Complete genome sequence of a tenth human polyomavirus

Nine polyomavirus (PyV) species are known to productively infect humans. The circular DNA genomes of PyVs are readily detectable using rolling circle amplification (RCA). RCA-based analysis of condyloma specimens from a patient with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome demonstrated the presence of a tenth apparently human-tropic polyomavirus species, which we name HPyV10.     

Identification of a UV-induced trans-acting protein that stimulates polyomavirus DNA replication.

Previous studies provided indirect evidence that the ability of a variety of DNA-damaging agents to induce asynchronous polyomavirus DNA replication in the H3 rat fibroblast cell line is mediated by a trans-acting factor. Using an erythrocyte insertion technique to introduce protein fractions from UV-irradiated cells into unirradiated H3 cells, we have now obtained evidence that this factor is a 60-kilodalton protein. These findings provide evidence that DNA damage in mammalian cells induces a factor that can alter the replication of a viral DNA.     

Identification of a novel polyomavirus from patients with acute respiratory tract infections

We report the identification of a novel polyomavirus present in respiratory secretions from human patients with symptoms of acute respiratory tract infection. The virus was initially detected in a nasopharyngeal aspirate from a 3-year-old child from Australia diagnosed with pneumonia. A random library was generated from nucleic acids extracted from the nasopharyngeal aspirate and analyzed by high throughput DNA sequencing. Multiple DNA fragments were cloned that possessed limited homology to known polyomaviruses. We subsequently sequenced the entire virus genome of 5,229 bp, henceforth referred to as WU virus, and found it to have genomic features characteristic of the family Polyomaviridae. The genome was predicted to encode small T antigen, large T antigen, and three capsid proteins: VP1, VP2, and VP3. Phylogenetic analysis clearly revealed that the WU virus was divergent from all known polyomaviruses. Screening of 2,135 patients with acute respiratory tract infections in Brisbane, Queensland, Australia, and St. Louis, Missouri, United States, using WU virus-specific PCR primers resulted in the detection of 43 additional specimens that contained WU virus. The presence of multiple instances of the virus in two continents suggests that this virus is geographically widespread in the human population and raises the possibility that the WU virus may be a human pathogen.     

A novel human polyomavirus closely related to the african green monkey-derived lymphotropic polyomavirus

We identified a novel human polyomavirus from a kidney transplant patient under immunosuppressive treatment, by use of a generic PCR. The genome of the virus was completely amplified and sequenced. In phylogenetic analyses, it appeared as the closest relative to the African green monkey-derived lymphotropic polyomavirus (LPV). Further investigation of clinical samples from immunocompromised patients with specific nested PCR revealed additional positive samples, indicating that the virus naturally infects humans. The virus was tentatively named human polyomavirus 9 (HPyV9). The previously observed seroreactivity to LPV in human populations might find a partial explanation in the circulation of HPyV9.     

Identification of key residues in the amino-terminal third of human interleukin-1 alpha

Two mutational approaches were used to perform a thorough structure-function analysis of the first 53 residues of the 159-residue cytokine human interleukin-1 alpha (hIL-1 alpha). In this study, a total of 26 deletions, 97 multiple amino acid substitutions, and 46 single amino acid substitutions were examined. A synthetic hIL-1 alpha gene with many unique restriction sites was constructed to facilitate the molecular manipulations that were performed. The mutational methods employed include: Bal-31 exonuclease-generated deletions at unique restriction sites and combinatorial cassette mutagenesis via segment replacement with synthetic DNA. The mutant hIL-1 alpha proteins were expressed at high levels in Escherichia coli and were assayed for biological activity in a mouse T cell proliferation assay. We observed that the activity of hIL-1 alpha was extraordinarily sensitive to deletion mutations. Most internal deletions of as few as 1 or 2 residues substantially reduced biological activity. Combinatorial cassette mutagenesis on residues 13-53 of hIL-1 alpha identified 15 important residue positions. Of these, 8 displayed strong preferences for residues with hydrophilic side chains, and the remainder preferred hydrophobic side chains. We found that functional hIL-1 alpha had an absolute requirement for a basic residue (Arg, Lys, or His) at either position 15 or 16, and that Leu was preferred at position 40.     

Identification and characterization of a third thioredoxin h in poplar

Three full-length sequences encoding thioredoxin h have been isolated in a leaf/root of Populus trichocarpa cv. Trichobel expressed sequence tag (EST) library. One of these, popCXXS1 exhibits the nontypical active site CXXS homologous to atCXXS1. The second one, named popTrxh4, is related to atTrxh9 which forms with several other plant thioredoxin h a distinct subgroup of thioredoxins h. The third one, named popTrxh3, displays 66% identity and also a high degree of homology (81%) vs. the previously described popTrxh1. Nevertheless, the active sites of both proteins differ, since the active site of popTrxh1 (WCPPC) is a variant of the canonical WCGPC found in popTrxh3. The cDNA sequence of popTrxh3 has been introduced in an expression plasmid (pET3d) in order to express the corresponding recombinant polypeptide. The protein has been expressed to a high level, purified from Escherichia coli cells with a high yield and its catalytic properties compared to popTrxh1. Furthermore, two mutants, popTrxh1P40G and popTrxh3G41P, have been engineered in order to explore the importance of the active site residues in the thioredoxin h catalytic properties. The results are discussed in relation with known biochemical properties of thioredoxins h.     

Identification and characterization of the hamster polyomavirus middle T antigen.

Hamster polyomavirus (HaPV) is associated with lymphoid and hair follicle tumors in Syrian hamsters. The early region of HaPV has the potential to encode three polypeptides (which are related to the mouse polyomavirus early proteins) and can transform fibroblasts in vitro. We identified the HaPV middle T antigen (HamT) as a 45-kDa protein. Like its murine counterpart, HamT was associated with serine/threonine phosphatase, phosphatidylinositol-3 kinase, and protein tyrosine kinase activities. However, whereas mouse middle T antigen associates predominantly with pp60c-src and pp62c-yes, HamT was associated with a different tyrosine kinase, p59fyn. The ability of HaPV to cause lymphoid tumors may therefore reside in its ability to associate with p59fyn, a potentially important tyrosine kinase in lymphocytes.     

Identification of jun-B as third member in human antioxidant response element-nuclear proteins complex.

Deletion mutagenesis in human NAD(P)H:Quinone Oxidoreductase (NQO1) gene and transfection studies into mammalian cells identified a segment of DNA designated as human Antioxidant Response Element (hARE) responsible for high basal expression in tumor cells and its induction by beta-naphthoflavone (beta-NF). The twenty four base pairs of the hARE contains an essential cis-element AP1 binding site and has been shown to bind to jun-D and c-fos proteins from mouse hepatoma (Hepa-1) nuclear extract. In the present report, we have identified jun-B as the third major protein in the hARE-Hepa-1 proteins complex observed in the band shift assays.     

The role of sialic acid in human polyomavirus infections

JC virus (JCV) and BK virus (BKV) are human polyomaviruses that infect approximately 85% of the population worldwide [1,2]. JCV is the underlying cause of the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML), a condition resulting from JCV induced lytic destruction of myelin producing oligodendrocytes in the brain [3]. BKV infection of kidneys in renal transplant recipients results in a gradual loss of graft function known as polyomavirus associated nephropathy (PVN) [4]. Following the identification of these viruses as the etiological agents of disease, there has been greater interest in understanding the basic biology of these human pathogens [5,6]. Recent advances in the field have shown that viral entry of both JCV and BKV is dependent on the ability to interact with sialic acid. This review focuses on what is known about the human polyomaviruses and the role that sialic acid plays in determining viral tropism.     

Identification of a third rat odorant-binding protein (OBP3)

From a rat olfactory epithelium cDNA library clones encoding a lipocalin were isolated with sequence identity to the previously described salivary-specific alpha-2u globulin and the N-terminal region of mouse odorant-binding proteins OBP-III and OBP-IV. In situ hybridization showed strong expression in nasal glands displaying a pattern equivalent to rat OBP1. Heterologously expressed protein was evaluated for its binding properties using spectroscopic approaches. The recombinant protein interacted with two fluorescent probes, 1-aminoanthracene (1-AMA) and 1,1'-bis(4-anilino-5-naphthalene)-sulfonic acid. 1-AMA binding was competed by several odorants with high affinity. The thermodynamic parameters of the protein-odorant interaction were determined using isothermal titration calorimetry. Due to its nasal expression and odorant-binding characteristics this protein was designated OBP3.     

Cloned human polyomavirus JC DNA can transform human amnion cells.

The genome of the human polyomavirus JC (Mad-1 strain) was molecularly cloned in Escherichia coli by using the plasmid vector pBR322. Recombinant DNA molecules were constructed with the entire JC genome inserted either at its unique EcoRI site at 0.0 map units or at its unique BamHI site at 0.51 map units. Viral DNA from each of these recombinant plasmids was capable of transforming human amnion cells, and cell lines established from transformed foci were positive for JC tumor antigen as assayed by indirect immunofluorescence.     

The human polyomavirus BK: Potential role in cancer

In human cancer, a role has been suggested for the human polyomavirus BK, primarily associated with tubulointerstitial nephritis and ureteric stenosis in renal transplant recipients, and with hemorrhagic cystitis in bone marrow transplant (BMT) recipients. After the initial infection, primarily unapparent and without clinical signs, the virus disseminates and establishes a persistent infection in the urinary tract and lymphocytes. There is correlative evidence regarding potential role of polyomavirus BK in cancer. In fact, the BK virus (BKV) DNA (complete genome and/or subgenomic fragments containing the early region) is able to transform embryonic fibroblasts and cells cultured from kidney and brain of hamster, mouse, rat, rabbit, and monkey. Nevertheless, transformation of human cells by BKV is inefficient and often abortive. Evidence supporting a possible role for BKV in human cancer has accumulated slowly in recent years, after the advent of polymerase chain reaction (PCR). BKV is known to commonly establish persistent infections in people and to be excreted in the urine by individuals who are asymptomatic, complicating the evaluation of its potential role in development of human cancer. Therefore, there is no certain proof that human polyomavirus BK directly causes the cancer in humans or acts as a cofactor in the pathogenesis of some types of human cancer.     

Identification and chromosomal mapping of a third mouse runt-like locus

The Drosophila runt gene, which controls early events in embryogenesis, has been shown to have homologues in human and mouse. The human gene on 21q22 is involved in the t(8;21) associated with acute myeloid leukemia. Two mouse runt-like loci encoding DNA-binding proteins have been identified. We report here the isolation and partial sequence of a molecular clone of a third mouse runt-like locus. By using a panel of somatic cell hybrids and interspecific backcross mice, we map the novel locus to the telomeric region of mouse chromosome 4.     

Identification and analysis of a third mouse Polycomb gene, MPc3

Betaine-homocysteine S-methyltransferase (BHMT) is one of the enzymes involved in the branch point metabolism of homocysteine. Elevated levels of plasma homocysteine may be a risk factor for the development of vascular disease; however, whether BHMT has a significant role in the regulation of plasma levels of homocysteine remains to be determined. As a prelude to creating a mouse strain deficient in BHMT activity, we screened a lambda library containing mouse SvJ 129 genomic DNA for the mouse BHMT gene using random probes made from the human cDNA. One genomic isolate was completely sequenced and found to encode an intronless BHMT pseudogene (mBHMT-ps). mBHMT-ps was then used as a template for the generation of random probes that were used to screen a BAC library containing mouse 129 Sv/Ev genomic DNA. In order to discriminate between pseudogenes and the authentic BHMT gene, a secondary PCR-based screen was employed which used primers designed from the pseudogene sequence that would predictably amplify across introns. Using this strategy, we isolated six mouse genomic clones that tested positive for the presence of all seven introns characteristic of the human gene, and the BHMT gene of one clone was completely sequenced. Like the human BHMT gene, the mouse gene spans 21 kb and is encoded by eight exons interrupted by seven introns. The structure of the mouse BHMT gene is described herein as well as the 5′-flanking region of the gene adjacent to exon 1, which we demonstrate is capable of conferring basal promoter activity in Chinese Hamster Ovary cells.