José Salas

Characterization of an African Swine Fever Virus 20-kDa DNA Polymerase Involved in DNA Repair

African swine fever virus (ASFV) encodes a novel DNA polymerase, constituted of only 174 amino acids, belonging to the polymerase (pol) X family of DNA polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA polymerase, highly distributive, lacking a proofreading 3′-5′-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA polymerases, together with the extrapolation to the crystal structure of mammalian DNA polymerase β (pol β), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol β-type DNA polymerase core, constituted by only the “palm” and “thumb” subdomains. It is worth noting that such an “unfingered” DNA polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle.

Role of the host cell nucleus in the replication of African swine fever virus DNA.

An examination by autoradiography of African swine fever virus-infected alveolar macrophages pulse labeled with [3H]thymidine showed that, at early times of viral DNA replication, the grains were localized exclusively in the nucleus in 20% of the cells, while in 45% the label was found in the cytoplasm. In the remaining 35%, newly synthesized DNA was detected in both the nucleus and the cytoplasm. At later times, the percentage of cells with grains in the nucleus decreased considerably. Pulse-chase experiments indicated that the DNA synthesized in the nucleus is then transported to the cytoplasm. The presence of virus-specific DNA sequences in the nucleus was confirmed by in situ hybridization of infected macrophages. Similar hybridization experiments with African swine fever virus-infected VERO cells followed by confocal microscopy also indicated the existence of a nuclear stage in the localization of the viral DNA. These results suggest a mechanism for African swine fever virus DNA replication with an initial stage in the nucleus followed by a cytoplasmic phase. Specific nuclear forms associated with the hybridization signal have been observed in African swine fever virus-infected macrophages and VERO cells. The nuclear forms seen in macrophages are consistent with a mechanism for the egress of the viral DNA from the nucleus that involves initial budding at the nuclear membrane.

Effect of neonatal and adult-onset hypothyroidism on pyramidal cells of the rat auditory cortex

Abstract Hearing dysfunctions of varying type and intensity are often found in endemic cretinism or associated with hypothyroidism, either congenital or acquired, and both in humans and experimental animals. We have studied the possible effect of thyroidectomy ( T ) of neonatal and adult rats on the morphology of pyramidal cells of the auditory cortex, the number and distribution of spines along their shaft being measured as parameters. The changes were compared to those found previously in the pyramids of the visual cortex of the same animals30–34. Both the number of apical shaft spines and their distribution change with age in pyramids of the auditory cortex, as studied between 10 and 220 days after birth, but the changes were not identical to those in the visual cortex. Results suggest a slower age-related evolution of the pyramidal cells of the auditory, as compared to the visual, cortex. T at 10 days of age resulted in a decreased number of apical shaft spines, the spine distribution curves also being affected. Treatment of these rats with 1.5 μg T 4/100 g body weight/day did not prevent these changes, even if started at 12 and 15 days of age. On the contrary, clear amelioration of visual cortex pyramidal cells was observed in the same animals. The number and distribution of spines along the shaft of the pyramidal cells of the auditory cortex was also affected by T of rats at 40 and 120 days of age, though longer exposure to hypothyroidism appears to be needed than to observe similar changes in neurons of the visual cortex. Present data therefore show a causal relationship between hypothyroidism and alterations of pyramidal cells of the auditory cortex. Some of the results also suggest that during the neonatal period hypothyroidism may induce changes in auditory cortex pyramids which are either more sensitive to a minor degree of hypothyroidism, or have a shorter ‘critical period’ during which treatment may reverse damage, than other cerebral or cerebellar neurons and structures.

Deep structure of an open sea eddy in the Algerian Basin

The Algerian Basin dynamics is dominated by the presence of very energetic mesoscale structures. Deep open sea eddies are key features that influence the regional circulation of water masses at all depths. In May 1998, an open sea eddy was sampled near 38j N2 jE by means of CTD, Acoustic Doppler Current Profiler (ADCP) and surface Lagrangian drifters. For the first time, it has been possible to characterise one of these mesoscale structures in its full depth, down to 2800 m. The density distribution indicated the presence of lighter water in the centre of the eddy from the surface to the bottom. The direct velocity measurements in the surface layer, plus the determination of the baroclinic shear from CTD data, evidenced that the anticyclonic motion was present in the whole water depth. D 2002 Elsevier Science B.V. All rights reserved.

Statistical analysis of the surface circulation in the Algerian Current using Lagrangian buoys

Abstract The Algerian Current (AC) is one of the most energetic flows in the Mediterranean basin. A characteristic picture of this current is formed by a series of mesoscale eddies at different scales. Here, statistical analysis of 15 surface Argos buoy tracks in 1996–1997 provides a complete Lagrangian view of the AC. The buoys, released upstream and across a coastal meander between 0°E and 1°E longitude, were followed for 3 months. They travelled eastward at an average speed of 14 cm/s and showed high energetic fluctuations related to several mesoscale eddies. The characteristic integral time and space scales are highly anisotropic. For the zonal component, these are about 4 days and 66 km, and for the meridional component, about 2 days and 26 km. Representative values of effective diffusivities from single dispersion statistics are within 0.7–1.3×10 8 and 1.5–6.0×10 7 cm 2 /s for the zonal and meridional directions, respectively. A local analysis shows that mesoscale motions are particularly relevant in the region 1–3°E and 7–8°E, provided the considerably high values of eddy kinetic energy in comparison with the mean kinetic energy. Eddy–mean current interactions are evidenced by the significant changes of sign of horizontal covariance from west to east. Finally, an Eulerian picture of the AC is built, exhibiting similar trends than previous and recent field observations.

Vaccinia virus-induced apoptosis in immature B lymphocytes: role of cellular Bcl-2.

Apoptosis is a form of physiological cell death which can be initiated in response to various stimuli including virus infections. We show that vaccinia virus (VV) infection induces apoptosis in an immature B lymphocyte line, WEHI-231. In these cells, several VV-specific proteins were synthesized during the infection, but neither virus production nor viral DNA synthesis were detected. The intracellular levels of the proto-oncogene Bcl-2, which effectively protects cells from programmed cell death, were found to be down-regulated by the VV infection, suggesting that this down-regulation might be involved in the viral induction of apoptosis in WEHI-231 cells. Stable transfectants overexpressing human Bcl-2 were shown to be resistant to the apoptosis produced by the infection, a finding consistent with the proposed role for the down-regulation of endogenous Bcl-2 in VV-induced apoptotic death.

Effect of inhibitors of the host cell RNA polymerase II on African swine fever virus multiplication

The role of the host cell RNA polymerase II in African swine fever (ASF) virus growth has been examined using inhibitors of this enzyme. The adenosine analog 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of mRNA precursor synthesis in mammalian cells, strongly inhibits the production of infectious progeny virus in Vero cells, but does not significantly affect the synthesis of virus-specific macromolecules. On the other hand, virion assembly seems to proceed normally in the presence of DRB, as virus particles can be seen in electron micrographs with a morphology indistinguishable from that observed in the absence of the inhibitor. However, taking into account the inhibition of the infectivity caused by the drug, most of these particles must be defective. In contrast with this effect of DRB on ASF virus replication, the toxin alpha-amanitin does not inhibit the production of infectious ASF virus in Vero cells or porcine alveolar macrophages. This result indicates that the host RNA polymerase II does not transcribe viral genes and that active transcription of the cell genome is not needed for ASF virus replication.

Poly(A) polymerases in normal and virus-transformed cells

The presence of poly(A) polymerase(s) has been studied in a clone of the established hamster embryo fibroblast line (NIL), and in a subclone of this line transformed by an RNA tumour virus, hamster sarcoma virus, (NIL-HS VIRUS). The results suggest the existence of three distinct poly(A) polymerases, designated I, IIA and IIB, in dense cultures of NIL and NIL-HS virus cells. Forms IIA and IIB have also been found in exponentially growing NIL and NIL-HS virus cells. Poly(A) polymerase I has not been detected in growing NIL cells, while growing and resting NIL-HS virus contain comparable amounts of this enzyme. The poly(A) polymerases differ in chromatographic behaviour and in their requirements for divalent cations. They are highly specific for ATP and require the presence of a primer. Cellular RNA or poly(A), but not the oligoribonucleotide (Ap)4A, can be utilized as primers. The products of the reactions have been identified as poly(A) chains (20-50 nucleotides long) by alkaline degradation and by their resistance to pancreatic RNAase.

Phosphorylation of African swine fever virus proteins in vitro and in vivo

Highly purified African swine fever virus contains a cyclic AMP-independent protein kinase which phosphorylates endogenous virus proteins with a specific activity of about 0.45 pmol/microgram of virus protein. The major substrates for the virion protein kinase in vitro were the structural proteins p10 and p9. Both proteins were phosphorylated preferentially at serine residues. A possible relationship between protein p10 phosphorylation and RNA synthesis in vitro by the virion-associated RNA polymerase is suggested by the finding that N-alpha-tosyl-L-lysyl-chloromethyl ketone inhibited both phosphorylation of p10 and transcription. Two phosphoproteins, with molecular masses of 35 and 17 kDa, were found in African swine fever virus purified from infected Vero cells labeled with [32P]phosphate. A phosphopolypeptide with a molecular mass of about 35 kDa was found in the cytoplasm of infected Vero cells.

African swine fever virus AP endonuclease is a redox-sensitive enzyme that repairs alkylating and oxidative damage to DNA.

African swine fever virus (ASFV) encodes an AP endonuclease (pE296R) which is essential for virus growth in swine macrophages. We show here that the DNA repair functions of pE296R (AP endonucleolytic, 3′ → 5′ exonuclease, 3′-diesterase and nucleotide incision repair (NIR) activities) and DNA binding are inhibited by reducing agents. Protein pE296R contains one intramolecular disulfide bond, whose disruption by reducing agents might perturb the interaction of the viral AP endonuclease with the DNA substrate. The characterization of the 3′ → 5′ exonuclease and 3′-repair diesterase activities of pE296R indicates that it has strong preference for mispaired and oxidative base lesions at the 3′-termini of single-strand breaks. Finally, the viral protein protects against DNA damaging agents in both prokaryotic and eukaryotic cells, emphasizing its importance in vivo. The biochemical and genetic properties of ASFV AP endonuclease are consistent with the repair of DNA damage generated by the genotoxic intracellular environment of the host macrophage.