Differences in regulatory sequences of naturally occurring JC virus variants.

The regulatory region was sequenced for DNAs representative of seven independent isolates of JC virus, the probable agent of progressive multifocal leukoencephalopathy. The isolates included an oncogenic variant (MAD-4), an antigenic variant (MAD-11), and two different isolates derived from the urine (MAD-7) and from the brain (MAD-8) of the same patient. The representative DNAs were molecularly cloned directly from diseased brain tissue and from human fetal glial cells infected with the corresponding isolated viruses. The regulatory sequences of these DNAs were compared with those of the prototype isolate, MAD-1, sequenced previously (R. J. Frisque, J. Virol. 46:170-176, 1983). We found that the regulatory region of JC viral DNA is highly variable due to complex alterations of the previously described 98-base-pair repeat of MAD-1 DNA. On the basis of these alterations, there are two general types of JC virus. There were no consistent alterations in regulatory sequences which could distinguish brain tissue DNAs from tissue culture DNAs. Furthermore, for each isolate except MAD-1 (R. J. Frisque, J. Virol. 46:170-176, 1983), the regulatory regions of brain tissue and tissue culture DNAs were not identical. The arrangement, sequence, or both of potential regulatory elements (TATA sequence, GGGXGGPuPu, tandem repeats) of JC viral DNAs are sufficiently different from those in other viral and eucaryotic systems that they may effect the unique properties of this slow virus.     

Naturally occurring BK virus variants (JL and Dik) with deletions in the putative early enhancer-promoter sequences.

The genomes of two independently isolated BK virus (BKV) variants (JL and Dik) were compared with prototype BKV DNA by restriction endonuclease mapping and sequence analysis. Differences were mainly detected in two regions: the BKV (JL) and BKV (Dik) putative early enhancer-promoter regions and the middle of the T-antigen-coding regions. Base sequence analysis of these two regions showed the following. (i) The putative enhancer-promoter regions of BKV (Dik) and BKV (JL) contained only one 68-base-pair (bp) unit of the 68-bp triplication (the central copy of which is missing 18 bp) present in prototype BKV. (ii) In the same region, BKV (JL) and BKV (Dik) contained unique stretches of DNA 33 and 63 bp long, respectively. In these 63 bp, a sequence which was very similar to the proposed simian virus 40 enhancer core sequence (GGAGTGGAAAG) was present. (iii) The altered restriction endonuclease recognition sites in the sequenced part of the T-antigen-coding region of BKV (JL) and BKV (Dik) were due to base sequence changes, leaving the amino acid sequence unchanged.     

Production of transmitochondrial cybrids containing naturally occurring pathogenic mtDNA variants

The human mitochondrial genome (mtDNA) encodes polypeptides that are critical for coupling oxidative phosphorylation. Our detailed understanding of the molecular processes that mediate mitochondrial gene expression and the structure–function relationships of the OXPHOS components could be greatly improved if we were able to transfect mitochondria and manipulate mtDNA in vivo. Increasing our knowledge of this process is not merely of fundamental importance, as mutations of the mitochondrial genome are known to cause a spectrum of clinical disorders and have been implicated in more common neurodegenerative disease and the ageing process. In organellar or in vitro reconstitution studies have identified many factors central to the mechanisms of mitochondrial gene expression, but being able to investigate the molecular aetiology of a limited number of cell lines from patients harbouring mutated mtDNA has been enormously beneficial. In the absence of a mechanism for manipulating mtDNA, a much larger pool of pathogenic mtDNA mutations would increase our knowledge of mitochondrial gene expression. Colonic crypts from ageing individuals harbour mutated mtDNA. Here we show that by generating cytoplasts from colonocytes, standard fusion techniques can be used to transfer mtDNA into rapidly dividing immortalized cells and, thereby, respiratory-deficient transmitochondrial cybrids can be isolated. A simple screen identified clones that carried putative pathogenic mutations in MTRNR1, MTRNR2, MTCOI and MTND2, MTND4 and MTND6. This method can therefore be exploited to produce a library of cell lines carrying pathogenic human mtDNA for further study.     

Cytotoxic T-lymphocyte clones specific for an immunodominant epitope display discerning antagonistic response to naturally occurring Epstein-Barr virus variants.

In the present study we have identified Epstein-Barr virus isolates which encode variant sequences within an HLA B35-restricted immunodominant cytotoxic T-lymphocyte (CTL) epitope that act as natural antagonists and can inhibit CTL activity on the wild-type epitope. This effect can be demonstrated if the wild-type epitope is presented as a synthetic peptide or when processed from a full-length Epstein-Barr virus protein expressed by recombinant vaccinia constructs. However, this antagonistic effect was only selectively seen with some CTL clones, while a strong agonistic effect was evident for other clones in the presence of the same variant peptide. The data presented in this study strongly suggest that it is unlikely that the variant viruses can completely antagonize a virus-specific CTL response by this mechanism since the host immune response is capable of generating CTLs expressing a diverse array of T-cell receptors. Moreover, many of these CTLs can recognize the variant sequences as efficiently as wild-type epitope.     

Susceptibility of sheep for scrapie as assessed by in vitro conversion of nine naturally occurring variants of PrP

Polymorphisms in the prion protein (PrP) gene are associated with phenotypic expression differences of transmissible spongiform encephalopathies in animals and humans. In sheep, at least 10 different mutually exclusive polymorphisms are present in PrP. In this study, we determined the efficiency of the in vitro formation of protease-resistant PrP of nine sheep PrP allelic variants in order to gauge the relative susceptibility of sheep for scrapie. No detectable spontaneous protease-resistant PrP formation occurred under the cell-free conditions used. All nine host-encoded cellular PrP (PrP(C)) variants had distinct conversion efficiencies induced by PrP(Sc) isolated from sheep with three different homozygous PrP genotypes. In general, PrP allelic variants with polymorphisms at either codon 136 (Ala to Val) or codon 141 (Leu to Phe) and phylogenetic wild-type sheep PrP(C) converted with highest efficiency to protease-resistant forms, which indicates a linkage with a high susceptibility of sheep for scrapie. PrP(C) variants with polymorphisms at codons 171 (Gln to Arg), 154 (Arg to His), and to a minor extent 112 (Met to Thr) converted with low efficiency to protease-resistant isoforms. This finding indicates a linkage of these alleles with a reduced susceptibility or resistance for scrapie. In addition, PrP(Sc) with the codon 171 (Gln-to-His) polymorphism is the first variant reported to induce higher conversion efficiencies with heterologous rather than homologous PrP variants. The results of this study strengthen our views on polymorphism barriers and have further implications for scrapie control programs by breeding strategies.     

Amino acid substitutions in naturally occurring variants of ail result in altered invasion activity.

Yersinia enterocolitica is the causative agent of a variety of gastrointestinal syndromes ranging from acute enteritis to mesenteric lymphadenitis. In addition, systemic infections resulting in high mortality rates can occur in elderly and immunocompromised patients. More than 50 serotypes of Y. enterocolitica have been identified, but only a few of them commonly cause disease in otherwise healthy hosts. Those serotypes that cause disease have been divided into two groups, American and non-American, based on their geographical distributions, biotypes, and pathogenicity. We have been studying two genes, inv and ail, from Y. enterocolitica that confer in tissue culture assays an invasive phenotype that strongly correlates with virulence. Some differences between the American and non-American serotypes at the ail locus were noted previously and have been investigated further in this report. The ail locus was cloned from seven Y. enterocolitica strains (seven different serotypes). Although the different clones produced similar amounts of Ail, the product of the ail gene from non-American serotypes (AilNA) was less able to promote invasion by Escherichia coli than was the product of the ail gene from American serotypes (AilA). This difference is probably due to one or more of the eight amino acid changes found in the derived amino acid sequence for the mature form of AilNA compared with that of AilA. Seven of these changes are predicted to be in cell surface domains of the protein (a model for the proposed folding of Ail within the outer membrane is presented). These results are discussed in relation to the growing family of outer membrane proteins, which includes Lom from bacteriophage lambda, PagC from salmonella typhimurium, and OmpX from Enterobacter cloacae.     

Myricetin and quercetin are naturally occurring co-substrates of cyclooxygenases in vivo

Bioflavonoids are ubiquitously present in the plant kingdom, and some of them are presently being sold as healthy dietary supplements around the world. Recently, it was shown that some of the dietary polyphenols were strong stimulators of the catalytic activity of cyclooxygenase I and II, resulting in increased formation of certain prostaglandin (PG) products in vitro and also in intact cells in culture. In the present study, we investigated the effect of two representative dietary compounds, quercetin and myricetin, on plasma and tissue levels of several PG products in normal Sprague-Dawley rats. We found that these two dietary bioflavonoids could strongly stimulate the formation of PG products in vivo in a time-dependent manner, and the stimulatory effect of these two bioflavonoids was dose-dependent with a unique biphasic pattern. At lower doses (<0.3 mg/kg b.w.), they strongly stimulated the formation of PGE₂, but at higher doses (>0.3 mg/kg b.w.), there was a dose-dependent reduction of the stimulatory effect. These results provide support for the hypothesis that some of the bioflavonoids are naturally occurring physiological co-substrates for the cyclooxygenases in vivo.     

Virus-associated RNAs of naturally occurring strains and variants of group C adenoviruses

We compared the sequences of the virus-associated (VA) RNAs of group C adenoviruses, serotypes 1, 2, 5, and 6, and of three variants of adenovirus type 2 (Ad2) selected for loss of the BamHI restriction site in the VA RNAI gene. In the naturally occurring strains. VA RNAI exists in two forms which differ by two nucleotides: one form is found in Ad2 and Ad6, and the other is found in Ad1 and Ad5. There are three sites of variation in Va RNAII, the Ad1, Ad2, and Ad5 forms each differing from Ad6 VA RNAII at one of the positions. One of the selected variants has a four-base duplication within the BamHI cleavage site, whereas the two others have acquired a VA RNAI sequence indistinguishable from that of Ad5. The findings are interpreted in terms of the secondary structures of the VA RNAs and the interrelationships among the viruses.     

Recombinant feline leukemia virus genes detected in naturally occurring feline lymphosarcomas.

Using a polymerase chain reaction strategy aimed at detecting recombinant feline leukemia virus (FeLV) genomes with 5' env sequences originating from an endogenous source and 3' env sequences resulting from FeLV subgroup A (FeLV-A), we detected recombinant proviruses in approximately three-fourths of naturally occurring thymic and alimentary feline lymphosarcomas (LSAs) and one-third of the multicentric LSAs from cats determined to be FeLV capsid antigen positive by immunofluorescence assay. In contrast, only 1 of 22 naturally arising FeLV-negative feline LSAs contained recombinant proviruses, and no recombinant env gene was detected in seven samples from normal tissues or tissues from FeLV-positive animals that died from other diseases. Four preferred structural motifs were identified in the recombinants; one is FeLV-B like (recognizing that FeLV-B itself is a product of recombination between FeLV-A and endogenous env genes), and three contain variable amounts of endogenous-like env gene before crossing over to FeLV-A-related sequences: (i) a combination of full-length and deleted env genes with recombination at sites in the middle of the surface glycoprotein (SU), (ii) the entire SU encoded by endogenous-like sequences, and (iii) the entire SU and approximately half of the transmembrane protein encoded by endogenous-like sequences. Additionally, three of the thymic tumors contained recombinant proviruses with mutations in the vicinity of the major neutralizing determinant for the SU protein. These molecular genetic analyses of the LSA DNAs correspond to our previous results in vitro and support the occurrence and association of viral recombinants and mutants in vivo in FeLV-induced leukemogenesis.     

Amyloidogenicity of naturally occurring full‐length animal IAPP variants

The aggregation of the 37‐amino acid polypeptide human islet amyloid polypeptide (hIAPP), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of human pancreatic β‐islet cells in type 2 diabetes. hIAPP is considered to be one of the most amyloidogenic proteins known. The quick aggregation of hIAPP leads to the formation of toxic species, such as oligomers and fibers, that damage mammalian cells (both human and rat pancreatic cells). Whether this toxicity is necessary for the progression of type 2 diabetes or merely a side effect of the disease remains unclear. If hIAPP aggregation into toxic amyloid is on‐path for developing type 2 diabetes in humans, islet amyloid polypeptide (IAPP) aggregation would likely need to play a similar role within other organisms known to develop the disease. In this work, we compared the aggregation potential and cellular toxicity of full‐length IAPP from several diabetic and nondiabetic organisms whose aggregation propensities had not yet been determined for full‐length IAPP.     

Prevalence of the archetypal regulatory region and sequence polymorphisms in nonpassaged BK virus variants.

Since the first isolation and characterization of BK virus (BKV), a number of BKV variants which differ in genomic structure or antigenic determinants have been described. The regulatory region, in particular, the enhancer elements, show the most divergent sequences among different isolates. The structural organization of a putative ancestral prototype or archetype, from which all of the variants are probably derived, has been proposed. By sequencing the regulatory regions of 13 different isolates from the urine of bone marrow transplant recipients, we determined the structures and sequences of BKV variants diffused in the human population. The enhancer region was amplified by polymerase chain reaction to avoid passage in culture, and the product was directly sequenced. The structure most frequently observed is in agreement with the postulated archetype, containing a single enhancer element with no repeats. By sequence analysis we identified four hot spots of nucleotide variation. These variations are consistent with the existence of two consensus sequences. One sequence motif, observed in about 85% of the isolates, is referred to as the archetypal BKV, while a second motif, observed in the remaining 15% of the variants, is highly reminiscent of the AS strain.     

Growth patterns and substrate requirements of naturally occurring obligate oligotrophs

Eighteen strains of obligately oligotrophic bacteria that grow in a medium containing 1 mg of organic carbon per liter and do not grow in a rich medium (5 g/liter of nutrient) were isolated as dominant organisms from the oligotrophic water of Lake Biwa. The growth properties of these, especially of five strains, were examined. The maximum cell yield ranged from 8.5×10⁴/ml to 2.3×10⁶/ml, and their doubling times ranged from 6.6/h to 11.8/h in LT10^–4 medium (0.5 mg trypticase and 0.05 mg yeast extract in 1 liter of filtered and aged lake water). They also showed good growth in lake water medium without adding nutrients. The optimum concentrations for their growth were 5 mg/1, 5–50 mg/1, 50 mg/1, or 500 mg/1, depending on the strains. They utilized glutamate, glycine, serine, and glycolate, but not acetate, proline, or leucine. Several properties were examined. Their growth properties were very different from those of oligotrophs or oligocarbophiles isolated by other researchers.     

A virus of tobacco rattle group naturally occurring inPlantago sp. in Czechoslovakia

The Czechoslovak isolate produced symptoms characteristic for viruses belonging to the rattle group. A virus variant comparable to Köhler's “Wintertyp” appeared to be present. Two predominant particle lengths of 90 and 191 nm were found in purified preparations. The virus reacted with Dutch TRV antiserum.     

The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts.

1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.     

Interaction of wild-type and naturally occurring deleted variants of hepatitis B virus core polypeptides leads to formation of mosaic particles

The simultaneous presence of hepatitis B virus (HBV) genomes carrying wild-type (wt) and in-frame deleted variants of the HBV core gene has been identified as a typical feature of HBV-infected renal transplant patients with severe liver disease. To investigate possible interactions of wt and deleted core polypeptides a two-vector Escherichia coli expression system ensuring their concomitant synthesis has been developed. Co-expression of wt and a mutant core lacking 17 amino acid residues (77-93) within the immunodominant region led to the formation of mosaic particles, whereas the mutant alone was incapable of self-assembly.     

Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA

The vaccinia virus genome is a single, linear, duplex DNA molecule whose complementary strands are naturally cross-linked. The molecular weight has been determined by contour length measurements from electron micrographs to be 122 ± 2.2 × 10⁶. Denaturation mapping techniques indicate that the nucleotide sequence arrangement of the DNA is unique. Two forms of cross-linked vaccinia DNA were observed in alkaline sucrose gradients. The relative S-values of the two cross-linked species were appropriate for a single-stranded circle and a linear single strand, each with a molecular weight twice that expected for an intact, linear, complementary strand of vaccinia DNA. The fraction of sheared vaccinia DNA able to “snap back” after denaturation suggested a minimum of two crosslinks per molecule. Full-length single-stranded circles were observed in the electron microscope after denaturation of vaccinia DNA. Partial denaturation produced single-stranded loops at the ends of all full-length molecules. Exposure of native vaccinia DNA to a single strand-specific endonuclease isolated from vaccinia virions caused disruption of the cross-links, as assayed by alkaline sedimentation, and produced free single-strand ends when partially denatured DNA was observed in the electron microscope. We conclude that vaccinia DNA contains two cross-links, one at or near (within 50 nucleotides) each end in a region of single-stranded DNA. Two models for the cross-links are presented.