Functional requirement of p23 and Hsp90 in telomerase complexes.
(9/2663)
Most normal human diploid cells have no detectable telomerase; however, expression of the catalytic subunit of telomerase is sufficient to induce telomerase activity and, in many cases, will bypass normal senescence. We and others have previously demonstrated in vitro assembly of active telomerase by combining the purified RNA component with the reverse transcriptase catalytic component synthesized in rabbit reticulocyte extract. Here we show that assembly of active telomerase from in vitro-synthesized components requires the contribution of proteins present in reticulocyte extracts. We have identified the molecular chaperones p23 and Hsp90 as proteins that bind to the catalytic subunit of telomerase. Blockade of this interaction inhibits assembly of active telomerase in vitro. Also, a significant fraction of active telomerase from cell extracts is associated with p23 and Hsp90. Consistent with in vitro results, inhibition of Hsp90 function in cells blocks assembly of active telomerase. To our knowledge, p23 and Hsp90 are the first telomerase-associated proteins demonstrated to contribute to telomerase activity. (+info)
Continuous in vitro propagation and differentiation of cultures of the intramolluscan stages of the human parasite Schistosoma mansoni.
(10/2663)
The metazoan parasitic blood flukes, Schistosoma spp., infect over 200 million people worldwide and cause extensive human morbidity and mortality. Research strategies for development of anti-schistosomal agents are impeded by the organism's complex molluscan-mammalian life cycle, which limits experimental approaches and availability of material. We derived long-term continuously proliferative cultures of Schistosoma mansoni sporocysts capable of generating cercariae in vitro. Cultured organisms retained the ability to parasitize the host, and they exhibited developmental regulation of candidate stage-specific genes in the host-free culture system. Evidence for expression of a reverse transcriptase also was found in the cultured organisms, pointing to this activity as a possible mechanistic contributor to the dynamic relationship between the parasite and its hosts. Continuous in vitro propagation of the asexual sporocyst stage allows isolation of clonally derived parasite populations and provides a means to study schistosomal molecular genetics, metabolism, and evasion of host defenses. (+info)
Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit.
(11/2663)
With the amino acid sequences of all reported Akt kinase physiological substrates, the possible Akt kinase substrate specificity has been suggested. The serine/threonine residue to be phosphorylated in these proteins is placed within stretches of amino acids with homology, and the arginine residues on the -5 and -3 positions and a hydrophobic amino acid on the +2 position are conserved relative to those of serine/threonine residues (XXRXRXXS/TXX). We noticed two putative Akt kinase phosphorylation sites (220GARRRGGSAS229) and (817AVRIRGKSYV826) in human telomerase reverse transcriptase (hTERT) subunit. To demonstrate that hTERT is an Akt kinase substrate protein, we performed the nonradioactive protein kinase assay with the fluorescein hTERT peptide (817AVRIRGKSYV826). We observed the phosphorylation of hTERT peptide by the human melanoma cell lysate or the activated recombinant Akt kinase proteins in vitro. With the treatment of the growth factor deprivation or okadaic acid, we also observed the up-regulation of both hTERT peptide phosphorylation and the telomerase activity. We noticed that Wortmannin down-regulates hTERT peptide phosphorylation and telomerase activity together. In addition, we observed the enhancement of telomerase activity with the pretreatment of Akt kinase in vitro. Thus, these observations suggest that Akt kinase enhances human telomerase activity through phosphorylation of hTERT subunit as one of its substrate proteins. (+info)
Novel endogenous type D retroviral particles expressed at high levels in a SCID mouse thymic lymphoma.
(12/2663)
A xenograft model of the human disease Langerhans cell histiocytosis (LCH) was investigated with severe combined immunodeficiency (SCID) mice. Transplantation of human LCH biopsy material into SCID mice resulted in the generation of mouse tumors resembling lymphomas. A thymoma cell line (ThyE1M6) was generated from one of these mice and found to display significant levels of Mg2+-dependent reverse transcriptase activity. Electron microscopy revealed particles with type D retroviral morphology budding from ThyE1M6 cells at a high frequency, whereas control cultures were negative. Reverse transcription-PCR of virion RNA with degenerate primers for conserved regions of various mouse, human, and primate retroviruses amplified novel sequences related to primate type D retroviruses, murine intracisternal A particles, Jaagsiekte sheep retrovirus, and murine long interspersed nuclear elements but not other retroviral classes. We demonstrate that these sequences represent a novel group of endogenous retroviruses expressed at low levels in mice but expressed at high levels in the ThyE1M6 cell line. Furthermore, we propose that the activation of endogenous retroviral elements may be associated with a high incidence of thymomas in SCID mice. (+info)
Improved Mg2+-based reverse transcriptase assay for detection of primate retroviruses.
(13/2663)
The reverse transcriptase (RT) assay is a simple, relatively inexpensive, widely used assay that can detect all retroviruses (known and novel retroviruses as well as infectious and defective retroviruses) on the basis of the divalent cation requirement of their RT enzyme, i.e., Mg2+ or Mn2+. Descriptions of various RT assays have been published; however, they cannot be directly applied to the analysis of biological products or clinical samples without further standardization to determine the lower limit of virus detection (sensitivity), assay variability (reproducibility), or ability to detect different retroviruses (specificity). We describe the detection of type E and type D primate retroviruses, which may be pathogenic for humans, by a new 32P-based, Mg2+-containing RT assay. The results show that the sensitivity of detection is <3.2 50% tissue culture infective doses (TCID50s) for human immunodeficiency virus type 1 (HIV-1) and <1 TCID50 for simian immunodeficiency virus isolated from a rhesus macaque (SIVmac). Analysis of recombinant HIV-1 RT enzyme indicated that 10(-5) U, which is equivalent to 4.25 x 10(4) virions, could be detected. Additionally, genetically distinct type D retroviruses such as simian AIDS retrovirus and squirrel monkey retrovirus were also detected in the assay with similar sensitivities. Thus, the improved RT assay can be used to detect genetically divergent Mg2+-dependent retroviruses of human and simian origin that can infect human cells and that therefore pose a potential health risk to humans. (+info)
The domain structure and retrotransposition mechanism of R2 elements are conserved throughout arthropods.
(14/2663)
R2 elements are non-LTR retrotransposons that insert in the 28S rRNA genes of arthropods. Partial sequence data from many species have previously suggested that these elements have been vertically inherited since the origin of this phylum. Here, we compare the complete sequences of nine R2 elements selected to represent the diversity of arthropods. All of the elements exhibited a uniform structure. Identification of their conserved sequence features, combined with our biochemical studies, allows us to make the following inferences concerning the retrotransposition mechanism of R2. While all R2 elements insert into the identical sequence of the 28S gene, it is only the location of the initial nick in the target DNA that is rigidly conserved across arthropods. Variation at the R2 5' junctions suggests that cleavage of the second strand of the target site is not conserved within or between species. The extreme 5' and 3' ends of the elements themselves are also poorly conserved, consistent with a target primed reverse transcription mechanism for attachment of the 3' end and a template switch model for the attachment of the 5' end. Comparison of the approximately 1,000-aa R2 ORF reveals that it can be divided into three domains. The central 450-aa domain can be folded by homology modeling into a tertiary structure resembling the fingers, palm, and thumb subdomains of retroviral reverse transcriptases. The carboxyl terminal end of the R2 protein appears to be the endonuclease domain, while the amino-terminal end contains zinc finger and c-myb-like DNA-binding motifs. (+info)
Evidence of avian leukosis virus subgroup E and endogenous avian virus in measles and mumps vaccines derived from chicken cells: investigation of transmission to vaccine recipients.
(15/2663)
Reverse transcriptase (RT) activity has been detected recently in all chicken cell-derived measles and mumps vaccines. A study of a vaccine manufactured in Europe indicated that the RT is associated with particles containing endogenous avian retrovirus (EAV-0) RNA and originates from the chicken embryonic fibroblasts (CEF) used as a substrate for propagation of the vaccine. We investigated the origin of RT in measles and mumps vaccines from a U.S. manufacturer and confirm the presence of RT and EAV RNA. Additionally, we provide new evidence for the presence of avian leukosis virus (ALV) in both CEF supernatants and vaccines. ALV pol sequences were first identified in particle-associated RNA by amplification with degenerate retroviral pol primers. ALV RNA sequences from both the gag and env regions were also detected. Analysis of hypervariable region 2 of env revealed a subgroup E sequence, an endogenous-type ALV. Both CEF- and vaccine-derived RT activity could be blocked by antibodies to ALV RT. Release of ALV-like virus particles from uninoculated CEF was also documented by electron microscopy. Nonetheless, infectivity studies on susceptible 15B1 chicken cells gave no evidence of infectious ALV, which is consistent with the phenotypes of the ev loci identified in the CEF. PCR analysis of ALV and EAV proviral sequences in peripheral blood mononuclear cells from 33 children after measles and mumps vaccination yielded negative results. Our data indicate that the sources of RT activity in all RT-positive measles and mumps vaccines may not be similar and depend on the particular endogenous retroviral loci present in the chicken cell substrate used. The present data do not support transmission of either ALV or EAV to recipients of the U.S.-made vaccine and provide reassurance for current immunization policies. (+info)
Catalytic features of the recombinant reverse transcriptase of bovine leukemia virus expressed in bacteria.
(16/2663)
We have expressed the recombinant reverse transcriptase (RT) of bovine leukemia virus (BLV) in bacteria. The gene encoding the RT was designed to start at its 5' end next to the last codon of the mature viral protease, namely the amino terminus of the RT matches the last 26 codons of the pro gene and is coded for by the pro reading frame. The RT sequence extends into the pol gene, utilizing the pol reading frame after overcoming the stop codon by adding an extra nucleotide (thus imitating the naturally occurring frameshift event). Hence we have generated a transframe polypeptide that is a 584-residues-long protein (see Rice, Stephens, Burny, and Gilden (1985) Virology 142, 357-377). This protein was partially purified after adding a six-histidine tag and studied biochemically testing a variety of parameters. The enzyme exhibits all activities typical of RTs, i.e., both RNA- and DNA-dependent DNA polymerase as well as a ribonuclease H (RNase H) activity. Unlike most RTs, the BLV RT is enzymatically active as a monomer even after binding a DNA substrate. The enzyme shows a preference for Mg2+ over Mn2+ in both its DNA polymerase and RNase H activities. BLV RT is relatively resistant to nucleoside triphosphate analogues, which are known to be potent inhibitors of other RTs such as that of HIV. (+info)