Wolfgang J. Neubert

Sendai virus vectors as an emerging negative‐strand RNA viral vector system

The power to manipulate the genome of negative‐strand RNA viruses, including the insertion of additional non‐viral genes, has led to the development of a new class of viral vectors for gene transfer approaches. The murine parainfluenza virus type I, or Sendai virus (SeV), has emerged as a prototype virus of this vector group, being employed in numerous in vitro as well as animal studies over the last few years. Extraordinary features of SeV are the remarkably brief contact time that is necessary for cellular uptake, a strong but adjustable expression of foreign genes, efficient infection in the respiratory tract despite a mucus layer, transduction of target cells being independent of the cell cycle, and an exclusively cytoplasmic replication cycle without any risk of chromosomal integration.

Measles Virus in Otosclerosis and the Specific Immune Response of the Inner Ear

Histologic and immunohistochemical studies of otosclerotic lesions have shown that there is a chronic inflammatory reaction of the otic capsule with bone resorption resulting from vascular invasion accompanied by inflammatory cells. During the active lytic stage of otosclerosis, paramyxoviral structures have been identified by electron microscopy and measles virus antigen expression by immunohistochemistry. Recently, measles virus related sequences have been detected in tissue of otosclerotic lesions. Because the otosclerotic focus has a close relation to the perilymphatic space, the expression of measles virus antigens within it should represent an immunologic challenge to the immune system of the endolymphatic sac. In this study, measles virus specific antibodies were detected in all of the perilymph samples from 19 patients suffering from otosclerosis, and the relative amount of these IgG antibodies was much higher than in serum samples of the same patients or in perilymph of control patients. These findings support the hypothesis that measles viruses play an crucial role in the pathogenesis of otosclerosis.

Caspase-8 and Apaf-1-independent Caspase-9 Activation in Sendai Virus-infected Cells

Apoptotic cell death is of central importance in the pathogenesis of viral infections. Activation of a cascade of cysteine proteases, i.e. caspases, plays a key role in the effector phase of virus-induced apoptosis. However, little is known about pathways leading to the activation of initiator caspases in virus-infected host cells. Recently, we have shown that Sendai virus (SeV) infection triggers apoptotic cell death by activation of the effector caspase-3 and initiator caspase-8. We now investigated mechanisms leading to the activation of another initiator caspase, caspase-9. Unexpectedly we found that caspase-9 cleavage is not dependent on the presence of active caspases-3 or -8. Furthermore, the presence of caspase-9 in mouse embryonic fibroblast (MEF) cells was a prerequisite for Sendai virus-induced apoptotic cell death. Caspase-9 activation occurred without the release of cytochrome cfrom mitochondria and was not dependent on the presence of Apaf-1 or reactive oxygen intermediates. Our results therefore suggest an alternative mechanism for caspase-9 activation in virally infected cells beside the well characterized pathways via death receptors or mitochondrial cytochrome c release.

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.

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.

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 gene expression in lytically and persistently infected cells

Sendai virus RNA species were quantitated in lytically and persistently infected cultured cells by Northern blot hybridization to region- and strand-specific cloned cDNA probes. Levels of NP, P and M mRNA in lytically infected cells were equally high, but F and HN mRNA were present in about 3-fold, and L mRNA in 30-fold, lower amounts, reflecting transcriptional attenuation especially at the M-F and HN-L gene junction. Two persistently infected cell lines, which release only 1% of the virus particles of lytically infected cells, were shown to contain only 4- to 8-fold-less amounts of each viral mRNA and 2- to 3-fold-less genomic RNA than lytically infected cells. Additionally, transcription was neither defective nor more attenuated as compared to the lytical infection. Taken together the results suggest the existence of an additional regulatory mechanism for the virus release. A cell-associated state of infection therefore seems to be achievable by a relatively weak general reduction of the copy numbers of viral mRNA and genomic RNA.

The Replicative Complex of Paramyxoviruses: Structure and Function

Publisher Summary In paramyxoviruses, the nucleocapsid is not only a structural component used to enwrap the genome but also a functional entity required for virus replication. Therefore, all protein components of the paramyxoviral nucleocapsid are involved in manifold activities. Not only is the three-dimensional structure of each entire protein subunit important in building up the viral replicative complex but also the specific protein-RNA and proteinprotein interactions are essential for bringing the components into the right contact with each other and to regulate enzymatic activities. This chapter discusses the methods to monitor the influence of defined nucleotide or amino acid exchanges on the assembly of nucleocapsids, transcription and replication of the viral RNA, and rescue of virus particles—that is, to study the regulation of paramyxoviruses replication during the whole viral life cycle. To explore domains involved in protein-protein interactions, viral wild-type proteins or specifically modified variants expressed in prokaryotic or eukaryotic cells were investigated using a variety of techniques such as protein-protein overlay assays, immunocoprecipitation, cosedimentation, and the two-hybrid system. Furthermore, as a prerequisite for functional investigations of the viral replicative complex— in vitro and in vivo —RNA transcription and/or replication assays were developed either by assembling nucleocapsids out of several protein-RNA and protein-protein complexes or by expressing all essential components together in eukaryotic cells, thus forming a functional viral nucleocapsid.

De Novo Synthesis of N and P Proteins as a Key Step in Sendai Virus Gene Expression

ABSTRACT Among the members of the paramyxovirus family, the transcription process and the components involved have been studied under in vitro conditions thus far. Here, we reexamined the function of the viral RNA-dependent RNA polymerase through infection studies with Sendai virus (SeV) N and P deletion (Δ) mutants. To elucidate solely transcription-specific processes, all virus mutants also were rendered deficient in genome replication. Using mutant SeV ΔP, the earlier suspected supplemental role of P protein was clearly demonstrated to be essential during viral gene expression. Moreover, when SeV ΔN or ΔN PΔ2-77 (with the 5′ end of the P gene deleted) mutant was used for infections, a completely unexpected new and essential role for N protein was discovered for viral gene expression. In the early phases of an infection and in the absence of de novo viral protein synthesis, primary transcription occurs at hardly detectable levels. In contrast, if newly synthesized N protein is present, primary viral transcription reaches normal levels. From our data, we conclude that de novo synthesis of SeV N and P proteins is a key step for viral gene expression that facilitates the transition from preliminary to normal primary transcriptional activity.

A chimeric respiratory syncytial virus fusion protein functionally replaces the F and HN glycoproteins in recombinant Sendai virus.

ABSTRACT Entry of most paramyxoviruses is accomplished by separate attachment and fusion proteins that function in a cooperative manner. Because of this close interdependence, it was not possible with most paramyxoviruses to replace either of the two protagonists by envelope glycoproteins from related paramyxoviruses. By using reverse genetics of Sendai virus (SeV), we demonstrate that chimeric respiratory syncytial virus (RSV) fusion proteins containing either the cytoplasmic domain of the SeV fusion protein or in addition the transmembrane domain were efficiently incorporated into SeV particles provided the homotypic SeV-F was deleted. In the presence of SeV-F, the chimeric glycoproteins were incorporated with significantly lower efficiency, indicating that determinants in the SeV-F ectodomain exist that contribute to glycoprotein uptake. Recombinant SeV in which the homotypic fusion protein was replaced with chimeric RSV fusion protein replicated in a trypsin-independent manner and was neutralized by antibodies directed to RSV-F. However, replication of this virus also relied on the hemagglutinin-neuraminidase (HN) as pretreatment of cells with neuraminidase significantly reduced the infection rate. Finally, recombinant SeV was generated with chimeric RSV-F as the only envelope glycoprotein. This virus was not neutralized by antibodies to SeV and did not use sialic acids for attachment. It replicated more slowly than hybrid virus containing HN and produced lower virus titers. Thus, on the one hand RSV-F can mediate infection in an autonomous way while on the other hand it accepts support by a heterologous attachment protein.