Synphilin-1 isoforms in Parkinson's disease: regulation by phosphorylation and ubiquitylation.

Parkinson's disease (PD) is characterized by the death of dopaminergic neurons and the presence of Lewy bodies in the substantia nigra pars compacta. The mechanisms involved in the death of neurons as well as the role of Lewy bodies in the pathogenesis of the disease are still unclear. Lewy bodies are made of aggregated proteins, in which alpha-synuclein represents their major component. Alpha-synuclein interacts with synphilin-1, a protein that is also present in Lewy bodies. When expressed in cells, synphilin-1 forms inclusions together with alpha-synuclein that resemble Lewy bodies. Synphilin-1 is ubiquitylated by various E3 ubiquitin-ligases, such as SIAH, parkin and dorfin. Ubiquitylation of synphilin-1 by SIAH is essential for its aggregation into inclusions. We recently identified a new synphilin-1 isoform, synphilin-1A, that is toxic to neurons, aggregation-prone and accumulates in detergent-insoluble fractions of brains from alpha-synucleinopathy patients. Synphilin-1A inclusions recruit both alpha-synuclein and synphilin-1. Aggregation of synphilin-1 and synphilin-1A seems to be protective to cells. We now discuss several aspects of the neurobiology and pathology of synphilin-1 isoforms, focusing on possible implications for PD.

Insertion of polydeoxyadenosine-rich sequences into an intergenic region increases transcription in Chlamydomonas reinhardtii chloroplasts.

In this study, chloroplast transformation in Chlamydomonas reinhardtii was used to insert a tract of polydeoxyadenosine, which is known to influence DNA structure and transcription in other systems, between the 3' end of the atpB gene, encoding the beta-subunit of the chloroplast ATP synthase, and a downstream chimeric gene, aadA, encoding antibiotic resistance. Run-on transcription and RNA analyses revealed that in cells containing (dA)40 and (dAAAGGG)8, aadA was transcribed at a higher rate, and its RNA accumulated to a relatively high level. It is concluded that poly(dA/dT) can function in the chloroplast as a transcription enhancer element. Therefore, the insertion of poly(dA/dT) sequence into the intergenic region of a multicistronic transcription unit may modulate gene expression at the transcriptional level.

Borna disease virus: a mystery as an emerging zoonotic pathogen.

For Central European veterinarians, Borna disease (BD) has been known for a long time as a sporadically occurring, progressive viral polioencephalomyelitis predominantly affecting horses and sheep and-as discovered in the last decade-an increasing number of domestic and zoo animals. The aetiological agent, the Borna disease virus (BDV), a negative-sense, single-stranded RNA virus classified in the new virus family Bornaviridae within the order Mononegavirales, can induce severe clinical signs typically of a viral encephalitis with striking behavioural disturbances. After an incubation period lasting a few weeks to several months, BDV-infection causes locomotor and sensory dysfunctions followed by paralysis and death. Natural infections seem to be subclinical in most cases. BD received world-wide attention when it was reported that sera and/or cerebrospinal fluids from neuro-psychiatric patients can contain BDV-specific antibodies. Since infected animals produce BDV-specific antibodies only after virus replication, it was assumed that the broad spectrum of BDV-susceptible species also includes man. However, reports describing the presence of other BDV-markers, i.e. BDV-RNA or BDV-antigen, in peripheral blood leukocytes or brain tissue of neuro-psychiatric patients are highly controversial and, therefore, the role of BDV in human neuro-psychiatric disorders is questionable. (c) 2001 Harcourt Publishers Ltd.

[Werner Schäfer. A life as researcher and teacher]. / Werner Schäfer. Ein Leben als Forscher und Lehrer.

The following short biography recalls Professor Dr. Dr. h.c. Werner Schäfer, emeritus professor and director of the Medical Biology Department of the Max-Planck-Institut für Virusforschung in Tübingen and scientific member of the Max-Planck Society who died on 25th April 2000. He was one of the most distinguished pioneers of animal virology and one of the great personalities who since the Second World War have helped German science to regain its international reputation. In a brief synopsis the important results of his work on the viruses he used as models to conduct his research have been portrayed. As a result of Schäfer's scientific conception to gain insights into the functional characteristics of viruses by looking at their structure, the field of virology has taken new directions and founded a school whose pupils try to continue his successful and much honoured life's work.

The post-Loeffler-Frosch era: contribution of German virologists.

This presentation dealt with the contributions of German virologists in the rapid development of virology following the Loeffler-Frosch era. Thereby, only research was included which was undertaken within German institutions, even though guest scientists from other countries or international cooperative efforts have in some cases contributed to the work. Contributions to the field of veterinary virology were not considered here, since this topic was treated separately during this centennial symposium. The overview includes contributions of the very early period when interest was focussed mainly on the determination of the physicochemical properties of the fast growing number of newly detected viruses, and of the pioneering period when fundamental discoveries of the nature of viruses were made. The concepts that derived from those studies made the development of modern virology possible. Some highlights of the present period were presented describing the findings of selected virus families. This part was followed by a description of the results which were relevant to problems of how viruses become pathogens, and the role of the immune response to virus infections. Finally, attention was drawn to the contributions of molecular studies which became important not only for the field of virology but also for life sciences in general.

Altering the 3 UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects 3-end maturation in vitro but not in vivo.

The 3' ends of chloroplast mRNAs are produced by the processing of longer precursors. The 3' ends of most plastid mRNAs are located at, or several nucleotides downstream of, stem-loop structures, which act as 3'-end-processing signals and RNA stability elements. In chloroplasts of the green alga Chlamydomonas reinhardtii, 3'-end maturation of atpB mRNA involves endonucleolytic cleavage of the pre-mRNA at an AU-rich site located about 10 nucleotides downstream of the stem-loop structure. This cleavage is followed by exonucleolytic resection to generate the mature 3' end. In order to define critical nucleotides of the endonucleolytic cleavage site, we mutated its sequence. Incubation of synthetic atpB pre-RNAs containing these mutations in a chloroplast protein extract resulted in the accumulation of 3'-end-processed products. However, in two cases where the AU-rich sequence of this site was replaced with a GC-rich one, the 3' end of the stable processing product differed from that of the wild-type product. To examine whether these mutations affected atpB mRNA processing or accumulation in vivo, the endogenous 3' UTR was replaced with mutated sequences by biolistic transformation of Chlamydomonas chloroplasts. Analysis of the resulting strains revealed that the accumulation of atpB mRNA was approximately equal to that of wild-type cells, and that a wild-type atpB 3' end was generated. These results imply that Chlamydomonas atpB 3' processing parallels the situation with other endonucleases such as Escherichia coli RNAse E, where specific sequences are required for correct in vitro processing, but in vivo these mutations can be overcome.

3'-Processed mRNA is preferentially translated in Chlamydomonas reinhardtii chloroplasts.

3'-end processing of nucleus-encoded mRNAs includes the addition of a poly(A) tail that is important for translation initiation. Since the vast majority of chloroplast mRNAs acquire their 3' termini by processing yet are not polyadenylated, we asked whether 3' end maturation plays a role in chloroplast translation. A general characteristic of the 3' untranslated regions of chloroplast mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops and their flanking sequences serve as RNA 3'-end formation signals. Deletion of the Chlamydomonas chloroplast atpB 3' IR in strain Delta26 results in reduced accumulation of atpB transcripts and the chloroplast ATPase beta-subunit, leading to weakly photosynthetic growth. Of the residual atpB mRNA in Delta26, approximately 1% accumulates as a discrete RNA of wild-type size, while the remainder is heterogeneous in length due to the lack of normal 3' end maturation. In this work, we have analyzed whether these unprocessed atpB transcripts are actively translated in vivo. We found that only the minority population of discrete transcripts of wild-type size is associated with polysomes and thus accounts for the ATPase beta-subunit which accumulates in Delta26. Analysis of chloroplast rbcL mRNA revealed that transcripts extending beyond the mature 3' end were not polysome associated. These results suggest that 3'-end processing of chloroplast mRNA is required for or strongly stimulates its translation.

The sequence and structure of the 3'-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability.

A general characteristic of the 3'-untranslated regions (3' UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops are RNA 3'-end processing signals and determinants of mRNA stability, not transcription terminators. Incubation of synthetic RNAs corresponding to the 3' UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products. Synthetic RNAs of the petA 3' UTR and the antisense strand of atpB 3' UTR were degraded in the extract. To examine 3' UTR function in vivo, the atpB 3' UTR was replaced with the 3' UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR plus trnR, rbcL, petA and E. coli thrA by biolistic transformation of Chlamydomonas chloroplasts. Each 3' UTR was inserted in both the sense and antisense orientations. The accumulation of both total atpB mRNA and ATPase beta-subunit protein in all transformants was increased compared to a strain in which the atpB 3' UTR had been deleted. However, the level of discrete atpB transcripts in transformants containing the antisense 3' UTR sequences was reduced to approximately one-half that of transformants containing the 3' UTRs in the sense orientation. These results imply that both the nucleotide sequences and the stem-loop structures of the 3' UTRs are important for transcript 3'-end processing, and for accumulation of the mature mRNAs.

Determinants of pantropism of the F1-R mutant of Sendai virus: specific mutations involved are in the F and M genes.

Mutations in the fusion, F, protein of Sendai virus resulting in increased cleavability by ubiquitous host protease(s), and mutations in the matrix, M, protein resulting in bipolar budding, are both important determinants for the systemic infection in mice caused by the protease activating pantropic mutant, F1-R. Several mutants of Sendai virus (BY, BF, and KD-M) with phenotypes of bipolar budding and/or increased cleavability of F protein were isolated. Genomic RNA sequence analysis of the F and M genes of the mutants revealed that several deduced amino acids in the F and M proteins were different from those of F1-R, T-5 (a revertant of F1-R), and wild-type viruses. The BF and KD-M mutants that budded bipolarly and were also activated by ubiquitous proteases were examined for replication in tissue culture cells and in mice. All of the mutants exhibited multiple-step replication in MDCK, MDBK, and LLC-MK2 cells without trypsin, but formed plaques only in MDCK cells. One of the mutants, designated KD-52M, was similar to F1-R in that it formed plaques in all three cell lines without addition of exogenous protease. However, none of the mutants viruses, including KD-52M, caused a systemic infection in mice. The mutated M protein of F1-R enhances the disruption of microtubles. However, none of the mutants with a bipolar budding phenotype (BY, BF, and KD-M), disrupted the microtubules to the same extent as F1-R. All of these mutants had mutations in the M protein that were different from those found in F1-R. Taken together, these results suggest that mutations at Ser115 to Pro in the F protein and at Asp 128 to Gly and Ile210 to Thr in the M protein of F1-R are the mutations specifically required for the systemic infection caused by F1-R.

[Influenza, a special form of zoonosis]. / Influenza, eine besondere Form einer Zoonose.

Findings based on molecular genetics and phylogeny indicate that avian species represent an important reservoir for influenza viruses and that virus strains of man and different mammals originated from avian influenza virus ancestors. In contrast to infectious agents causing classical zoonoses, influenza viruses have to alter their genetic make up in order to change their host range. The special, segmented structure of the viral RNA allows an exchange of gene(s) between two different influenza viruses (reassortment) resulting in viruses with different combinations of genome segments and thereby creating new biological properties. Under the selective pressure of the new host the most adapted virus variants will succeed which arose from a genetically heterogeneous virus population with additional mutations. In particular mutations of the genes encoding the polymerase complex (mutator mutations) would be advantageous for rapid adaptation in a hostile environment. The generation of influenza viruses capable of overcoming the species barrier is a rare event since only virus variants will succeed which are genetically stable and transmissible and which replicate efficiently in the new host. It is considered likely that pigs act as intermediate hosts for adaptation of avian viruses to man.

Failure to detect Borna disease virus infection in peripheral blood leukocytes from humans with psychiatric disorders.

The presence of antibodies reactive with Borna disease virus (BDV) in the sera of some patients with certain psychiatric illnesses has been taken as evidence that this veterinary neurotrophic virus may occasionally infect and cause psychiatric disorders in humans. In this paper, we report the results of our studies concerning the detection of BDV-specific RNA in blood cells from patients with psychiatric diseases. Contrary to the results obtained by others, we have found no evidence for the presence of BDV-RNA in such cells. Prior work with BDV sequences in the assay environment, together with the exquisite sensitivity of RT-PCR, may account for the sporadic appearance of false positive evidence that BDV-specific RNA is present in human blood cells.

The 3' untranslated regions of chloroplast genes in Chlamydomonas reinhardtii do not serve as efficient transcriptional terminators.

A general characteristic of the 3' untranslated regions of plastid mRNAs is an inverted repeat sequence that can fold into a stem-loop structure. These stem-loops are superficially similar to structures involved in prokaryotic transcription termination, but were found instead to serve as RNA 3' end processing signals in spinach chloroplasts, and in the atpB mRNA of Chlamydomonas reinhardtii chloroplasts. In order to carry out a broad study of the efficiency of the untranslated sequences at the 3' ends of chloroplast genes in Chlamydomonas to function as transcription terminators, we performed in vivo run-on transcription experiments using Chlamydomonas chloroplast transformants in which different 3' ends were inserted into the chloroplast genome between a petD promoter and a reporter gene. The results showed that none of the 3' ends that were tested, in either sense or antisense orientation, prevented readthrough transcription, and thus were not highly efficient transcription terminators. Therefore, we suggest that most or all of the 3' ends of mature mRNAs in Chlamydomonas chloroplasts are formed by 3' end processing of longer precursors.

Involvement of the mutated M protein in altered budding polarity of a pantropic mutant, F1-R, of Sendai virus.

Wild-type Sendai virus buds at the apical plasma membrane domain of polarized epithelial MDCK cells, whereas a pantropic mutant, F1-R, buds at both the apical and basolateral domains. In F1-R-infected cells, polarized protein transport and the microtubule network are impaired. It has been suggested that the mutated F and/or M proteins in F1-R are responsible for these changes (M. Tashiro, J. T. Seto, H.-D. Klenk, and R. Rott, J. Virol. 67:5902-5910, 1993). To clarify which gene or mutation(s) was responsible for the microtubule disruption which leads to altered budding of F1-R, MDCK cell lines containing the M gene of either the wild type or F1-R were established. When wild-type M protein was expressed at a level corresponding to that synthesized in virus-infected cells, cellular polarity and the integrity of the microtubules were affected to some extent. On the other hand, expression of the mutated F1-R M protein resulted in the formation of giant cells about 40 times larger than normal MDCK cells. Under these conditions, the effects on the microtubule network were enhanced. The microtubules were disrupted and polarized protein transport was impaired as indicated by the nonpolarized secretion of gp80, a host cell glycoprotein normally secreted from the apical domain, and bipolar budding of wild-type and F1-R Sendai viruses. The mutated F glycoprotein of F1-R was transported bipolarly in cells expressing the F1-R M protein, whereas it was transported predominantly to the apical domain when expressed alone or in cells coexpressing the wild-type M protein. These findings indicate that the M protein of F1-R is involved in the disruption of the microtubular network, leading to impairment of cellular polarity, bipolar transport of the F glycoprotein, and bipolar budding of the virus.

Influenza viruses, cell enzymes, and pathogenicity.

Proteolytic cleavage of the influenza virus hemagglutinin glycoprotein (HA) by cellular proteases is a prerequisite for virus infectivity, spread of the virus in the infected organism, tissue tropism, and viral pathogenicity. Production of infectious virus depends upon the structure at the HA cleavage site as well as the substrate specificity and the distribution of appropriate enzymes. Differences exist in the specificities of the endoproteases that recognize the different sequence motifs at the cleavage site. With avian influenza viruses that cause lethal systemic infections, the cleavage site consists of multibasic amino acids. Furin, which activates this type of HA, is a member of the subtilisin family and represents the prototype of ubiquitously occurring membrane-bound proteases. On the other hand, serine proteases secreted from a restricted number of cell types and some bacterial enzymes recognize a monobasic cleavage signal at HA of the mammalian and the apathogenic avian influenza viruses. The limited occurrence of these proteases results in only localized infection. Implementation of these defined conditions for virus activation may represent a novel type of disease control.

Trypsin-resistant protease activation mutants of an influenza virus.

New classes of mutants of influenza virus A/seal/Mass/1/80 are described in which the haemagglutinins (HA) have lost their protease cleavability by trypsin, but can be activated by elastase, chymotrypsin or thermolysin in different cell types. The same proteases that were required for activation of infectivity of the mutants also activated haemolysis and cell-fusing properties. The protease activation (pa)-mutants were non-pathogenic for chickens, but induced a protective immune response against a highly pathogenic challenge virus. The failure of the mutants to be activated by trypsin, but instead to be activated by the other proteases employed, was related to amino acid exchanges around the HA cleavage site. The cleavability of the chymotrypsin and elastase pa-mutants is most likely determined by replacement of Arg-1 by neutral amino acids such as Ile, Thr, Met or Leu, depending on the substrate specificity of the activating proteases. Cleavage activation of the thermolysin pa-mutants, on the other hand, became possible by insertion of a single Leu residue at position 4 of the HA2 polypeptide, which compensates for the loss of the Gly residue at the N terminus of the fusion peptide due to thermolysin cleavage. The correction of the mutations in revertants confirmed the conclusions drawn from sequence analyses of the pa-mutants.

Pathogenicity of influenza A/Seal/Mass/1/80 virus mutants for mammalian species.

Increases in infectiousness, neurotropism and virulence were found in a laboratory variant of influenza A/Seal/Massachussets/1/80 (H7N7) virus having a highly cleavable hemagglutinin. Sequential passage from host to host further increased pathogenicity of the H7N7 virus in mice, ferrets and rats.

Induction of protection against Borna disease by inoculation with high-dose-attenuated Borna disease virus.

Borna disease is a chronic neurological disease caused by an enveloped negative-strand RNA virus (BDV). Experimental disease can be reproduced in rats with brain homogenates derived from infected animals or with virus derived from infected cells in culture. The virus replicates in cultured cells without evidence of cytopathic effect or production of significant levels of cell-free virus. Borna disease is caused by an immunopathological response to viral infection of neural cells. To further investigate the pathogenesis of Borna disease, rats were inoculated with different doses of BDV attenuated by culture in MDCK cells. Low doses of attenuated BDV (10(2)-10(4) TCID50) resulted in typical clinical disease and severe encephalitis; however, the lag period between inoculation and disease was considerably longer than that with virulent BDV. In contrast, animals inoculated with a high dose of attenuated BDV (10(5)-10(6) TCID50) did not develop clinical disease, although a mild encephalitic response was present that did not progress beyond the mild encephalitis. Animals inoculated with a high dose of BDV developed high titers of anti-BDV antibody and were protected against virulent challenge. Protection was correlated with the rapid induction of an immune response in the animals and the lack of any biologically detectable virus in the CNS.

Thermolysin activation mutants with changes in the fusogenic region of an influenza virus hemagglutinin.

Influenza virus A/seal/Mass/1/80 (H7N7) mutants were obtained; the hemagglutinins (HAs) of the mutants were not activated by trypsin, as in the wild-type virus, but by thermolysin. The mutants grew efficiently under multiple replication cycle conditions and formed plaques in chicken embryo cells only when thermolysin was added to the culture medium. They exhibited hemolytic activity and induced protective immunity in chickens after an asymptomatic course of infection. Nucleotide sequencing of the HA gene and direct amino acid sequencing showed that insertion of a single leucine into the fusion peptide of the HA2 chain close to the cleavage site and a shift of the cleavage site toward the C terminus by one amino acid were responsible for the changes in the biological properties of the thermolysin activation mutants. Revertants could be obtained when trypsin or trypsin-like endoproteases were present in the virus-producing system.