mutations at the hypoxanthine phosphoribosyltransferase gene of a human control population.

The ability to recognize a change in mutation spectrum after an exposure to a toxic substance and then relate that exposure to health risk depends on the knowledge of mutations that occur in the absence of exposure. Toward this end, we have been studying both the frequency and molecular nature of mutations of the hypoxanthine phosphoribosyltransferase (hprt) gene in peripheral blood lymphocytes as surrogate reporters of genetic damage. We have analyzed mutants, one per donor to ensure independence, from a control population in which the quantitative effects of smoking and age on mutant frequency have been well defined. Analyses of cDNA and genomic DNA by polymerase chain reaction and sequencing have identified the mutations in 63 mutants, 45 from males and 18 from females, of which 34 were smokers and 29 were nonsmokers. Slightly less than half of the mutations were base substitutions; they were predominantly at GC base pairs. Different mutations at the same site indicated that there are features of the hprt polypeptide that affect the mutation spectrum. Two pairs of identical mutations indicated that there may also be hot spots. Mutations not previously reported have been detected, indicating that the mutation spectrum is only partly defined. The remainder of the mutations were deletions or insertions/duplications; deletions ranged from one base pair to complete loss of the locus. Despite a small average increase in mutant frequency for smokers, an increased proportion of base substitutions at AT base pairs in smokers (p = 0.2) hinted at a smoking-associated shift in the mutation spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping the end points of large deletions affecting the hprt locus in human peripheral blood cells and cell lines.

We have examined the extent of HPRT- total gene deletions in three mutant collections: spontaneous and X-ray-induced deletions in TK6 human B lymphoblasts, and HPRT- deletions arising in vivo in T cells. A set of 13 Xq26 STS markers surrounding hprt and spanning approximately 3.3 Mb was used. Each marker used was observed to be missing in at least one of the hprt deletion mutants analyzed. The largest deletion observed encompassed at least 3 Mb. Nine deletions extended outside of the mapped region in the centromeric direction (> 1.7 Mb). In contrast, only two telomeric deletions extended to marker 342R (1.26 Mb), and both exhibited slowed or limited cell growth. These data suggest the existence of a gene, within the vicinity of 342R, which establishes the telomeric limit of recoverable deletions. Most (25/41) X-ray-induced total gene deletion mutants exhibited marker loss, but only 1/8 of the spontaneous deletions encompassed any Xq26 markers (P = 0.0187). Furthermore, nearly half (3/8) of the spontaneous 3' total deletion breakpoints were within 14 kb of the hprt coding sequence. In contrast, 40/41 X-ray-induced HPRT- total deletions extended beyond this point (P = 0.011). Although the overall representation of total gene deletions in the in vivo spectrum is low, 4/5 encompass Xq26 markers flanking hprt. This pattern differs significantly from spontaneous HPRT- large deletions occurring in vitro (P = 0.032) but resembles the spectrum of X-ray-induced deletions.

A study of the effects of exposure on cleanup workers at the Chernobyl nuclear reactor accident using multiple end points.

Blood samples were collected from 192 exposed workers who participated in the cleanup after the April 26, 1986, nuclear reactor accident at Chernobyl, Ukraine. These samples, together with samples from 73 individuals living in Russia but not involved in Chernobyl cleanup activities, were collected during September 1991 to May 1996 and shipped to the U.S. for evaluation by three bioassays: cytogenetic analysis based on chromosome painting, HPRT mutation analysis and glycophorin A (GPA) variant analysis. Univariate statistical analyses of the results of each bioassay (including adjustments for age, smoking status and estimated precision of the bioassay) found greater frequencies of chromosome translocations and HPRT mutant T lymphocytes among the exposed individuals compared to the controls (P < or = 0.01). GPA analyses showed no significant difference for exposed compared to controls for either hemizygous, N/O, or homozygous, N/N, variant cell frequency. Multivariate analysis of variance of the subset of 44 exposed and 14 unexposed individuals with measurements from all three bioassays found elevated frequencies of chromosomal translocations and HPRT mutants, and reduced frequencies for both GPA end points among the exposed persons compared to the controls. However, none of these differences, considered singly or in combination, was statistically significant (although statistical power is low due to small sample sizes). Mean estimated dose, based on cytogenetic response, for those exposed was 9 cGy (range 0 to 51 cGy) and was less than that estimated by physical dosimetry (25 cGy). Correlation between the end points of the bioassays and estimated physical dosimetry was low (r < 0.2); the only significant correlation found was for physical dose estimate and dates worked at Chernobyl (r = 0.4, P < 0.01), with those working soon after the accident receiving greater estimated doses.