Gerd Hobom

Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade

Influenza A viruses are important worldwide pathogens in humans and different animal species. The functions of most of the ten different viral proteins of this negative-strand RNA virus have been well elucidated. However, little is known about the virus-induced intracellular signalling events that support viral replication. The Raf/MEK/ERK cascade is the prototype of mitogen-activated protein (MAP) kinase cascades and has an important role in cell growth, differentiation and survival. Investigation of the function of this pathway has been facilitated by the identification of specific inhibitors such as U0126, which blocks the cascade at the level of MAPK/ERK kinase (MEK). Here we show that infection of cells with influenza A virus leads to biphasic activation of the Raf/MEK/ERK cascade. Inhibition of Raf signalling results in nuclear retention of viral ribonucleoprotein complexes (RNPs), impaired function of the nuclear-export protein (NEP/NS2) and concomitant inhibition of virus production. Thus, signalling through the mitogenic cascade seems to be essential for virus production and RNP export from the nucleus during the viral life cycle.

Mutational analysis of influenza virus promoter elements in vivo

RNA polymerase I transcription in vivo in transiently DNA-transfected cells has been used to express influenza virus vRNA molecules coding for chloramphenicol acetyltransferase (CAT) in an antisense orientation. Influenza virus superinfection provided viral RNA polymerase and other proteins required for transcriptional conversion of minus-strand vRNA into plus-strand viral mRNA molecules expressing CAT activity. This system has been used for analysis of the vRNA sequences which cooperatively constitute the vRNA promoter structure via nucleotide exchanges as well as deletions and insertions of both terminal segments. Several mutants caused greatly enhanced expression over wild-type levels, which was transmitted during serial passage of progeny virus. The data obtained for the mutations in various promoter elements support a model implicating double-stranded vRNA promoter structures in binding of viral polymerase, and in consecutive steps during initiation of RNA synthesis.

Molecular and biological characteristics of avian polyomaviruses: isolates from different species of birds indicate that avian polyomaviruses form a distinct subgenus within the polyomavirus genus.

The isolation and characterization of two avian polyomaviruses, from chicken (BFDV-2) and a parrot (BFDV-3), is reported. Both isolates are closely related to the non-mammalian polyomavirus budgerigar fledgling disease virus (BFDV) isolated from budgerigars (now called BFDV-1), and all three viral genomes are shown to have the same basic size of 4981 bp. A 151 bp insertion was, however, observed in the non-coding region of BFDV-2 which represented an exact duplication of the left half of the non-coding region, including the putative early promoter and amino terminus of the large T antigen. With a further 15 base pairs exchanged elsewhere throughout the three genomes, these viruses have distinct degrees of tropism for various avian species. The production of antibodies directed against a beta-galactosidase-large T antigen fusion protein of BFDV-1 is described. These antibodies detected the large T antigen, with an M(r) of approximately 80K, and the small t antigen, with an M(r) of approximately 24K, in cells infected with BFDV isolates. Whereas these antibodies bind with low affinity to the large T antigen of simian virus 40 (SV40), SV40- or mouse polyomavirus-specific antibodies will not bind to the BFDV large T antigen. Antibodies directed against BFDV structural polypeptides exhibit broad, reciprocal cross-reactivities with all three structural proteins of mammalian polyomaviruses. The significance of polyomavirus infections in various avian species is discussed. Based on unique structural and biological properties we propose that these viruses should be placed in a distinct subgenus (Avipolyomavirus) within the polyomaviruses.

Structure of the bovine lactoferrin-encoding gene and its promoter

Lactoferrin (Lf), a ferric ion (Fe3+)-binding glycoprotein, is found most notably in milk, probably to mediate protection against microbial infection of the mammary gland. Based on an initial isolation and sequencing of a complete cDNA of the bovine Lf gene (bLf), the complete gene was obtained from genomic libraries on five overlapping phage lambda EMBL3 clones. A detailed restriction map and the complete exon/intron structure of the gene are presented, together with 1 kb of sequence data of the promoter upstream from the proximal exon. The coding sequence is dispersed over 17 exons spanning 34.5 kb of genomic DNA. While the exons are of similar size, as in other members of the transferrin gene family (Tf), some of the intron sizes are very different. Evolutionary conservation of both exon sizes and their contribution to the various domains of the protein molecule add to the evidence that Lf originated via an internal sequence duplication. The promoter sequence lacks some of the sequence motifs for transcriptional enhancers found in the promoters of human and mouse Lf, suggesting a potential reason for the relatively weak expression of bLf.

The genome of budgerigar fledgling disease virus, an avian polyomavirus

Budgerigar fledgling disease virus (BFDV) represents the first avian member of the Polyomavirus family. In contrast to mammalian polyomaviruses BFDV exhibits unique biological properties, in particular it is able to cause an acute disease with distinct organ manifestations in affected birds. Here we present the complete nucleotide sequence of the BFDV genome, consisting of 4980 bp. When compared to published nucleotide sequences of other polyomaviruses, the BFDV genome exposes a number of very similar structural features, and undoubtedly qualifies as a member of that family of viruses. The most important differences include a large T antigen remarkably reduced in size, and an origin of replication region with fundamental deviations from the origin structure of all other polyomaviruses. The specific characteristics of the BFDV genome may be used to place this virus into a distinct subgroup within the Polyomavirus family and may give a clue to the elucidation of its extraordinary biological properties.

Efficient Expression of the Tumor-Associated Antigen MAGE-3 in Human Dendritic Cells, Using an Avian Influenza Virus Vector

Dendritic cells (DCs) are the most potent inducers of immune reactions. Genetically modified DCs, which express tumor-associated antigens (TAA), can efficiently induce antitumor immunity and thus have a high potential as tools in cancer therapy. The gene delivery is most efficiently achieved by viral vectors. Here, we explored the capacity of influenza virus vectors to transduce TAA genes. These viruses abortively infect DCs without interfering with their antigen-presenting capacity. In contrast to other viruses used for DC transduction, influenza viruses can be efficiently controlled by antiviral pharmaceuticals, lack the ability to integrate into host chromosomes, and fail to establish persistent infections. Genes encoding a melanoma-derived TAA (MAGE-3), or the green fluorescence protein (GFP), were introduced into a high-expression avian influenza virus vector. Monocyte-derived mature DCs infected by these recombinants efficiently produced GFP or MAGE-3. More than 90% of the infected DCs can express a transduced gene. Importantly, these transduced DCs retained their characteristic phenotype and their potent allogeneic T cell stimulatory capacity, and were able to stimulate MAGE-3-specific CD8(+) cytotoxic T cells. Thus influenza virus vectors provide a highly efficient gene delivery system in order to transduce human DCs with TAA, which consequently stimulate TAA-specific T cells.

OmpA—FMDV VP1 fusion proteins: production, cell surface exposure and immune responses to the major antigenic domain of foot-and-mouth disease virus

Exposure at the bacterial outer surface of the major antigenic epitope of the foot-and-mouth disease (FMDV) viral protein VP1 was studied using protein fusion with outer membrane protein A (OmpA) of Shigella dysenteriae for production and transport of the foreign polypeptide to the outer membrane of Escherichia coli. Fusion constructs with VP1 peptide insertions of up to 56 amino acids in the third outer domain of OmpA could be demonstrated on the bacterial surface by indirect immunofluorescence and immunogold labelling. OmpA fusion proteins with large insertions from sequences of the FMDV protein VP1 were shown to elicit virus-specific immune responses in rabbits.

A single point mutation results in A allele-specific exon skipping in the bovine αs1-casein mRNA

Bovine alpha s1-casein (alpha s1-CN) allele A is found in low allelic frequencies among different cattle breeds and is known to be characterized by the deletion of amino-acid residues 14 to 26 of the mature protein (as defined via the most common allele B), and a corresponding deletion of 39 bp from its cDNA. Based upon the genomic sequence of bovine alpha s1-CN [Koczan et al., Nucleic Acids Res. 19 (1991) 5591-5596], this allelic deviation can be interpreted as an absence of exon 4 from the A allele mRNA and protein product. We demonstrate that this allelic aberration is not caused by a genomic deletion across the exon-4 DNA, but is correlated with a single point mutation at position +6 in the splice donor sequence distal of exon 4, which results in upstream exon skipping during the serial splice reactions of the A allele alpha s1-CN pre-mRNA. The A-allele-specific mutation at position +6 is able to interrupt the perfect complementarity of the intron-4 splice donor signal (positions one to eight) with U1-snRNA, which may then no longer be able to compensate for a rather weak exon-4 upstream splice acceptor sequence in facilitating the initial binding of U2 auxiliary factor/65-kDa (U2AF65) to that polypyrimidine tract. This interpretation of the exon skipping mechanism in alpha s1-CN allele A is in agreement with similar results obtained [Hoffmann and Grabowski, Genes Dev. 6 (1992) 2554-2568] in an analysis of the rat preprotachykinin-encoding gene and in vitro experiments.

Efficient generation and expansion of antigen‐specific CD4+ T cells by recombinant influenza viruses

Adoptive transfer of in vitro generated antigen‐specific T cells has been successfully used to treat viral infections in immunodeficient patients. Therefore, methods for the rapid in vitro expansion of antigen‐specific T cells are needed. Influenza virus efficiently infects dendritic cells, and peptides derived from viral proteins are processed and presented to CD8+ cytotoxic T cells. However, both, CD4+ and CD8+ T cells are necessary for the efficient control of viral infections, and it is becoming increasingly clear that a T helper cell response is very important for the maintenance and strength of the immune response. Here we show that recombinant influenza virus efficiently infects a wide range of professional antigen‐presenting cells and does not interfere with antigen presentation pathways. Using T cell clones for three different MHC class II‐restricted antigens we demonstrate that peptides derived from these antigens are efficiently presented on MHC class II molecules. Importantly, it was possible to generate and expand antigen‐specific CD4+ T cells following in vitro infection of professional antigen‐presenting cells with recombinant influenza virus. These findings support the notion that recombinant influenza virus is a valuable tool for the expansion of antigen‐specific CD4+ T cells in vitro.

Host restriction in the productive cycle of avian polyomavirus budgerigar fledgling disease virus type 3 depends on a single amino acid change in the common region of structural proteins VP2(VP3

The three avian polyomaviruses budgerigar fledgling disease virus types 1 to 3 (BFDV-1 to -3) contain genomes of identical size, 4981 bp. With differences of up to only 15 bp between the three genomes, these viruses show distinct tropism for cultured cells of various avian species: infection of chicken embryo (CE) cells with BFDV-1 and -2 results in virus propagation, whereas BFDV-3 is not replicated; all three viruses replicate, with different efficiencies, in infected Muscovy duck cells. Transfection of CE cells with BFDV-3 DNA results in a single productive cycle. As shown by construction of hybrid virus genomes and site-directed mutagenesis, a single amino acid difference (glycine instead of valine or alanine) within the common region of the minor structural proteins VP2/VP3 is responsible for this type of abortive infection of CE cells. Further experiments indicate a defect in one of the early steps during infection, at or prior to uncoating.