Self-Replicating RNA Derived from the Genomes of Positive-Strand RNA Viruses.

Self-replicating RNA derived from the genomes of positive-strand RNA viruses represents a powerful tool for both molecular studies on virus biology and approaches to novel safe and effective vaccines. The following chapter summarizes the principles how such RNAs can be established and used for design of vaccines. Due to the large variety of strategies needed to circumvent specific pitfalls in the design of such constructs the technical details of the experiments are not described here but can be found in the cited literature.

A double deletion prevents replication of the pestivirus bovine viral diarrhea virus in the placenta of pregnant heifers.

In contrast to wild type bovine viral diarhea virus (BVDV) specific double deletion mutants are not able to establish persistent infection upon infection of a pregnant heifer. Our data shows that this finding results from a defect in transfer of the virus from the mother animal to the fetus. Pregnant heifers were inoculated with such a double deletion mutant or the parental wild type virus and slaughtered pairwise on days 6, 9, 10 and 13 post infection.

The Molecular Basis for E Dimerization in Classical Swine Fever Virus.

The pestivirus classical swine fever virus (CSFV) represents one of the most important pathogens of swine. Its virulence is dependent on the RNase activity of the essential structural glycoprotein E that uses an amphipathic helix as a membrane anchor and forms homodimers via disulfide bonds employing cysteine 171. Dimerization is not necessary for CSFV viability but for its virulence.

Interaction of Pestiviral E1 and E2 Sequences in Dimer Formation and Intracellular Retention.

Pestiviruses contain three envelope proteins: E, E1, and E2. Expression of HA-tagged E1 or mutants thereof showed that E1 forms homodimers and -trimers. C123 and, to a lesser extent, C171, affected the oligomerization of E1 with a double mutant C123S/C171S preventing oligomerization completely.

Generation and first characterization of TRDC-knockout pigs lacking γδ T cells.

The TRDC-locus encodes the T cell receptor delta constant region, one component of the γδ T cell receptor which is essential for development of γδ T cells. In contrast to peptide recognition by αβ T cells, antigens activating γδ T cells are mostly MHC independent and not well characterized. Therefore, the function of γδ T cells and their contribution to protection against infections is still unclear.

The E Carboxyterminus: Much More Than a Membrane Anchor.

Pestiviruses express the unique essential envelope protein E, which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of E is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host's innate immune response to pestivirus infection.

Characterization of Membrane Topology and Retention Signal of Pestiviral Glycoprotein E1.

Pestiviruses are members of the family , a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, E, E1, and E2. Among them, E1 is the least characterized concerning both biochemical features and function.

Charged Residues in the Membrane Anchor of the Pestiviral E Protein Are Important for Processing and Secretion of E and Recovery of Infectious Viruses.

The pestivirus envelope protein E is anchored in membranes via a long amphipathic helix. Despite the unusual membrane topology of the E membrane anchor, it is cleaved from the following glycoprotein E1 by cellular signal peptidase. This was proposed to be enabled by a salt bridge-stabilized hairpin structure (so-called charge zipper) formed by conserved charged residues in the membrane anchor.

Downstream Sequences Control the Processing of the Pestivirus E-E1 Precursor.

Like other enveloped viruses, pestiviruses employ cellular proteases for processing of their structural proteins. While typical signal peptidase cleavage motifs are present at the carboxy terminus of the signal sequence preceding E and the E1/E2 and E2/P7 sites, the E-E1 precursor is cleaved by signal peptidase at a highly unusual structure, in which the transmembrane sequence upstream of the cleavage site is replaced by an amphipathic helix. As shown before, the integrity of the amphipathic helix is crucial for efficient processing.

Structure-function relationship in the 'termination upstream ribosomal binding site' of the calicivirus rabbit hemorrhagic disease virus.

Caliciviruses use a termination/reinitiation mechanism for translation of their minor capsid protein VP2. A sequence element of about 80 nucleotides denoted 'termination upstream ribosomal binding site' (TURBS) is crucial for reinitiation. RNA secondary structure probing and computer aided secondary structure prediction revealed a rather low degree of secondary structure determinants for the TURBS of the rabbit hermorrhagic disease virus.

Restoration of glycoprotein E dimerization via pseudoreversion partially restores virulence of classical swine fever virus.

The classical swine fever virus (CSFV) represents one of the most important pathogens of swine. The CSFV glycoprotein E is an essential structural protein and an important virulence factor. The latter is dependent on the RNase activity of this envelope protein and, most likely, its secretion from the infected cell.

Proposed revision to the taxonomy of the genus Pestivirus, family Flaviviridae.

We propose the creation of seven new species in the genus Pestivirus (family Flaviviridae) in addition to the four existing species, and naming species in a host-independent manner using the format Pestivirus X. Only the virus species names would change; virus isolates would still be referred to by their original names. The original species would be re-designated as Pestivirus A (original designation Bovine viral diarrhea virus 1), Pestivirus B (Bovine viral diarrhea virus 2), Pestivirus C (Classical swine fever virus) and Pestivirus D (Border disease virus).

Type 2 BVDV N suppresses IFN-1 pathway signaling in bovine cells and augments BRSV replication.

Bovine viral diarrhea virus (BVDV) infection induces immunosuppression and in conjunction with bovine respiratory syncytial virus (BRSV) contributes to the bovine respiratory disease complex. Bovine turbinate cells were single or co-infected with type 2 BVDV wild-type (BVDV2-wt), its dysfunctional N mutant (BVDV2-E), and/or BRSV. BVDV2-E significantly up-regulated PKR, IRF-7, TBK-1, IRF-3, and IFN-β mRNAs based on real-time Q-RT-PCR.

Self-Replicating RNA.

Self-replicating RNA derived from the genomes of positive strand RNA viruses represents a powerful tool for both molecular studies on virus biology and approaches to novel safe and effective vaccines. The following chapter summarizes the principles how such RNAs can be established and used for design of vaccines. Due to the large variety of strategies needed to circumvent specific pitfalls in the design of such constructs the technical details of the experiments are not described here but can be found in the cited literature.

Proposed update to the taxonomy of the genera Hepacivirus and Pegivirus within the Flaviviridae family.

Proposals are described for the assignment of recently reported viruses, infecting rodents, bats and other mammalian species, to new species within the Hepacivirus and Pegivirus genera (family Flaviviridae). Assignments into 14 Hepacivirus species (Hepacivirus A-N) and 11 Pegivirus species (Pegivirus A-K) are based on phylogenetic relationships and sequence distances between conserved regions extracted from complete coding sequences for members of each proposed taxon. We propose that the species Hepatitis C virus is renamed Hepacivirus C in order to acknowledge its unique historical position and so as to minimize confusion.

Type I and III IFNs Produced by Plasmacytoid Dendritic Cells in Response to a Member of the Flaviviridae Suppress Cellular Immune Responses.

The pestivirus noncytopathic bovine viral diarrhea virus (BVDV) can suppress IFN production in the majority of cell types in vitro. However, IFN is detectable in serum during acute infection in vivo for ∼5-7 d, which correlates with a period of leucopoenia and immunosuppression. In this study, we demonstrate that a highly enriched population of bovine plasmacytoid dendritic cells (DCs) produced IFN in response to BVDV in vitro.

Lipid Binding of the Amphipathic Helix Serving as Membrane Anchor of Pestivirus Glycoprotein Erns.

Pestiviruses express a peculiar protein named Erns representing envelope glycoprotein and RNase, which is important for control of the innate immune response and persistent infection. The latter functions are connected with secretion of a certain amount of Erns from the infected cell. Retention/secretion of Erns is most likely controlled by its unusual membrane anchor, a long amphipathic helix attached in plane to the membrane.

The Molecular Biology of Pestiviruses.

Pestiviruses are among the economically most important pathogens of livestock. The biology of these viruses is characterized by unique and interesting features that are both crucial for their success as pathogens and challenging from a scientific point of view. Elucidation of these features at the molecular level has made striking progress during recent years.

Feline calicivirus can tolerate gross changes of its minor capsid protein expression levels induced by changing translation reinitiation frequency or use of a separate VP2-coding mRNA.

Caliciviruses use reinitiation of translation governed by a 'termination upstream ribosomal binding site' (TURBS) for expression of their minor capsid protein VP2. Mutation analysis allowed to identify sequences surrounding the translational start/stop site of the feline calicivirus (FCV) that fine tune reinitiation frequency. A selection of these changes was introduced into the infectious FCV cDNA clone to check the influence of altered VP2 levels on virus replication.

Two alternative ways of start site selection in human norovirus reinitiation of translation.

The calicivirus minor capsid protein VP2 is expressed via termination/reinitiation. This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). We have shown for feline calicivirus and rabbit hemorrhagic disease virus that the TURBS contains three sequence motifs essential for reinitiation.

Structure of the membrane anchor of pestivirus glycoprotein E(rns), a long tilted amphipathic helix.

E(rns) is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms.

A new type of intracellular retention signal identified in a pestivirus structural glycoprotein.

Sorting of membrane proteins into intracellular organelles is crucial for cell function. Viruses exploit intracellular transport and retention systems to concentrate envelope proteins at the site of virus budding. In pestiviruses, a group of important pathogens of pigs and ruminants closely related to human hepatitis C virus, the E(rns) protein translated from the viral RNA is secreted from the infected cells and found in the serum of infected animals.

A new type of signal peptidase cleavage site identified in an RNA virus polyprotein.

Pestiviruses, a group of enveloped positive strand RNA viruses belonging to the family Flaviviridae, express their genes via a polyprotein that is subsequently processed by proteases. The structural protein region contains typical signal peptidase cleavage sites. Only the site at the C terminus of the glycoprotein E(rns) is different because it does not contain a hydrophobic transmembrane region but an amphipathic helix functioning as the E(rns) membrane anchor.

Mutation of cysteine 171 of pestivirus E rns RNase prevents homodimer formation and leads to attenuation of classical swine fever virus.

Pestiviruses represent important pathogens of farm animals that have evolved unique strategies and functions to stay within their host populations. E(rns), a structural glycoprotein of pestiviruses, exhibits RNase activity and represents a virulence factor of the viruses. E(rns) forms disulfide linked homodimers that are found in virions and virus-infected cells.