FLAVIVIRUSES 1103 Background and Classification 1103 Structure and Physical Properties of the Virion 1104 Binding and Entry 1105 Genome Structure 1106 Translation and Proteolytic Processing 1107 Features of the Structural Proteins 1108 Features of the Nonstructural Proteins 1109 RNA Replication 1112 Membrane Reorganization and the Compartmentalization of Flavivirus Replication 1112 Assembly and Release of Particles from Flavivirus-infected Cells 1112 Host Resistance to Flavivirus Infection 1113

Flaviviridae :T he Viruses and Their Replication

https://www.aphis.usda.gov/animal_health/acah/downloads/documents/control_eradication_fmd_virus_res_2003_vo-91.pdf

Control and eradication of foot-and-mouth disease

Viruses have evolved a multitude of strategies to subvert the innate immune system by interfering with components of the alpha/beta interferon (IFN-alpha/beta) induction and signaling pathway. It is well established that the pestiviruses prevent IFN-alpha/beta induction in their primary target cells, such as epitheloidal and endothelial cells, macrophages, and conventional dendritic cells, a phenotype mediated by the viral protein N(pro). Central players in the IFN-alpha/beta induction cascade are interferon regulatory factor 3 (IRF3) and IRF7. Recently, it was proposed that classical swine fever virus (CSFV), the porcine pestivirus, induced the loss of IRF3 by inhibiting the transcription of IRF3 mRNA. In the present study, we show that endogenous IRF3 and IRF3 expressed from a cytomegalovirus (CMV) promoter are depleted in the presence of CSFV by means of N(pro), while CSFV does not inhibit CMV promoter-driven protein expression. We also demonstrate that CSFV does not reduce the transcriptional activity of the IRF3 promoter and does not affect the stability of IRF3 mRNA. In fact, CSFV N(pro) induces proteasomal degradation of IRF3, as demonstrated by proteasome inhibition studies. Furthermore, N(pro) coprecipitates with IRF3, suggesting that the proteasomal degradation of IRF3 is induced by a direct or indirect interaction with N(pro). Finally, we show that N(pro) does not downregulate IRF7 expression.

Classical Swine Fever Virus Npro Interacts with Interferon Regulatory Factor 3 and Induces Its Proteasomal Degradation

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. The analyses revealed that major aspects of pestivirus biology show significant similarity to the biology of human hepatitis C virus (HCV). The detailed molecular analyses conducted for pestiviruses and HCV supported and complemented each other during the last three decades resulting in elucidation of the functions of viral proteins and RNA elements in replication and virus-host interaction. For pestiviruses, the analyses also helped to shed light on the molecular basis of persistent infection, a special strategy these viruses have evolved to be maintained within their host population. The results of these investigations are summarized in this chapter.

The Molecular Biology of Pestiviruses.

The genes encoding pestivirus E2 and NS2-3 are separated by a sequence that encodes a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa (p7). It has been shown that cleavage between E2 and p7 is incomplete, resulting in proteins E2-p7, E2, and p7. We found no precursor-product relationship between E2-p7 and E2, which indicates a stable nature of E2-p7. To study the function of the E2-p7 region of the polyprotein, mutations were introduced into an infectious cDNA of bovine viral diarrhea virus (BVDV). When cleavage between E2 and p7 was abolished, viral RNA replication occurred; however, no infectious virus could be recovered. A corresponding result was obtained with a construct encompassing a large in-frame deletion of p7. To prevent synthesis of E2-p7, a translational stop codon was introduced after the last codon of the E2 gene and an internal ribosome entry site element followed by a signal peptide coding sequence was inserted upstream of the p7 gene. Transfection of RNA transcribed from the bicistronic construct led to the release of infectious virus particles. Thus, synthesis of E2-p7 is not essential for the generation of infectious virions. Cell lines constitutively expressing BVDV p7 and/or E2 were generated for complementation studies. Transfection of BVDV RNAs with point mutations or a deletion in the E2-p7 region into the complementing cell lines led to the generation of infectious virions. According to our studies, p7 as well as E2 can be complemented in trans.

E2-p7 Region of the Bovine Viral Diarrhea Virus Polyprotein: Processing and Functional Studies

Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease in pigs. Virulent strains cause characteristic disease symptoms such as fever, neurological disorders, hemorrhages, and high mortality rates, whereas infection with avirulent strains remains clinically inapparent but induces a protective immunity. Furthermore, prenatal infection of fetuses can lead to persistently infected animals which shed virus over a long period, as do pigs which exhibit the chronic form of disease (3, 4, 29, 31).

CSFV, bovine viral diarrhea virus (BVDV), and border disease virus form the pestivirus genus within the family Flaviviridae (33). CSFV is a small enveloped virus containing a positive-strand RNA genome of 12.3 kb, which consists of a 5′ untranslated region (5′UTR), a large open reading frame (ORF) encoding a single polyprotein, and a 3′ untranslated region (3′UTR) (16, 29). The 5′UTR contains an internal ribosomal entry site for cap-independent translation initiation of the viral polyprotein which is co- and posttranslationally processed by host cell and viral proteases (reviewed in reference 16). The cleavage sites of the polyprotein have been determined for BVDV, except for p7-NS2 (5, 16, 23, 26). C, Erns, E1, and E2 represent the structural proteins found in mature virions. As demonstrated for other flaviviruses (2), RNA replication of pestiviruses is thought to occur on cytoplasmatic membranes via the synthesis of a negative-stranded full-length genome (6, 7, 23), while the components of the viral replication complex have not been identified. It has been shown recently that the viral protein NS5B of BVDV has RNA-dependent RNA polymerase activity (37). The NS3 protease is responsible for the cleavage of the viral polyprotein downstream of NS3 and requires NS4A as a cofactor (28, 35). Furthermore, it possesses both helicase and NTPase activity (27, 32), suggesting a role in RNA replication.

According to their behavior in tissue culture pestiviruses can be divided into two biotypes, noncytopathogenic (NCP) and cytopathogenic (CP). The mechanism responsible for the cytopathic effect (CPE) is poorly understood, but the overexpression of NS3 is a common feature of all CP pestiviruses (16). Furthermore, it has been shown that cells infected with CP BVDV undergo apoptosis (10, 36). CP pestiviruses represent mutants which arise from NCP viruses, presumably by RNA recombination during replication (16). Several mutants have been described for BVDV. Most of them contain rearrangements of viral sequences and/or insertions of host cell sequences (reviewed in reference 16). However, some CP BVDV and all CP CSFV isolates described so far are composed of defective interfering (DI) particles and NCP helper virus. CSFV DI genomes, with the identical deletion of 4,764 nucleotides (nt) corresponding to the genes encoding Npro through NS2, have been isolated from different sources (12, 14, 17). A genome with a similar deletion of 4,746 nt has been described recently (12). Furthermore, a defective genome was reported which lacks the 4,263 nt encoding C through NS2 but retains the foreign murine ubiquitin gene which had been used to replace Npro at the 5′ terminus of the ORF in the parental genome (30). After transfection into susceptible cells, such defective CSFV genomes, obtained either by extraction from infected cells or by transcription in vitro from the respective cloned cDNA, replicate and are packaged efficiently in the presence of helper virus RNA, thereby causing a CPE (14, 15).

An autonomously replicating defective BVDV genome which lacks the genes encoding C, Erns, E1, E2, p7, and NS2 has been described recently by Behrens et al. (1). It demonstrates that none of these proteins is essential for RNA replication. In the same study it was suggested that the 5′ terminal region of the ORF contains a cis signal required for RNA replication, since the Npro gene could neither be deleted completely nor be replaced by a ubiquitin gene. However, the replacement of the Npro gene by ubiquitin in the CSFV Alfort/187 genome yielded infectious virus (30).

In this report a series of in vitro-constructed defective CSFV genomes derived from strain Alfort/187 were analyzed with respect to autonomous RNA replication in SK-6 cells. The question of whether these defective genomes can be packaged in the presence of a helper virus and whether they are cytopathogenic was also addressed.

Cytopathogenic and Noncytopathogenic RNA Replicons of Classical Swine Fever Virus

Oronasal vaccination with classical swine fever virus (CSFV) replicon particles with either partial or complete deletion of the E2 gene induces partial protection against lethal challenge with highly virulent CSFV.

Rescue of infectious classical swine fever and foot-and-mouth disease virus by RNA transfection and virus detection by RT-PCR after extended storage of samples in Trizol.

Establishment and application of bicistronic classical swine fever virus genomes for foreign gene expression and complementation of E2 deletion mutants

Peter Stettler

Papers

Cytopathogenic and Noncytopathogenic RNA Replicons of Classical Swine Fever Virus

Oronasal vaccination with classical swine fever virus (CSFV) replicon particles with either partial or complete deletion of the E2 gene induces partial protection against lethal challenge with highly virulent CSFV.

Rescue of infectious classical swine fever and foot-and-mouth disease virus by RNA transfection and virus detection by RT-PCR after extended storage of samples in Trizol.

Establishment and application of bicistronic classical swine fever virus genomes for foreign gene expression and complementation of E2 deletion mutants