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PubMed1997

The enigma of pX: A host-dependent cis-acting element with variable effects on tombusvirus RNA accumulation.

Tomato bushy stunt virus (TBSV) is a small isometric virus that contains a single-stranded RNA genome with five major genes. In this study, we have analyzed the importance of an additional small sixth open reading frame (ORF) of 207 nucleotides, designated pX, which resides at the 3' end of the genome. Bioassays showed that deletions or additions of nucleotides at the 5' end of the pX gene that were designed to disrupt the ORF, or site-specific inactivation of its start codon, all gave rise to TBSV mutants which were unable to accumulate to detectable levels in cucumber or Nicotiana benthamiana protoplasts. Although these results suggested a role for the putative pX protein, introduction of a premature stop codon in the pX gene had no strong negative effect. However, a comparable mutation that affected the same nucleotides without changing the predicted amino acid sequence greatly reduced RNA accumulation. Therefore, we hypothesize that cis-acting RNA sequences within the pX gene, rather than the predicted protein influence genome accumulation. The requirement of the cis-acting pX ORF sequences appears to be host-dependent because comparisons revealed that subtle pX gene mutations that prohibited accumulation of TBSV RNA in cucumber or N. benthamiana, failed to interfere substantially with replication in Chenopodium quinoa protoplasts or plants. Irrespective of the host, the cis-acting pX gene sequences were dispensable on replicase-deficient RNAs that require helper TBSV for replication in trans. In addition, the pX gene was not essential for in vitro translation of replicase proteins from genomic RNA. These results suggest that neither translation nor polymerase activity of the replicase proteins require pX gene sequences. However, it is possible that very early in the replication cycle of genomic RNA in vivo, the pX gene cis-acting element is essential for some other unidentified function which involves interaction with one or more host components whose composition varies slightly between different plants.

Enhancer Elements, Genetic↗
PubMed1994

Hepatitis B virus X gene product acts as a transactivator in vivo.

It has previously been shown that the hepatitis B virus X gene product, pX, transactivates homologous and heterologous transcriptional regulatory sequences of viruses and various cellular genes in vitro. However, there is no evidence about the reproducibility and the relevance of this phenomenon in vivo. In this study we crossbred transgenic mice expressing the X gene under the control of the human antithrombin III (ATIII) gene regulatory sequences with transgenics carrying either the chloramphenicol acetyl-transferase or the LacZ bacterial reporter genes driven by the HIV1-LTR, which is known to be activated in trans by pX. Expression of pX in the liver stimulates the HIV1-LTR driven expression of both chloramphenicol acetyl-transferase and beta-galactosidase reporter genes in double transgenic mice. No detectable increase in chloramphenicol acetyl-transferase expression was observed in tissues, such as the spleen, brain and heart, that do not express pX. Our results confirm the transactivating properties of pX in vivo for the first time and support the hypothesis that pX might indeed modify gene expression in HBV-infected hepatocytes and influence viral pathogenesis.

Animals↗
PubMed2006

Loss of parasympathetic innervation leads to sustained expression of pro-inflammatory genes in the rat lacrimal gland.

It has been shown that removal of parasympathetic innervation to the lacrimal gland (LG) leads to rapid reduction in tear flow. Additionally, removal of the neural input resulted in disorganization of LG structure and changes in the expression of genes associated with the secretory pathway and inflammation. The goal of this study was to investigate the change in pro-inflammatory and pro-apoptotic gene expression in the rat LG following parasympathetic denervation. Male Long-Evans rats underwent unilateral sectioning of the greater superficial petrosal nerve and were sacrificed 7 days or 2.5 months later. cDNA was synthesized from LG RNA from the contralateral control (Ctla) and parasympathectomized (Px) glands and comparative real-time PCR was performed. Mean threshold cycles (MC(T)) for the Ctla and Px LG genes were normalized to 18S rRNA MC(T) values, and the relative fold change was calculated for each gene using the 2(-DeltaDeltaC)(T) method. The expression of nuclear factor kappa B1, caspase 1, eotaxin, leukocyte antigen MRC-OX44, allograft inflammatory factor-1, MHC class II molecules RT.1B and RT.1D, IgG receptor FcRn, and macrophage metalloelastase was increased and remained elevated in the Px LG, compared with the Ctla LG. Increased expression of the initiator of apoptosis gene, caspase 2, was confirmed, but expression of the executor gene, caspase 6, was not elevated in the Px LG. Reduced expression of genes associated with post-translational protein processing-furin convertase, protein disulfide isomerase, and UDP-gal transporter isozyme 1-was noted in the Px LG. No significant changes in the expression of genes associated with lysosomal and non-lysosomal-mediated protein degradation were found. Removal of parasympathetic input may lead to decreased capacity for protein synthesis and elevated immune responses in the Px LG. These changes occur without increases in expression of the muscarinic acetylcholine receptor subtype 3, and may suggest the early changes in LG acinar cells and the pathophysiology of autoimmune responses.

Animals↗
PubMed1991

Polymerase chain reaction analysis of defective human T-cell leukemia virus type I proviral genomes in leukemic cells of patients with adult T-cell leukemia.

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia, and the clonally derived leukemic cells all contain proviral genomes. Polymerase chain reaction with a variety of primers which span the HTLV-I genome was used to determine that a significant fraction of patients (at least 32%) carry deleted viral genomes in their leukemic cells. The pX region of the HTLV-I genome encoding the regulatory genes tax and rex was preferentially retained. The fact that the tax coding region was retained provides supporting evidence that the tax protein contributes to leukemogenesis in vivo. The reasonably high fraction of patients with adult T-cell leukemia carrying deleted genomes in their tumor cells suggests that the deletions have a role in leukemogenesis.

Adult↗
PubMed1999

[Mechanism of selenium protecting against free radical damages during myocardial ischemia/reperfusion in rats].

OBJECTIVE: To investigate the mechanism of selenium (Se) protecting against free radical damages during myocardial ischemia/reperfusion (I/R) in rats. METHODS: All rats were separated into three groups: control, organic Se and inorganic Se supplemented groups. Myocardial I/R was established by operation in all three groups. Biochemical assays, atomic absorption spectrophotometry, RT-PCR and cDNA sequence scanning were performed to examine the changes in myocardial MDA level, GSH-Px activity and gene expression, GSH-Px cDNA nucleotide sequence, as well as Se and Ca2+ and Mg2+ concentrations in the heart muscle during I/R. RESULTS: The administration of Se for 7 days did not significantly change the content of Se in the plasma and blood cells. In the two Se-supplemented groups, after reperfusion for 30 minutes, myocardial Se concentration and GSH-Px activity were increased significantly, and GSH-Px expression was elevated with a normal cDNA sequence, and at the same time, MDA contents in the two Se-supplemented groups were lower than that in the control group. CONCLUSION: Se supplementation can protect against free radical damages during myocardial I/R by increasing myocardial Se content and improving both the expression and activity of GSH-Px.

Animals↗
PubMed1990

Transactivation by the hepatitis B virus X protein depends on AP-2 and other transcription factors.

The hepatitis B virus (HBV) X gene product (pX) could be important in disease pathogenesis because it is known to transactivate transcription from many viral and cellular gene promoters, including the HBV core gene promoter, the human immunodeficiency virus (HIV-1) long terminal repeat, and the c-myc promoter. We have previously shown that only a subset of the promoters that can be transactivated by pX is transactivated in any particular cell line, and have proposed that pX acts through multiple, cell type-specific transcription factors. We show here that pX acts through both AP-1 and AP-2 sites, and that pX has a transcription activation domain. We conclude that transactivation by pX depends on at least two distinct cellular DNA-binding transcription factors and we present a model for the action of pX.

Animals↗
PubMed2003

A developmentally regulated and psychostimulant-inducible novel rat gene mrt1 encoding PDZ-PX proteins isolated in the neocortex.

Single or repeated exposure to psychostimulants such as amphetamines and cocaine after postnatal week 3 leads to an enduring enhancement in the psychotomimetic responses elicited by a subsequent challenge of a stimulant in rodents. This behavioral sensitization phenomenon has been considered to be the neural consequences of stimulant-induced alterations in gene expression in the brain after a critical period of postnatal development. Using a differential cloning technique, RNA arbitrarily primed PCR, we have now identified from the rat neocortex a novel and developmentally regulated methamphetamine (MAP)-inducible gene mrt1 (MAP responsive transcript 1). mrt1 encodes two major types of PDZ- and PX-domains containing proteins of approximately 62 kDa in size with different carboxy termini, Mrt1a and Mrt1b. The mrt1 mRNAs for Mrt1a, mrt1a, and for Mrt1b, mrt1b, are predominantly expressed in various brain regions and the testes, respectively. Acute MAP injection upregulated mrt1b expression in the neocortex after postnatal week 3 in a D1 receptor antagonist-sensitive manner without affecting mrt1a expression. This upregulation was mimicked by another stimulant, cocaine, whereas pentobarbital and D1 antagonist failed to change the mrt1b transcript levels. Moreover, repeated daily treatment of MAP, but not MAP plus D1 antagonist, for 5 days caused an augmentation of the basal expression of mrt1b 2 and 3 weeks after the drug discontinuation. These late-developing, cocaine-crossreactive, D1 antagonist-sensitive and long-term regulations of mrt1b by MAP are similar to the pharmacological profiles of stimulant-induced behavioral sensitization, and therefore may be associated with the initiation and/or maintenance of the long-term neuronal adaptation.

Aging↗
PubMed1995

Autoimmunity induction by human T cell leukemia virus type 1 in transgenic mice that develop chronic inflammatory arthropathy resembling rheumatoid arthritis in humans.

We recently reported on an inflammatory arthropathy resembling rheumatoid arthritis that develops in high incidence among transgenic mice that carry the env-pX region of the human T cell leukemia virus type 1 genome. In an effort to elucidate the pathogenesis of this disease, we found that genes for inflammatory cytokines, including IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, transforming growth factor-beta 1, IFN-gamma, and IL-2, as well as MHC genes were activated in transgenic joints. Serum levels of IL-1 beta and IL-6 were also elevated. Interestingly, these mice produced Ab against IgG, type II collagen (IIC), and heat shock proteins accompanied by IgG hypergammaglobulinemia. The cellular immune response to IIC as well as that to heat shock proteins were activated. Moreover, these mice became immunologically responsive to exogenously administered IIC and developed arthritis, in contrast to their nontransgenic littermates, which showed little response to IIC. Taken together, the results suggest that human T cell leukemia virus type 1 can cause immune system hyperreactivity and induce autoimmunity. The possibility that elevated cytokine and/or MHC gene expression are involved in the development of autoimmunity and arthropathy are discussed.

Animals↗
PubMed1997

[Immunological analysis of HTLV-I env-pX transgenic rat with various collagen vascular diseases].

Human T lymphocyte virus type-I (HTLV-I) is an etiologic agent of not only adult T cell leukemia but also HTLV-I associated myelopathy/tropical spastic paraparesis, HTLV-I associated arthropathy and other immunological disorders, such as Sjögren syndrome, dermatitis, polymyositis, and uveitis. We recently reported that a wide spectrum of collagen vascular diseases, including inflammatory arthropathy resembling rheumatoid arthritis, myocarditis, dermatitis, necrotizing arteritis, myositis and sialoadenitis similar to Sjögren's syndrome, developed in the transgenic rat carrying HTLV-I env-pX region under the control of its own long terminal repeat (env-pX rat). To investigate the pathogenetic role of these diseases, cell surface molecules and functions of lymphocytes from env-pX rats were examined in this study. In env-pX rats with diseases, not only infiltrating lymphocytes in the inflammatory sites but also peripheral lymphocytes expressed a large number of co-stimulating molecules such as CD80/86 and ICAM-1, compared with those of normal rats or rats with adjuvant-induced arthritis or myosin-induced myocarditis. Moreover, the expression of CD80/86 and ICAM-1 was already up-regulated on the surface of peripheral lymphocytes in env-pX rat before developing any diseases. Lymphocytes, taken from env-pX rats immunized with myosin in vivo, showed significantly higher cellular response against myosin in vitro than those of normal rats. Lymphocytes from env-pX rats also showed the hyper-reactivity to the stimulation by super-antigens, but no significant difference of the usage of T cell receptor V beta was found compared with those of normal rats. These results suggest that the env-pX transgene may induce the high expression of CD80/86 and ICAM-1 on the lymphocytes and the resulting hyper-immune responsiveness in env-pX rats.

Animals↗
PubMed1983

Auditory and vestibular pathology of seizure-prone chicks.

The mutant sex-linked lethal recessive px (paroxysmal) gene, expressed in White Leghorn chicks (Gallus domesticus) causes seizures beginning on approximately day 8 post-hatching. Seizures are spontaneous and inducible by auditory but not by photic stimulation. Prior to seizure onset px chicks are indistinguishable from non-px siblings. With seizure onset is a decrease in food intake which causes deterioration and death by 4-10 weeks of age. In a preliminary histological study conducted on 22-day-old seizure-prone chicks, brains were perfused and sections treated according to a modified cupric-silver staining technique. Nuclei and fiber tracts of px auditory and vestibular systems were extensively degenerated; control brains showed essentially no degeneration. A second experiment was conducted with preseizure chicks to determine whether and to what extent degeneration occurs prior to seizure onset. Deep nuclei of px cerebellum appeared to be the first seriously affected (5 days of age). Extent of degeneration progressed steadily over time through 20 days of age, by which time all components of the two sensory systems were maximally affected. Ambient noise did not affect onset or extent of degeneration, nor did it affect onset of seizures.

Acoustic Stimulation↗
PubMed2000

px(2), the newly identified gene in Rhizobium leguminosarum, is characterized to enhance its adjacent nodF expression.

nodFEL operon is one of the NodD-dependent inducible nod operons that is clustered on the symbiosis plasmid of Rhizobium leguminosarum biovar viciae. A recent study on the upstream regulatory region of nodFEL operon had identified a new promoter, which was responsible for the transcription of a 0.72 Kb RNA molecule, called px(2), in the opposite direction to nodF. This new promoter was further characterized to overlap with that of nodF and its in vitro transcription was inhibited by another newly identified nod regulator, Px. In this paper we report that the sequence analysis of the px(2) transcript revealed only one open reading frame (ORF(66)), corresponding to a polypeptide of 66 amino acids. Moreover, the increase in px(2) copy numbers enhanced the in vivo inducible expression level of nodF, whereas the frame-shift mutation of ORF(66) eliminated such effect, providing evidence that px(2) is responsible for specifically upregulating nodF expression. This result also raises the big possibility that px(2) encodes this polypeptide. A model for coordinated expression of px(2) and nodF, transcribed divergently from each other, is proposed.

Amino Acid Sequence↗
PubMed1978

Brain noradrenaline concentration in seizure-prone chicks, Gallus domesticus.

The mutant, sex-linked recessive px (paroxysm) gene, expressed in female White Leghorn chicks (Gallus domesticus), causes seizures beginning on approximately day 9 after hatching. In an attempt to determine possible central nervous system involvement in the seizures, brain levels of the putative transmitter noradrenaline were assayed. Brains of px chicks and controls (normal female siblings) were removed at 7, 14, and 21 days, weighed, and frozen immediately in liquid nitrogen for later analysis by the alumina-trihydroxyindoleamine method. Noradrenaline was assayed in whole brain and in 4 brain parts: optic lobes, brainstem, cerebral hemispheres, and cerebellum. No differences between px and controls were found in whole brain or optic lobe levels. Brainstem levels of noradrenaline increased significantly with time in both groups, though px increases were more rapid. Px chicks at 21 days of age had significantly higher levels or noradrenaline in the cerebral hemispheres, while cerebellar levels were significantly lower at the same age. The differences found in brainstem, cerebral, and cerebellar noradrenaline are of interest as possible explanations for the lowered body temperature, hyperexcitability, and lack of motor coordination seen in the px chicks.

Animals↗
PubMed2003

Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs.

The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex mRNA compared to pX-tax-orfII and pX-rex-orfI mRNAs, but did not affect exon skipping. Ectopic expression of hnRNP A1 did not affect pX splice site utilization, but increased exon skipping, as the level of pX-p21rex mRNA was increased by almost 10-fold.

Alternative Splicing↗
PubMed1996

Human T lymphocyte virus type I-induced myeloneuropathy in rats: implication of local activation of the pX and tumor necrosis factor-alpha genes in pathogenesis.

The pathogenetic roles of human T lymphocyte virus type I (HTLV-I) and cytokines were investigated in HTLV-I-induced myeloneuropathy in Wistar-King-Aptekman-Hokudai rats. In the nervous system, pX messenger RNAs of HTLV-I were selectively expressed in the diseased spinal cord and peripheral nerves but not in the unaffected cerebrum and cerebellum, even though proviral DNAs were consistently identified in these tissues. Among several cytokines examined, mRNA expression and production of tumor necrosis factor (TNF)-alpha in the spinal cord and cerebrospinal fluid correlated positively with the development of spinal cord lesions. The collective evidence strongly suggests that selective activation of HTLV-I, in particular Tax expression and production of TNF-alpha induced by HTLV-I infection in target spinal cord and peripheral nerves, is causally related to apoptotic death of oligodendrocytes and Schwann cells, a major pathogenetic pathway of the HTLV-I-induced myeloneuropathy.

Animals↗
PubMed1998

Deleted HTLV-1 provirus in cord-blood samples of babies born to HTLV-1-carrier mothers.

We screened 596 cord-blood samples by nested short PCRs for the gag and pX regions of HTLV-1 which are capable of detecting a single copy. The samples were derived from 449 and 147 babies born to seropositive and seronegative mothers respectively. Of these, 20 samples were positive in at least one of the PCRs: 9 (45%) were positive in both PCRs, but 10 and 1 samples in either the pX or the gag PCR respectively. These samples were tested further in nested long PCRs directed for gag-pX, gag-pol and pol-pX regions capable of detecting 8, 1 and 2 copies respectively. Of 9 dually positive samples, 7 (77%) showed the predicted 6.2-kbp band in the gag-pX PCR; only 2 of them had the predicted band alone; 7 samples had discrete bands shorter than the predicted size. In the gag-pol PCR, all 9 samples showed the predicted 2.2-kbp band alone. In the pol-pX PCR, 819 samples showed the predicted 4.2-kbp band, including one with an additional 2.1-kbp band, and the last a 1.0-kbp band alone. Thus, all of the dually positive samples had proviruses harboring gag, pol and pX priming sites. In contrast, none of the 11 singly positive samples showed the predicted band in the gag-pX PCR: 5 had no visible band, and the other 6 had shorter bands only. None of these 11 samples showed any positive signal in either gag-pol or pol-pX PCR. Our results suggest that HTLV-1 proviruses in the cord blood are frequently defective.

Base Sequence↗
PubMed1999

Titration of cellular export factors, but not heteromultimerization, is the molecular mechanism of trans-dominant HTLV-1 rex mutants.

The HTLV-1 Rex protein is an essential shuttle protein required for nuclear export of unspliced and incompletely-spliced viral RNAs. Several trans-dominant (TD) mutant Rex proteins have been reported, however, the mechanism of trans-dominance is not known. We compared TD Rex mutants and found that a natural occurring Rex mutant, Rexp21, lacking the RNA binding domain, was highly TD and inhibited also HIV-1 Rev function. Using fusions to the green fluorescent protein (GFP) we observed that Rexp21-GFP displayed a cytoplasmic localization but was actively shuttling between the nucleus and the cytoplasm in live human cells. The presence of Rexp21-GFP inhibited the nuclear export of Rex and HIV-1 Rev as assayed by cotransfection and microinjection experiments. However, Rex-GFP or Rexp21-GFP did not form heteromultimers with nuclear Rex mutants in vivo. In contrast, shuttling was essential for trans-dominance. Thus, we propose that TD Rex mutants do not function by retaining WT Rex in the nucleus by protein-protein interactions, as demonstrated for Rev, but to titrate factors essential for Rex/Rev export. Our findings demonstrate differences between the regulatory proteins Rex and Rev and implicate a novel strategy to generate highly TD Rex mutants also applicable to other proteins.

Animals↗
PubMed1997

HTLV-I induced myeloneuropathy in WKAH rats: apoptosis and local activation of the HTLV-I pX and TNF-alpha genes implicated in the pathogenesis.

To investigate the pathogenesis of HTLV-I associated diseases, we established a rat model for HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in WKAH rats. In the spinal cords of WKAH rats carrying HTLV-I, chronological histopathology revealed the occurrence of apoptotic cell death starting at 9 months after the infection, followed by demyelination, macrophage infiltration, and the activation of astrocytes starting at 12, 15 and 20 months, respectively. Apoptosis of the Schwann cells was also observed in the peripheral nerves of these rats. By RT-PCR, pX mRNA of HTLV-I was selectively expressed in the diseased spinal cords and peripheral nerves, but not in the unaffected cerebra, cerebella, even though provirus DNAs were consistently identified in these tissues. Among several cytokines examined, mRNA expression and production of TNF-alpha were frequently detected in the spinal cord and the cerebrospinal fluid. The collective evidence suggests that the selective activation of HTLV-I, in particular Tax expression, and/or the production of TNF-alpha in target spinal cord and peripheral nerves are causally related to apoptotic death of the oligodendrocytes and Schwann cells, a major pathogenetic pathway of HTLV-I induced myeloneuropathy in the WKAH rat.

Animals↗