Institute of Molecular Biology - University of Zürich
Schneider, H., Kaelin, K., and Billeter, M.A.
The measles virus (MV) phosphoprotein (P) gene encodes three proteins, P, C, and V. The V protein is synthesized by pseudo-templated transcription, also designated as RNA editing: during P gene transcription one G residue is inserted at a defined position in about 50% of the mRNAs. To study the importance of sequence elements for the nontemplated G insertion, we generated recombinant MVs in which six different mutations were introduced within the region where editing occurs (3' UUUUUCCC, template strand). These viruses were then analyzed for their ability to edit their P mRNA and to produce V protein. Single U to C changes within the U stretch abolished editing. Extending the template by three C residues at the site of G insertion resulted in a less precise editing phenotype and overproduction of V. None of these mutants were impaired in their multiplication behavior when analyzed in cultured cells. However, the syncytia of a recombinant MV overproducing V protein were in general smaller and lysed 1 to 2 days later than usual.
Scheider, H., Spielhofer, P., Kaelin, K., Dötsch, C., Radecke, F., Sutter, G. and Billeter, M.A.
A system which allows the reconstitution of measles virus (MV) from cloned cDNA is described. The severely host cell restricted vaccinia vector MVA-T7 expressing bacteriophage T7 RNA polymerase was used to generate full-length antigenomic MV RNA and simultaneously the mRNAs encoding the viral N, P and L proteins in order to produce replicationally and transcriptionally active nucleocapsids. The functionality of the N, P and L proteins was demonstrated first by their ability to rescue MV specific subgenomic RNAs. Assembly and budding of reconstituted MV was shown by syncytia formation and subsequently by virus isolation. The inability of MVA-T7 to produce progeny virus in most mammalian cells circumvents the necessity to separate the reconstituted MV from the MVA-T7 helper virus. Since all components are expressed transiently, this system is especially suitable for studying the functions of N, P and L. Furthermore, it is useful for investigating later steps in the MV life cycle.
Schlender J. Schnorr JJ. Spielhofer P. Cathomen T. Cattaneo R. Billeter MA. Termeulen V. Schneiderschaulies S.
A marked suppression of immune function has long been recognized as a major cause of the high morbidity and mortality rate associated with acute measles, As a hallmark of measles virus (MV)-induced immunosuppression, peripheral blood lymphocytes (PBLs) isolated from patients exhibit a significantly reduced capacity to proliferate in response to mitogens, allogens, or recall antigens, In an in vitro system we show that proliferation of naive PBLs [responder cells (RCs)] in response to a variety of stimuli was significantly impaired after cocultivation with MV-infected, UV-irradiated autologous PBLs [presenter cells (PCs)], We further observed that a 50% reduction in proliferation of RCs could still be observed when the ratio of PC to RC was 1:100, The effect was completely abolished after physical separation of the two populations, which suggests that soluble factors were not involved, Proliferative inhibition of the RCs was observed after short cocultivation with MV-infected cells, which indicates that surface contact between one or more viral proteins and the RC population was required, We identified that the complex of both MV glycoproteins, F and H, is critically involved in triggering MV-induced suppression of mitogen-dependent proliferation, since the effect was not observed (i) using a recombinant MV in which F and H were replaced with vesicular stomatitis virus G or (ii) when either of these proteins,vas expressed alone, Coexpression of F and H, however, lead to a significant proliferative inhibition in the RC population, Our data indicate that a small number of MV-infected PBLs can induce a general nonresponsiveness in uninfected PBLs by surface contact, which may, in turn, account for the general suppression of immune responses observed in patients with acute measles.
Bankamp, B., Horikami, S.M., Thompson, P.D., Huber, M., Billeter, M.A. and Moyer, S.A.
Radecke F. Billeter MA.
Measles virus (MV) is a highly contagious agent which causes a major health problem in developing countries. Efficacious and safe live attenuated vaccine strains are available, but for the elimination of measles a better knowledge about the molecular biology of MV appears crucial. Whereas the roles of the six structural proteins in the replication cycle are known, the functions of the two nonstructural proteins C and V are unclear, which is also true for related viruses. In vitro studies implicating Sendai virus suggest that the C protein might be involved in downregulating viral mRNA synthesis (J. Curran, J.B. Marq, and D. Kolakofsky, Virology 189, 647-656, 1992). However, not all members of the Paramyxovirinae subfamily encode this protein, raising the question about its importance for the viral replication cycle. Taking advantage of a recently developed reverse genetics system allowing MV recovery from cloned DNA (F. Radecke, P. Spielhofer, H. Schneider, K. Kaelin, M. Huber, C. Dotsch, G. Christiansen, and M.A. Billeter, EMBO J: 14, 5773-5784, 1995), the question was addressed whether the C protein is essential for the life cycle of MV. A plasmid was constructed to produce a derivative of the Edmonston B vaccine strain, MV C- EdB, having its C reading frame silenced by two point mutations. The C- mutant MV could indeed be rescued, and it multiplies in cultured cells without obvious impairment.
Radecke F. Spielhofer P. Schneider H. Kaelin K. Huber M. Dotsch C. Christiansen G. Billeter MA.
A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.
Sidhu MS. Chan J. Kaelin K. Spielhofer P. Radecke F. Schneider H. Masurekar M. Dowling PC. Billeter MA. Udem SA.
Measles virus (MV) mRNA transcription and replication are thought to be controlled by cis-acting sequence elements contained within the terminal MV genomic noncoding nucleotides. To validate these promoter and regulatory signal assignments, cDNAs were constructed allowing synthesis of RNAs corresponding to a MV genome in which all coding and intercistronic regions were replaced by the chloramphenicol acetyl transferase (CAT) coding sequence. Transcript production by T7 polymerase starting and ending precisely with the MV genome terminal residues was achieved by fusing the T7 polymerase promoter and the hepatitis delta virus genome ribozyme followed by tandem T7 polymerase termination sequences to the MV genomic 5' and 3' ends, respectively. Transfection of these negative polarity transcripts, mimicking natural defective interfering RNAs of the internal deletion type, into MV-infected 293 cells gave rise to CAT activity which could be serially transferred and massively amplified together with progeny helper virus in fresh cells. Transfer was blocked only by antibodies able to neutralize MV infectivity, indicating that the chimeric RNA not only was encapsidated, transcribed, and replicated, but also packaged into virions. Sequence analyses confirmed that both the expected chimeric antigenome and mRNA products were transcribed and replicated with fidelity during serial passage. Minor changes introduced in the transcription promoter markedly compromised function. This system now can be exploited to examine MV genomic cis-acting regulatory elements and extended to the development of full-length MV cDNAs.
Komase K. Rima BK. Pardowitz I. Kunz C. Billeter MA. ter Meulen V. Baczko K.
The nucleotide sequences of the large protein (L) gene derived from two wild-type measles viruses (MV) and two SSPE brain-derived viruses have been determined. All sequences have single large open reading frames encoding 2183 amino acid residues. The deduced L proteins are well conserved and the proposed functional domains which have been identified for rhabdo- and paramyxoviruses are completely conserved in all strains. The degree of variability of L proteins is the lowest of all structural proteins of MV, reflecting its role in virus reproduction and persistence. Biased hypermutation was not observed in the L genes derived from SSPE brain tissue. None of the nucleotide changes can be associated with the attenuated phenotype of the Edmonston vaccine viruses.