Measles virus and otosclerosis.

Measles virus (MeV) might play an important role as an environmental stimulus in the etiopathogenesis of otosclerosis. Chronic inflammation was shown in morphologic investigations of otosclerotic foci and MeV N, P, and F proteins were detected within cells of the otosclerotic focus by immunohistochemical investigations. MeV RNA was extracted from fresh-frozen otosclerotic tissue by the use of in vitro RT-PCR. This result was validated through amplification of MeV genome sequences by RT-PCR from celloidin-embedded sections with morphologically ascertained otosclerotic foci. In searching for an immune response of the inner ear immune system against MeV proteins, elevated anti-MeV IgG levels were detected in the perilymph of patients with otosclerosis in comparison with the serum levels. In situ RT-PCR allowed the localization of MeV sequences in osteoclasts, osteoblasts, chondrocytes, macrophages, and epithelial cells in middle ear mucosa of otosclerotic tissue. Further evidence for MeV persistence has recently been given. Genotyping of MeV in otosclerotic foci demonstrated the presence of MeV genotype A, which circulated in Europe around 1960. All the above results confirm a strong association between MeV and otosclerosis.

Reduced toxicity of F-deficient Sendai virus vector in the mouse fetus.

Current concerns over insertional mutagenesis by retroviral vectors mitigate investigations into alternative, potentially persistent gene therapy vector systems not dependent on genomic integration, such as Sendai virus vectors (SeVV). Prenatal gene therapy requires efficient gene delivery to several tissues, which may not be achievable by somatic gene transfer to the adult. Initially, to test the potential and tropism of the SeVV for gene delivery to fetal tissues, first-generation (replication- and propagation-competent) recombinant SeVV, expressing beta-galactosidase was introduced into late gestation immunocompetent mice via the amniotic and peritoneal cavities and the yolk sac vessels. At 2 days, this resulted in very high levels of expression particularly in the airway epithelium, mesothelium and vascular endothelium, respectively. However, as expected, substantial vector toxicity was observed. The efficiency of gene transfer and the level of gene expression were then examined using a second-generation SeVV. The second generation was developed to be still capable of cytoplasmic RNA replication and therefore high-level gene expression, but incapable of vector spread due to lack of the gene for viral F-protein. Vector was introduced into the fetal amniotic and peritoneal cavities, intravascularly, intramuscularly and intraspinally; at 2 days, expression was observed in the airway epithelia, peritoneal mesothelia, unidentified cells in the gut wall, locally at the site of muscle injection and in the dorsal root ganglia, respectively. Mortality was dramatically diminished compared with the first-generation vector.

Persistent measles virus infection and otosclerosis.

The cause of otosclerosis is still unknown. Recently, measles virus involvement has been implicated. The aim of this study was to analyze the presence of measles virus RNA within the otosclerotic focus and to evaluate the perilymphatic antibody pattern. Bone and perilymph specimens from 40 patients with the spontaneous form of otosclerosis and from control patients were investigated by reverse transcription polymerase chain reaction (RT-PCR), Western blot techniques, and cell culture. By the use of RT-PCR, measles virus RNA could be detected in 32 patients, but not in controls. Analysis of perilymph revealed the presence of antibodies to N, F1, and M measles virus proteins in all cases, and antibodies against H protein in 2 additional cases. In preosteoblasts cultured from otosclerotic bone chips, no measles virus RNA could be amplified. We conclude that the spontaneous form of otosclerosis is, in the vast majority of cases, a measles virus-associated disease of the otic capsule.

Localization of a new neutralizing epitope on the mumps virus hemagglutinin-neuraminidase protein.

Four protein fragments which span the entire hemagglutinin-neuraminidase protein (HN) of mumps virus were expressed in HeLa cells and cell extracts were tested for their capability to induce neutralizing antibodies in mice. Fragment HN3 (aa 213-372) was able to induce the production of hemagglutination-inhibiting and neutralizing antibodies. When a subfragment of HN3, the synthetic peptide NSTLGVKSAREF (aa 329-340 of HN) was used for immunization, hemagglutination-inhibiting and neutralizing antibodies against mumps wild type virus but not against the Urabe Am9 vaccine virus were raised. The peptide could, therefore, contain a new epitope, which may be critical for protective host humoral immune response.

Persistent measles virus infection as a possible cause of otosclerosis: state of the art.

The etiopathogenesis of otosclerosis is still largely unexplained and remains controversial. Morphologic examinations have shown the presence of a chronic inflammation in otosclerotic tissue. Among the proposed explanations for this inflammation are an immunologic reaction against collagen, mutations of collagen gene 1A1, and a viral infection. In this paper, we focus on the role of measles virus in otosclerosis, and we review the current literature, devoting particular attention to a suspected paramyxoviral etiopathogenesis in Paget's disease. Our examination of footplate fragments by reverse transcription polymerase chain reaction testing in 95 patients with otosclerosis revealed the presence of measles virus RNA in 83% of cases. Quantification of measles virus immunoglobulin G (IgG) in otosclerosis patients indicated that the ratio of antimeasles virus IgG in total IgG was higher in perilymph than in serum. Furthermore, an almost identical incidence of otosclerosis and measles virus-caused mortality in women suggests that women are more susceptible to measles virus infection. Finally, since the introduction of the measles virus vaccination program in Europe, there has been a decline in the incidence of otosclerosis. Moreover, the average age of patients at diagnosis and surgery at our hospital has increased to 54 years. Our findings, when they are considered along with findings regarding the presence of paramyxoviral RNA in Paget's disease, support the hypothesis that measles virus is involved in the etiopathogenesis of otosclerosis.

Sendai virus infection induces apoptosis through activation of caspase-8 (FLICE) and caspase-3 (CPP32).

Sendai virus (SV) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. We now show that SV infection triggers an apoptotic program in target cells. Incubation of infected cells with the peptide inhibitor z-VAD-fmk abrogated SV-induced apoptosis, indicating that proteases of the caspase family were involved. Moreover, proteolytic activation of two distinct caspases, CPP32/caspase-3 and, as shown for the first time in virus-infected cells, FLICE/caspase-8, could be detected. So far, activation of FLICE/caspase-8 has been described in apoptosis triggered by death receptors, including CD95 and tumor necrosis factor (TNF)-R1. In contrast, we could show that SV-induced apoptosis did not require TNF or CD95 ligand. We further found that apoptosis of infected cells did not influence the maturation and budding of SV progeny. In conclusion, SV-induced cell injury is mediated by CD95- and TNF-R1-independent activation of caspases, leading to the death of host cells without impairment of the viral life cycle.

Rapid and efficient recovery of Sendai virus from cDNA: factors influencing recombinant virus rescue.

In a comparative study the factors influencing the recovery of recombinant Sendai viruses (SeV) from plasmid based cDNA were analysed systematically in order to establish an efficient and robust method for virus rescue. The amounts and ratios of transfected helper plasmids encoding the viral N, P and L proteins proved to be crucial for virus rescue, and they were optimised step-by-step for enhanced virus release. When the C open reading frame from the P gene was expressed at low level, virus rescue was generally possible but virus release could be improved when C gene expression was abolished completely. SeV particle formation could be increased greatly when the transcription initiation site for T7 polymerase in the cDNA was modified or when the genomic ribozyme instead of the antigenomic ribozyme of hepatitis delta virus was used for processing the 3'end of the viral RNA transcript. Heterologous helper viruses vTF7-3 and MVA-T7, which are necessary for T7 polymerase production in transfected cells, were compared for their use in SeV recovery and subsequent elimination of the helper virus from recombinant SeV. Interference with SeV replication was less severe with MVA-T7, and MVA-T7 was eliminated efficiently without the need for any inhibitors by serial passages in Vero cells. Optimal combination of all parameters led to a highly efficient generation of recombinant SeV from cDNA. Titres of the released virus particles are high enough to enable analysis of the recombinant SeV directly on test cells or propagation in cell cultures without the need for amplification in embryonated chicken eggs. The system is very robust and allows rapid generation of defined SeV mutants that require specialised host cells for propagation.

Pseudotype formation of Moloney murine leukemia virus with Sendai virus glycoprotein F.

Mixed infection of cells with both Moloney murine leukemia virus (MoMLV) and related or heterologous viruses produces progeny pseudotype virions bearing the MoMLV genome encapsulated by the envelope of the other virus. In this study, pseudotype formation between MoMLV and the prototype parainfluenza virus Sendai virus (SV) was investigated. We report for the first time that SV infection of MoMLV producer cells results in the formation of MoMLV(SV) pseudotypes, which display a largely extended host range compared to that of MoMLV particles. This could be associated with SV hemagglutinin-neuraminidase (SV-HN) glycoprotein incorporation into MoMLV envelopes. In contrast, solitary incorporation of the other SV glycoprotein, SV fusion protein (SV-F), resulted in a distinct and narrow extension of the MoMLV host range to asialoglycoprotein receptor (ASGP-R)-positive cells (e.g., cultured human hepatoma cells). Since stably ASGP-R cDNA-transfected MDCK cells, but not parental ASGP-R-negative MDCK cells, were found to be transduced by MoMLV(SV-F) pseudotypes and transduction of ASGP-R-expressing cells was found to be inhibited by ASGP-R antiserum, a direct proof for the ASGP-R-restricted tropism of MoMLV(SV-F) pseudotypes was provided. Cultivation of ASGP-R-positive HepG2 hepatoma cells on Transwell-COL membranes led to a significant enhancement of MoMLV(SV-F) titers in subsequent flowthrough transduction experiments, thereby suggesting the importance of ASGP-R accessibility at the basolateral domain for MoMLV(SV-F) pseudotype transduction. The availability of such ASGP-R-restricted MoMLV(SV-F)-pseudotyped vectors opens up new perspectives for future liver-restricted therapeutic gene transfer applications.

Sendai virus-like particles devoid of haemagglutinin-neuraminidase protein infect cells via the human asialoglycoprotein receptor.

Virus-like particles with genetically defined envelope proteins were generated from cDNA in order to examine the requirement of Sendai virus haemagglutinin-neuraminidase (HN) protein for particle formation, and the role of fusion protein (F) in receptor binding and membrane fusion. Characterization of particles devoid of HN protein showed that particle formation was unimpaired by the absence of HN protein, indicating that HN protein is dispensable for virus assembly and budding. Infection studies further demonstrated that virus adsorption and penetration can be mediated solely by the F protein when the human asialoglycoprotein receptor is present at the surface of host cells.

Sendai virus efficiently infects cells via the asialoglycoprotein receptor and requires the presence of cleaved F0 precursor proteins for this alternative route of cell entry.

Biochemical evidence suggests that the asialoglycoprotein receptor (ASGP-R) can be used as an alternative receptor for a temperature-sensitive Sendai virus (SV) mutant. We now have investigated this possible alternative route of infection for SV wild-type (SV-wt) strain Fushimi by using a pair of cell lines which differ only with regard to ASGP-R expression. Infection studies after enzymatic destruction of conventional sialic acid-containing SV receptors (SA-R) revealed that only ASGP-R-expressing cells could be infected by SV-wt. This alternative route of cell entry could be completely blocked by incubation of cells with ASGP-R-specific antibodies prior to infection. Furthermore, cleavage of SV-F0 precursor protein into the subunits F1 and F2 was necessary to establish infection via ASGP-R, suggesting a fusion-mediated cell entry after binding of SV-wt to the ASGP-R on host cells. Interestingly, infection via ASGP-R was found to be nearly as efficient as infection via conventional sialic acid-containing SV receptors. A possible physiological role of the ASGP-R-mediated route of SV infection is discussed.

The pathogenesis of multiple sclerosis: reconsideration of the role of viral agents and defence mechanisms.

Numerous ubiquitous ribonucleic acid and deoxyribonucleic acid viruses, inducing acute, monophasic infections (mostly childhood diseases) have been considered as potential causes of multiple sclerosis. The present hypothesis reconsiders the role of the viral agent: not the virus, but the reaction of the defense system to the viral persistence, appearing after the acute phase, is postulated as a key factor. A prerequisite of multiple sclerosis is polygenetically determined or acquired immunodeficiency; the defense system is not able to stop repeated viral reactivations induced by a set of exogenous and/or endogenous factors. Thus, an aberrant virus production can appear repeatedly. If the virus spreads from primary target--the lymphoreticular system--into the central nervous system, the multiple sclerosis process can be initiated. Activated T cells and endothelial cells serve as first-host cells. Their infection triggers a set of reactive events: multiple microthrombosis and inflammation play a key role, both of which can result in nonspecific degradation of the myelin. An increased release of myelin antigens induces a homeostatic autoimmunity. Long-term repetition of the shifts and the infection of inflammatory cells can lead to disturbances in self-tolerance. A dysregulated pathological autoimmunity can develop, which acts as a main effector of the specific demyelination.

[Pathogenesis of otosclerosis. "State of the art"]. / Zur Pathogenese der Otosklerose. "State of the Art".

Women suffer from otosclerosis 1.6 times more often than males. Histologically, otosclerotic foci can be found in temporal bones of females 1.9 times more often than in those of males. Characteristic topographic regions are the oval window, round window niche and promontory. Otosclerosis can also occur principally in any area of the enchondral/periosteal layer of the otic capsule. Evidence is presented that otosclerosis is an inflammatory tissue reaction associated with macrophages, T- and B-lymphocytes, HLA-DR positive cells and plasma cells. Dependent on the stage of the osteolytic bone disease present deposits of complement and immunoglobulins (IgG, IgA) can be found. These immunoglobulins have been identified as antibodies to measles virus proteins. Using the polymerase chain reaction we were successful in demonstrating RNA sequences of measles viruses in otosclerotic bone from footplates removed during stapes surgery. Since most of the otosclerotic lesions were in direct contact to the perilymphatic space, it may be expected that the endolymphatic sac--as the immune competent organ of the inner ear--specifically reacts to antigens delivered from the otosclerosis focus into the perilymph. Perilymph samples from patients were collected during stapes surgery and their antibody titers against measles were compared with that in corresponding blood serum. All samples revealed a significantly elevated-specific anti-measles IgG amount which was significantly higher than in the corresponding serum. In contrast, antibody titer in the perilymph against herpes simplex or cytomegalo viruses did not differ from that of the serum. These findings indicate that otosclerosis is a measles virus associated inflammatory osteolytic disease of the temporal bone. Since women suffer from severe measles virus infections more often than males, it can be hypothesized that females have a higher susceptibility of their cochleo-vestibular tissues to these infections (organotropism). In addition, estrogens are well-known stimulators of osteocytic activity and may play a dominant role during ossification of an otospongeotic bone lesion. This may explain the onset of a conductive hearing loss due to otosclerosis during pregnancy.

Conserved high-affinity NF-kappa B binding site in the interferon regulatory factor-1 promoter is not occupied by NF-kappa B in vivo and is transcriptionally inactive.

The promoter of the interferon regulatory factor-1 (IRF-1) gene contains at position -47 to -38 an evolutionary conserved binding sequence for the inducible transcription factor NF-kappa B. This site is highly homologous to a transcriptionally active site from the MHC class I enhancer. In this study, we show by in vitro assays using purified NF-kappa B that the kappa B motif in the IRF-1 promoter binds the factor specifically and with high affinity, comparable to various other cis-acting kappa B elements. Two copies of the IRF-1 kappa B site fused to the heterologous c-fos promoter conferred induction of a chloramphenicol acetyl transferase (CAT) reported gene in response to stimulation of L929 fibroblasts with various NF-kappa B inducers, such as tumor necrosis factor alpha (TNF alpha) or phorbol 12-myristate 13-acetate (PMA). Mutation of the binding site completely abolished transcriptional inducibility of the heterologous promoter. Surprisingly, the same IRF-1 kappa B motif in context of the homologous IRF-1 promoter was transcriptionally inactive in CAT assays. The very weak induction of the IRF-1 promoter in response to TNF treatment or infection of fibroblasts with Newcastle disease virus (NDV) was barely affected by point mutation of the kappa B site or loss of the site by truncation of the promoter. Analysis of the occupational state of the chromosomal IRF-1 kappa B site by in vivo foot-printing revealed that no footprint was induced over the kappa B motif in the IRF-1 promoter after PMA treatment of L929 fibroblast cells, despite the simultaneous induction of IRF-1 mRNA and NF-kappa B binding activity. Constitutive footprints were detected at a CCAAT and GC-rich region in the promoter. This is the first example of a high-affinity NF-kappa B binding site within a promoter which may not participate in transcriptional regulation under conditions activating NF-kappa B DNA binding and gene expression.

The influence of imperfectly paired helices I and III on the catalytic activity of hammerhead ribozymes.

Several catalytic antisense RNAs directed against different regions of the genomic or antigenomic RNA of Sendai virus were constructed. All RNAs contained the same catalytic domain based on hammerhead ribozymes but some had deletions or mutations resulting in imperfect helices I and III. Pre-annealed substrate/ribozyme complexes were used to determine the rates of the cleavage process for the different ribozymes under single-turnover conditions. It was found that the sequence context surrounding the cleavable motif influenced the cleavage efficiencies. Deletions or mutations of nucleotides 2.1 or 15.1 and 15.2 according to the numbering system for hammerhead ribozymes of Hertel et al. destroyed catalytic activity. Deletions of nucleotide 2.2 or additional nucleotides in the helix I-forming region of the ribozyme did not destruct, but only reduced the cleavage efficiencies. Similar results were observed for a deletion of nucleotide 15.3. Simultaneous deletions within helices I and III resulted in alternative cleavage sites. The potential consequences for the specificity of the ribozyme reaction are discussed.

Long-term replication of Sendai virus defective interfering particle nucleocapsids in stable helper cell lines.

An essential prerequisite for generating a stable helper cell line, which constitutively expresses functional Sendai virus RNA-dependent RNA polymerase, is the expression of all three Sendai virus nucleocapsid (NC) proteins, NP, P, and L, simulataneously. Generating a stable helper cell line was accomplished by cotransfecting cell line 293 with all three corresponding viral genes under the control of cytomegalovirus promoter-enhancer elements. Cotransfection with a dominant selectable marker enabled selection for stably transfected cells. The levels of the expressed P and NP proteins reached up to 1/10th and 1/20th of the protein levels in Sendai virus-infected cells, respectively. The Sendai virus polymerase activity of the coexpressed proteins was demonstrated by an in vivo polymerase assay. The cell clone H29 gave the strongest signal and produced DI genomes continuously for at least 3 months. This result demonstrates that it is possible to stably express adequate levels of all three viral NC proteins to form Sendai virus polymerase activity, thereby performing the replication and encapsidation of viral RNA, essential prerequisites for a helper cell line to be competent in producing recombinant viruses.

The carboxy-terminal domain of Sendai virus nucleocapsid protein is involved in complex formation between phosphoprotein and nucleocapsid-like particles.

The replicative unit of Sendai virus, the nucleocapsid, is composed of the viral genomic RNA and the viral proteins NP, P, and L. P and L proteins form a polymerase complex and are associated with the nucleocapsid core (NP/RNA complex). The NP protein has recently been shown to be composed of two domains. While the amino-terminal domain I encapsidates RNA and gives rise to the characteristic helical nucleocapsid structure, the precise function of the carboxy-terminal domain II remained unclear. It was however supposed to be involved in binding the polymerase complex. To test this hypothesis, we established a cosedimentation assay which detects complex formation between P protein and nucleocapsid-like (NC-like) particles. Four mutant NC-like particles all carrying amino acid deletions in domain II of the NP protein were tested. Complex formation was abolished after deletion of amino acids 426-497 or 456-524, while deletions of amino acids 400-415 or 414-439 had no influence on the interaction. The results indicate that domain II of NP protein is involved in binding P protein to nucleocapsids. The function and position of a putative P protein binding site in domain II are discussed.

Evidence of measles virus RNA in otosclerotic tissue.

By use of the very sensitive polymerase chain reaction technique, evidence is given that otosclerosis is a measles-virus-associated disease of the otic capsule. These results support recent observations which demonstrated the expression of measles antigen within active otosclerotic foci by immunohistochemical methods. Thus it is hypothesized that the viral infection acts as at least one pathogenetic factor during the development of this obscure, locally restricted inflammatory bone disease.

The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly.

Sendai virus nucleocapsid protein NP synthesized in the absence of other viral components assembled into nucleocapsid-like particles. They were identical in density and morphology to authentic nucleocapsids but were smaller in size. The reduction in size was probably due to the fact that they contained RNA only 0.5 to 2 kb in length. Nucleocapsid assembly requires NP-NP and NP-RNA interactions. To identify domains on NP protein involved in nucleocapsid formation, 29 NP protein mutants were tested for the ability to assemble. Any deletion between amino acid residues 1 and 399 abolished formation of nucleocapsid-like particles, but mutants within this region exhibited two different phenotypes. Deletions between positions 83 and 384 completely abolished all interactions. Deletions between residues 1 and 82 and between residues 385 and 399, at the N- and C-terminal ends of the region from 1 to 399, resulted in unstructured aggregates of NP protein, indicating only a partial loss of function. Deletions within the C-terminal 124 amino acids were the only ones that did not affect assembly. The results suggest that NP protein can be divided into at least two separate domains which function independently of each other. Domain I (residues 1 to 399) seems to contain all of the structural information necessary for assembly, while domain II (residues 400 to 524) is not involved in nucleocapsid formation.