Inactivation of the RNase Activity of Glycoprotein Erns of Classical Swine Fever Virus Results in a Cytopathogenic Virus

Envelope glycoprotein E^rns of classical swine fever virus (CSFV) has been shown to contain RNase activity and is involved in virus infection. Two short regions of amino acids in the sequence of E^rns are responsible for RNase activity. In both regions, histidine residues appear to be essential for catalysis. They were replaced by lysine residues to inactivate the RNase activity. The mutated sequence of E^rns was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact E^rns, the mutated proteins had lost their RNase activity. The mutated proteins reacted with E^rns-specific neutralizing monoclonal and polyclonal antibodies and were still able to inhibit infection of swine kidney cells (SK6) with CSFV, but at a concentration higher than that measured for intact E^rns. This result indicated that the conformation of the mutated proteins was not severely affected by the inactivation. To study the effect of these mutations on virus infection and replication, a CSFV mutant with an inactivated E^rns (FLc13) was generated with an infectious DNA copy of CSFV strain C. The mutant virus showed the same growth kinetics as the parent virus in cell culture. However, in contrast to the parent virus, the RNase-negative virus induced a cytopathic effect in swine kidney cells. This effect could be neutralized by rescue of the inactivated E^rns gene and by neutralizing polyclonal antibodies directed against E^rns, indicating that this effect was an inherent property of the RNase-negative virus. Analyses of cellular DNA of swine kidney cells showed that the RNase-negative CSFV induced apoptosis. We conclude that the RNase activity of envelope protein E^rns plays an important role in the replication of pestiviruses and speculate that this RNase activity might be responsible for the persistence of these viruses in their natural host.Classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV), and border disease virus belong to the genus Pestivirus within the family Flaviviridae (10). The viruses are structurally, antigenically, and genetically closely related. BVDV and border disease virus can infect ruminants and pigs. CSFV infections are restricted to pigs (6). Pestiviruses are small, enveloped, positive-stranded RNA viruses (23). The genome of pestiviruses varies in length from 12.5 to 16.5 kb (1, 2, 7, 17, 19, 25, 26, 28, 32) and contains a single large open reading frame (ORF) (1, 7, 8, 17, 26). The ORF is translated into a polyprotein which is processed into mature proteins by viral and host cell proteases (30). The envelope of the pestivirus virion contains three glycoproteins, E^rns, E1, and E2 (35). Animals infected with pestiviruses raise antibodies against at least two viral glycoproteins, namely, E^rns and E2 (16, 34, 42). Inhibition studies with E2 and E^rns produced in insect cells showed that both envelope proteins are indispensable for viral attachment and entry of pestiviruses into susceptible cells (13). In the virion, E^rns is present as a homodimer with a molecular mass of about 100 kDa (35). E^rns lacks a membrane anchor, and association with the envelope is accomplished by an as-yet-unknown mechanism. Significant amounts of E^rns are secreted from infected cells (30). A unique feature is that E^rns, besides being an envelope protein, possesses RNase activity (12, 31). E^rns belongs to the family of extracellular RNases consisting of several fungal (e.g., RNase T₂ and Rh) and plant (e.g., S glycoproteins of Nicotiana alata) RNases (12, 31). These RNases contain two homologous regions of 8 amino acids each which are spaced by 38 (E^rns) nonhomologous amino acids and which form the RNase active site. Histidine residues in both regions appear to be essential for RNase catalysis (15).The role of this RNase activity in the replication of pestiviruses or in the pathogenesis of a pestivirus infection is an interesting issue that, as yet, has not been studied. The availability of a recently generated infectious DNA copy of CSFV strain C (24) has given us the opportunity to study the effect of defined mutations in a pestivirus genome. In this paper, we report the inactivation of the RNase activity of E^rns by mutagenesis. To characterize the mutated proteins, we produced large amounts of them in insect cells (12). By reverse genetics, we generated an RNase-negative CSFV recombinant. The effect of the inactivation of the RNase activity of E^rns on the replication of CSFV in vitro was studied.     

B30.2-like domain proteins: update and new insights into a rapidly expanding family of proteins

The B30.2 domain is a conserved region of around 170 amino acids associated with several different protein domains, including the immunoglobulin folds of butyrophilin and the RING finger domain of ret finger protein. We recently reported several novel members of this family as well as previously undescribed protein families possessing the B30.2 domain. Many proteins have subsequently been found to possess this domain, including pyrin/marenostrin and the midline 1 (MID1) protein. Mutations in the B30.2 domain of pyrin/marenostrin are implicated in familial Mediterranean fever, and partial loss of the B30.2 domain of MID1 is responsible for Opitz G/BBB syndrome, characterized by developmental midline defects. In this study, we scrutinized the available sequence data bases for the identification of novel B30.2 domain proteins using highly sensitive database-searching tools. In addition, we discuss the chromosomal localization of genes in the B30.2 family, since the encoded proteins are likely to be involved in other forms of periodic fever, autoimmune, and genetic diseases.      

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Placentation in dolphins from the Amazon River Basin: the Boto, Inia geoffrensis, and the Tucuxi, Sotalia fluviatilis

A recent reassessment of the phylogenetic affinities of cetaceans makes it timely to compare their placentation with that of the artiodactyls. We studied the placentae of two sympatric species of dolphin from the Amazon River Basin, representing two distinct families. The umbilical cord branched to supply a bilobed allantoic sac. Small blood vessels and smooth muscle bundles were found within the stroma of the cord. Foci of squamous metaplasia occurred in the allanto-amnion and allantochorion. The interhemal membrane of the placenta was of the epitheliochorial type. Two different types of trophoblastic epithelium were seen. Most was of the simple columnar type and indented by fetal capillaries. However, there were also areolar regions with tall columnar trophoblast and these were more sparsely supplied with capillaries. The endometrium was well vascularised and richly supplied with actively secreting glands. These findings are consistent with the current view that Cetacea are nested within Artiodactyla as sister group to the hippopotamids.     

Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides

Background  

Lignin and hemicelluloses are the major components limiting enzyme infiltration into cell walls. Determination of the topochemical distribution of lignin and aromatics in sugar cane might provide important data on the recalcitrance of specific cells. We used cellular ultraviolet (UV) microspectrophotometry (UMSP) to topochemically detect lignin and hydroxycinnamic acids in individual fiber, vessel and parenchyma cell walls of untreated and chlorite-treated sugar cane. Internodes, presenting typical vascular bundles and sucrose-storing parenchyma cells, were divided into rind and pith fractions.     

Identification of mouse brain proteins associated with isoform 3 of metallothionein

Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and beta actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.     

Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors

Background  

The tear film is a thin layer of fluid that covers the ocular surface and is involved in lubrication and protection of the eye. Little is known about the protein composition of tear fluid but its deregulation is associated with disease states, such as diabetic dry eyes. This makes this body fluid an interesting candidate for in-depth proteomic analysis.     

Ecosystem linkages revealed by experimental lake-derived isotope signal in heathland food webs

Cross-ecosystem movement of nutrients and biomass can have important effects on recipient systems. Emerging aquatic insects are subsidies to terrestrial ecosystems and can influence foodweb interactions in riparian systems. In a 2-year field experiment, we simulated aquatic insect deposition by adding adult midge carcasses (150?g dry mass m^?2 year^?1) to 1-m² heathland plots at a site with low natural midge deposition. We established four levels of midge-addition treatments and measured stable isotopes (??¹³C and ??¹⁵N) in plants and arthropods within each treatment. We used a multiple-source isotope Bayesian mixing model to estimate the terrestrial versus aquatic contribution to the diets of arthropods. Aquatic resources were incorporated into plant, detritivore, and predator biomass. Detritivorous Collembola showed the greatest difference in isotope values (+3??? ??¹⁵N and +4??? ??¹³C) between midge-addition and reference treatments. Isotope values of small spiders followed the same trend of enrichment as Collembola while other arthropods (mites and large spiders) were only enriched after 2?years of midge addition. Although predator diets did not change, they became isotopically enriched via their likely prey (Collembola). Plants also had elevated ??¹⁵N (+1???) in midge-addition treatments. The time required and amount of midge-derived C and N detected varied and depended on trophic position. Midge-derived nutrients were no longer present in arthropod biomass in the year following midge addition. Aquatic insect carcasses can be rapidly incorporated into terrestrial food webs in nearshore habitats, and repeated inputs can be detected at multiple trophic levels, thus highlighting the importance of the detrital pathway for aquatic to terrestrial cross-ecosystem subsidies.