Different genetically engineered mutants of bovine viral diarrhea virus (BVDV) were analyzed for the ability to establish infection in the fetuses of pregnant heifers. The virus mutants exhibited either a deletion of the overwhelming part of the genomic region coding for the N-terminal protease N^pro, a deletion of codon 349, which abrogates the RNase activity of the structural glycoprotein E^rns, or a combination of both mutations. Two months after infection of pregnant cattle with wild-type virus or either of the single mutants, the majority of the fetuses contained virus or were aborted or found dead in the uterus. In contrast, the double mutant was not recovered from fetal tissues after a similar challenge, and no dead fetuses were found. This result was verified with a nonrelated BVDV containing similar mutations. After intrauterine challenge with wild-type virus, mutated viruses, and cytopathogenic BVDV, all viruses could be detected in fetal tissue after 5, 7, and 14 days. Type 1 interferon (IFN) could be detected in fetal serum after challenge, except with wild-type noncytopathogenic BVDV. On days 7 and 14 after challenge, the largest quantities of IFN in fetal serum were induced by the N^proand RNase-negative double mutant virus. The longer duration of fetal infection with the double mutant resulted in abortion. Therefore, for the first time, we have demonstrated the essential role of both N^proand E^rnsRNase in blocking interferon induction and establishing persistent infection by a pestivirus in the natural host.

Calicivirus structure proteins are expressed from a subgenomic mRNA with two overlapping cistrons. The first ORF of this RNA codes for the viral major capsid protein VP1, and the second for the minor capsid protein VP2. Translation of VP2 is mediated by a termination/reinitiation mechanism, which depends on an upstream sequence element of ∼70 nucleotides denoted “termination upstream ribosomal binding site” (TURBS). Two short sequence motifs within the TURBS were found to be essential for reinitiation. By a whole set of single site mutations and reciprocal base exchanges we demonstrate here for the first time conclusive evidence for the necessity of mRNA/18S rRNA hybridization for translation reinitiation in an eukaryotic system. Moreover, we show that motif 2 exhibits intramolecular hybridization with a complementary region upstream of motif 1, thus forming a secondary structure that positions post-termination ribosomes in an optimal distance to the VP2 start codon. Analysis of the essential elements of the TURBS led to a better understanding of the requirements for translation termination/reinitiation in eukaryotes.

The calicivirus minor capsid protein VP2 is expressed via reinitiation of protein synthesis after termination of translation of the preceding VP1 gene. A sequence element of about 80 nucleotides denoted "termination upstream ribosomal binding site" (TURBS) (25) is crucial for reinitiation. Deletion mapping in the TURBS of a rabbit calicivirus identified two short sequence motifs that were crucial for VP2 expression. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18S rRNA. Single-residue exchanges in this motif severely impaired reinitiation when they affected the putative rRNA binding, whereas an exchange preserving complementarity had only a minor effect. Single exchanges in motif 2 were rather well tolerated, but the introduction of double exchanges almost blocked VP2 expression. In contrast, the deletion analyses showed that the RNA between the two motifs is of minor importance. The distance between motif 2 and the start site was found to be important, since deletions of increasing length in this sequence or upstream positioning of the start codon reduced VP2 expression stepwise to low levels, whereas multiple-nucleotide exchanges in this region were tolerated. The low flexibility of the arrangement of TURBS motif 2 and the start codon stand in marked contrast to the requirements with regard to the location of the stop codon of the preceding VP1 gene, which could be moved far downstream with continuous reduction, but without loss, of VP2 translation. The sequence mapping resulted in a refined model of the reinitiation mechanism leading to VP2 expression.

The E ^rns protein is a structural glycoprotein of pestiviruses that lacks a typical membrane anchor sequence and is known to be secreted from the infected cell. However, major amounts of the protein are retained within the cell and attached to the virion by a so far unknown mechanism. Transient-expression studies with cDNA constructs showed that in a steady-state situation, 16% of the protein is found in the supernatant of the transfected cells while 84% appears as intracellular protein. We show here that E ^rns represents a membrane-bound protein. Membrane binding occurs via the carboxy-terminal region of E ^rns . By fusion of this sequence to the carboxy terminus of green fluorescent protein (GFP), the subcellular localization of the reporter protein switched from cytosolic to membrane bound. A core sequence of 11 amino acids necessary for membrane binding was elicited in truncation experiments with GFP constructs. However, this peptide is not sufficient to confer membrane anchoring but needs either upstream or downstream accessory sequences. Analyses with different extraction procedures showed that E ^rns is neither easily stripped from the membrane, like a peripheral membrane protein, nor as tightly membrane bound as a transmembrane protein.

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^rnsforms disulfide linked homodimers that are found in virions and virus-infected cells. Mutation or deletion of cysteine 171, the residue engaged in intermolecular disulfide bond formation, results in loss of dimerization as tested in coprecipitation and native protein gel electrophoresis analyses. Nevertheless, stable virus mutants with changes affecting cysteine codon 171 could be recovered in tissue culture. These mutants grew almost as well as the parental viruses and exhibited an RNase-positive phenotype. E^rnsdimerization-negative mutants of classical swine fever virus were found to be attenuated in pigs even though the virus clearly replicated and induced a significant neutralizing antibody response in the animals.