Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE) functions in a position-dependent manner

The Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE) is a cis-acting RNA element located in the 3' untranslated region (UTR) of the viral genome. The HIV-1 and SIV Rev/RRE regulatory system can be replaced with MPMV CTE (Bray et al., 1994; Zolotukhin et al., 1994; Rizvi et al., 1996a); similarly, CTE function can also be replaced by the HIV or SIV Rev/RRE regulatory system (Rizvi et al., 1996b; Ernst et al., 1997). In addition, we have shown that in the context of the SIV genome, position is important for CTE function (Rizvi et al., 1996a). To determine the importance of position for CTE function in the context of the MPMV genome, MPMV molecular clones were generated by deleting CTE or removing it from the 3' UTR and placing it in the approximately 40 bp of intervening sequences between the pol termination codon and env initiation codon. A test of these molecular clones in a single round of replication assay revealed that deletion or displacement of CTE in the intervening sequences between pol and env completely abrogated virus replication. Western blot analysis of cell lysates and pelleted culture supernatants revealed negligible amounts of Pr78 Gag/Pol precursor and the processed p27(gag) when CTE was deleted or displaced. Slot blot analysis of fractionated RNAs revealed entrapment of the viral Gag/Pol mRNA in the nucleus with CTE deletion or displacement. Upon reinsertion of CTE in the original genomic position of clones with the deleted or displaced CTE, virus replication, Gag/Pol protein production, and nucleocytoplasmic transport of viral mRNA were restored to normal levels. Displacement of CTE to the 5' UTR immediately upstream of the Gag initiation codon also resulted in aberrant Gag/Pol protein production and nucleocytoplasmic transport of viral RNA. Reinsertion of CTE at the original genomic position of the clone with CTE displacement at the 5' UTR restored normal Gag/Pol protein production and RNA transport, demonstrating that the 3' terminal position of CTE is important for its function. To explore why the 3' terminal location of CTE is important, heterologous DNA sequences of increasing lengths were inserted between CTE and the polyadenylation (poly(A)) signal of the virus to augment the distance between the two cis-acting elements. Test of these constructs revealed that CTE function was progressively lost with incremental increase in distance between CTE and poly(A). To explore this relationship further, CTE was displaced to the env region approximately 2000 bp upstream of the poly(A) signal which abrogated CTE function. However, cloning of poly(A) signal to approximately 200 bp downstream of CTE in the env region (the natural distance between CTE and poly(A)) restored CTE function. Together, these results demonstrate that the close proximity of CTE to the poly(A) signal is important for CTE function, suggesting a functional interaction between CTE and the polyadenylation machinery.     

Solution structure and backbone dynamics of Mason-Pfizer monkey virus (MPMV) nucleocapsid protein

Retroviral nucleocapsid proteins (NCPs) are CCHC-type zinc finger proteins that mediate virion RNA binding activities associated with retrovirus assembly and genomic RNA encapsidation. Mason-Pfizer monkey virus (MPMV), a type D retrovirus, encodes a 96-amino acid nucleocapsid protein, which contains two Cys-X2-Cys-X4-His-X4-Cys (CCHC) zinc fingers connected by an unusually long 15-amino acid linker. Homonuclear, two-dimensional sensitivity-enhanced 15N-1H, three-dimensional 15N-1H, and triple resonance NMR spectroscopy have been used to determine the solution structure and residue-specific backbone dynamics of the structured core domain of MPMV NCP containing residues 21-80. Structure calculations and spectral density mapping of N-H bond vector mobility reveal that MPMV NCP 21-80 is best described as two independently folded, rotationally uncorrelated globular domains connected by a seven-residue flexible linker consisting of residues 42-48. The N-terminal CCHC zinc finger domain (residues 24-37) appears to adopt a fold like that described previously for HIV-1 NCP; however, residues within this domain and the immediately adjacent linker region (residues 38-41) are characterized by extensive conformational averaging on the micros-ms time scale at 25 degrees C. In contrast to other NCPs, residues 49-77, which includes the C-terminal CCHC zinc-finger (residues 53-66), comprise a well-folded globular domain with the Val49-Pro-Gly-Leu52 sequence and C-terminal tail residues 67-77 characterized by amide proton exchange properties and 15N R1, R2, and (1H-15N) NOE values indistinguishable to residues in the core C-terminal finger. Twelve refined structural models of MPMV NCP residues 49-80 (pairwise backbone RMSD of 0.77 A) reveal that the side chains of the conserved Pro50 and Trp62 are in van der Waals contact with one another. Residues 70-73 in the C-terminal tail adopt a reverse turn-like structure. Ile77 is involved in extensive van der Waals contact with the core finger domain, while the side chains of Ser68 and Asn75 appear to form hydrogen bonds that stabilize the overall fold of this domain. These residues outside of the core finger structure are conserved in D-type and related retroviral NCPs, e.g., MMTV NCP, suggesting that the structure of MPMV NCP may be representative of this subclass of retroviral NCPs.     

Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1

The hypothesis that the cellular protein Crm1 mediates human immunodeficiency virus type 1 (HIV-1) Rev-dependent nuclear export posits that Crm1 can directly interact both with the Rev nuclear export signal (NES) and with cellular nucleoporins. Here, we demonstrate that Crm1 is indeed able to interact with active but not defective forms of the HIV-1 Rev NES and of NESs found in other retroviral nuclear export factors. In addition, we demonstrate that Crm1 can bind the Rev NES when Rev is assembled onto the Rev response element RNA target and that Crm1, like Rev, is a nucleocytoplasmic shuttle protein. Crm1 also specifically binds the Rev NES in vitro, although this latter interaction is detectable only in the presence of added Ran . GTP. Overexpression of a truncated, defective form of the nucleoporin Nup214/CAN, termed DeltaCAN, that retains Crm1 binding ability resulted in the effective inhibition of HIV-1 Rev or human T-cell leukemia virus Rex-dependent gene expression. In contrast, DeltaCAN had no significant affect on Mason-Pfizer monkey virus constitutive transport element (MPMV CTE)-dependent nuclear RNA export or on the expression of RNAs dependent on the cellular mRNA export pathway. As a result, DeltaCAN specifically blocked late, but not early, HIV-1 gene expression in HIV-1-infected cells. These data strongly validate Crm1 as a cellular cofactor for HIV-1 Rev and demonstrate that the MPMV CTE nuclear RNA export pathway uses a distinct, Crm1-independent mechanism. In addition, these data identify a novel and highly potent inhibitor of leucine-rich NES-dependent nuclear export.     

A proline-rich motif (PPPY) in the Gag polyprotein of Mason-Pfizer monkey virus plays a maturation-independent role in virion release

Virus assembly represents one of the last steps in the retrovirus life cycle. During this process, Gag polyproteins assemble at specific sites within the cell to form viral capsids and induce membrane extrusion (viral budding) either as assembly progresses (type C virus) or following formation of a complete capsid (type B and type D viruses). Finally, the membrane must undergo a fusion event to pinch off the particle in order to release a complete enveloped virion. Structural elements within the MA region of the Gag polyprotein define the route taken to the plasma membrane and direct the process of virus budding. Results presented here suggest that a distinct region of Gag is necessary for virus release. The pp24 and pp16 proteins of the type D retrovirus Mason-Pfizer monkey virus (M-PMV) are phosphoproteins that are encoded in the gag gene of the virus. The pp16 protein is a C-terminally located cleavage product of pp24 and contains a proline-rich motif (PPPY) that is conserved among the Gag proteins of a wide variety of retroviruses. By performing a functional analysis of this coding region with deletion mutants, we have shown that the pp16 protein is dispensable for capsid assembly but essential for virion release. Moreover, additional experiments indicated that the virus release function of pp16 was abolished by the deletion of only the PPPY motif and could be restored when this motif alone was reinserted into a Gag polyprotein lacking the entire pp16 domain. Single-amino-acid substitutions for any of the residues within this motif confer a similar virion release-defective phenotype. It is unlikely that the function of the proline-rich motif is simply to inhibit premature activation of protease, since the PPPY deletion blocked virion release in the context of a protease-defective provirus. These results demonstrate that in type D retroviruses a PPPY motif plays a key role in a late stage of virus budding that is independent of and occurs prior to virion maturation.     

The apolipoprotein(a) promoter contains a retinoid response element

OBJECTIVE AND IMPORTANCE: Dural arteriovenous fistulas of the superior sagittal sinus (SSS) account for 8% of intracranial dural fistulas. Their association with a thrombosis of the posterior part of the SSS is rare. In such cases, the usual neurosurgical and endovascular approaches cannot provide a good technical solution for treatment of the lesion, and a combined neurosurgical and neuroradiological approach is therefore needed. CLINICAL PRESENTATION: A 68-year-old man presented with rapidly evolving dementia. Cerebral angiography revealed a dural arteriovenous fistula of the SSS associated with thrombosis of the posterior part of the SSS. Various endovascular and neurosurgical approaches failed to cure the fistula. INTERVENTION: A burr hole was drilled in the frontal region, in the neurosurgical room. The patient was then transferred to the angiographic room, and the SSS was occluded using free spirals. CONCLUSION: This procedure led to a complete anatomic cure of the fistula, and a slow clinical improvement was observed.     

Encephalomyelitis due to a Sarcocystis neurona-like protozoan in a rhesus monkey (Macaca mulatta) infected with simian immunodeficiency virus

A captive-born rhesus monkey (Macaca mulatta) experimentally infected with simian immunodeficiency virus developed neurologic abnormalities approximately seven months postinoculation. A chronic necrotizing encephalomyelitis with intralesional protozoal schizonts was diagnosed histologically. The protozoa was identified as Sarcocystis neurona based on its morphologic characteristics by light and electron microscopic examination, the developmental stages of the schizonts, and positive staining with antisera against Sarcocystis cruzi by immunocytochemical techniques. Although S. neurona may be confused with Toxoplasma gondii by light microscopy, the former lacks rhoptries, is in direct contact with the host cell cytoplasm, and divides by endopolygeny. Sarcocystis neurona has recently been identified as an etiologic agent of encephalomyelitis in horses, raccoons, and mink.     

Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system

The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.     

Expression of sodium-dependent purine nucleoside carrier (SPNT) mRNA correlates with nucleoside transport activity in rat liver

Co-ligation of antigen receptor and complement receptor 2 (CD21) in the B cell membrane is important in the immune response to T-dependent antigens. Four CD21 ligands have so far been identified, but only the activated products of the third component of complement (C3) are known to augment the immune response to specific antigens. The most recently discovered ligand for CD21 is CD23. We have generated a CD32+ CD23+ fibroblast cell line which presents a surrogate antigen (anti-IgM) to human tonsil B cells in vitro. Incubation with these cells causes a 10- to 100-fold reduction in the threshold concentration of anti-IgM required for B cell proliferation. Anti-CD19 further enhances the response to antigen and induces proliferation in the absence of anti-IgM. Addition of soluble CD21 totally inhibits the effect of CD23, suggesting that CD21 mediates synergistic signaling by CD23.     

The parameters of cellular proliferation in acute (immature-cell) leukemias

The standard distribution of the fatty acids in the total lipids of serum was gaschromatographically defined in the respective umbilical cord blood of 24 mothers and their newborn babies, as well as in 20 placentas. The same examinations were made with 30 newborn babies on the day of birth, 14 children in the newborn stage, 22 young babies between 3 and 10 weeks old and 16 older babies between 4 and 12 months old. In the umbilical cord mixed blood of another collective (n = 10) the separation of the total serum lipids in the individual fatty fractions (neutral lipids, phospholipids, cholesterol esters) was also undertaken. The results were compared and existing differences were discussed. It was further established that an advance towards the adult's fatty acid pattern already takes place at an age between 4 and 12 months.     

Renal cellular transport, metabolism, and cytotoxicity of S-(6-purinyl)glutathione, a prodrug of 6-mercaptopurine, and analogues

Plasmids currently used for nonviral gene transfer have the disadvantage of carrying a bacterial origin of replication and an antibiotic resistance gene. There is, therefore, a risk of uncontrolled dissemination of the therapeutic gene and the antibiotic resistance gene. Minicircles are new DNA delivery vehicles which do not have such elements and are consequently safer as they exhibit a high level of biological containment. They are obtained in E. coli by att site-specific recombination mediated by the phage lambda integrase. The desired eukaryotic expression cassette, bounded by the lambda attP and attB sites was cloned on a recombinant plasmid. The expression cassette was excised in vivo after thermoinduction of the integrase gene leading to the formation of two supercoiled molecules the minicircle and the starting plasmid lacking the expression cassette. In various cell lines, purified minicircles exhibited a two- to 10-fold higher luciferase reporter gene activity than the unrecombined plasmid. This could be due to either the removal of unnecessary plasmid sequences, which could affect gene expression or the smaller size of mini-circle which may confer better extracellular and intracellular bioavailability and result in improved gene delivery properties.     

p21(waf1) mRNA contains a conserved element in its 3'-untranslated region that is bound by the Elav-like mRNA-stabilizing proteins

The Elav-like proteins are specific mRNA-binding proteins that regulate mRNA stability. The neuronal members of this family (HuD, HuC, and Hel-N1) are required for neuronal differentiation. In this report, using purified HuD protein we have localized a high affinity HuD binding site to a 42-nucleotide region within a U-rich tract in the 3'-untranslated region p21(waf1) mRNA. The binding of HuD to this site is readily displaced by an RNA oligonucleotide encoding the HuD binding site of c-fos. The sequence of this binding site is well conserved in human, mouse, and rat p21(waf1) mRNA. p21(waf1) is an inhibitor of cyclin-dependent kinases and proliferating cell nuclear antigen and induces cell cycle arrest at G1/S, a requisite early step in cell differentiation. The identification of an Elav-like protein binding site in the 3'-untranslated region of p21(waf1) provides a novel link between the induction of differentiation, mRNA stability, and the termination of the cell cycle.     

The 3'-untranslated region of human type 2 iodothyronine deiodinase mRNA contains a functional selenocysteine insertion sequence element

Type 2 deiodinase (D2) catalyzes the 5'-deiodination of thyroxine to form 3,5,3'-triiodothyronine. Two mammalian D2 cDNAs have been identified containing 2 kilobases (kb) of the 7-kb mRNA including the complete coding sequence. Both contain in-frame TGA codons, which should serve as selenocysteine codons. However, the selenocysteine insertion sequence (SECIS) elements required for the decoding of UGA as a selenocysteine in the 3'-untranslated region (UTR) of the mRNA are not present. We have identified two overlapping expressed sequence tag clones, which contain the missing 4.4-kb 3'-UTR of the human D2 (hD2) cDNA. Computer analysis predicts a stem loop structure 280 base pairs 5' to the polyadenylation site, which has potent SECIS activity. A fragment containing these sequences hybridizes to D2 mRNA in human thyroid. A G to A mutation in the essential AUGA motif of this element abolished its function. Transfection of the hD2 coding region plus the 3'-UTR results in the expression of D2, and its in vitro transcribed mRNA programs D2 activity in Xenopus oocytes. This is the first identification of a SECIS element in a mammalian D2 cDNA and establishes that hD2 is a bona fide selenoprotein.     

The development of auditory event related potentials in the rhesus monkey (Macaca mulatta)

Auditory event related potentials were recorded from neonatal, 3-month, and 3-year old rhesus monkeys. Auditory brainstem evoked responses (ABRs) were reliably recorded at all ages. ABR latencies decreased with age. Age effects were greater the more centrally generated the wave. Wave I amplitude decreased with age, Wave II increased, and Wave IV remained about the same. Stimulus rate effects were greater in neonates than older monkeys. Stimulus frequency also affected the ABR, but not differentially as a function of age. Recording montage had a significant effect on the recorded waveform. Wave I tended to be larger in amplitude in horizontal recordings and front-back recordings, while the later waves were relatively more prominent in more vertical montages. Middle latency evoked responses and late potentials were less reliably recorded than the ABR. Their reproducibility improved with age. Auditory event related potentials are promising measures of auditory function for research requiring nonhuman primate models of the developing human.     

The ultrastructural features of progestagen-induced decidual cells in the rhesus monkey (Macaca mulatta)

Five rhesus monkeys were treated with levonorgestrel-releasing intravaginal rings for 52 weeks. The rings were designed to provide a sustained release of ten times the human dose level of the hormone. Histological examination of the uterine endometrium showed widespread decidualisation of endometrial stromal cells. Ultrastructurally, two distinct cell types were identified: decidual and granular. Some of the decidual cells were ultrastructurally comparable with those reported elsewhere; however, in aproportion of these cells, intracisternal sequestration of the rough endoplasmic reticulum was widespread. The granular cells resembled those described in the metrial gland of rats and, as their name suggests, contained variable numbers of electron-dense granules.     

The Philippine cynomolgus monkey (Macaca fasicularis) provides a new nonhuman primate model of tuberculosis that resembles human disease

A nonhuman primate model of tuberculosis that closely resembles human disease is urgently needed. We have evaluated the Philippine cynomolgus monkey, Macaca fasicularis, as a model of TB. Cynomolgus monkeys challenged intratracheally with extremely high doses of Mycobacterium tuberculosis (10(5) or 10(4) CFU) developed an acute, rapidly progressive, highly fatal multilobar pneumonia. However, monkeys challenged with moderate or low doses of M. tuberculosis (相似文献