Tillmann Rümenapf

Molecular cloning and nucleotide sequence of the genome of hog cholera virus

A cDNA clone derived from genomic RNA of hog cholera virus (HCV) was identified using an oligonucleotide complementary to the RNA encoding a hexapeptide from the putative RNA-dependent RNA polymerase of the closely related bovine viral diarrhea virus (BVDV). This clone served as a probe for screening different size-selected cDNA libraries. After molecular cloning and nucleotide sequencing the HCV genome was shown to consist of 12,284 nucleotides containing one long open reading frame. Sequence comparison revealed a high degree of homology between HCV and BVDV genomic RNAs. With respect to HCV the genome of BVDV contains an insertion coding for 90 amino acids.

Polypeptide requirements for assembly of functional Sindbis virus replication complexes: a model for the temporal regulation of minus- and plus-strand RNA synthesis.

Proteolytic processing of the Sindbis virus non‐structural polyproteins (P123 and P1234) and synthesis of minus‐ and plus‐strand RNAs are highly regulated during virus infection. Although their precise roles have not been defined, these polyproteins, processing intermediates or mature cleavage products (nsP1‐4) are believed to be essential components of viral replication and transcription complexes. In this study, we have shown that nsP4 can function as the polymerase for both minus‐ and plus‐strand RNA synthesis. Mutations inactivating the nsP2 proteinase, resulting in uncleaved P123, led to enhanced accumulation of minus‐strand RNAs and reduced accumulation of genomic and subgenomic plus‐strand RNAs. In contrast, no RNA synthesis was observed with a mutation which increased the efficiency of P123 processing. Inclusion of this mutation in a P123 polyprotein with cleavage sites 1/2 and 2/3 blocked allowed synthesis of both minus‐ and plus‐strand RNAs. We conclude that nsP4 and uncleaved P123 normally function as the minus‐strand replication complex, and propose that processing of P123 switches the template preference of the complex to minus‐strands, resulting in efficient synthesis of plus‐strand genomic and subgenomic RNAs and shut‐off of minus‐strand RNA synthesis.

Hog cholera virus--characterization of specific antiserum and identification of cDNA clones.

A specific antiserum was raised against the pestivirus inducing hog cholera (hog cholera virus, HCV). Using immunoprecipitation and SDS-PAGE, this antiserum served for comparison of HCV-induced proteins with those from a related and better characterized pestivirus, bovine viral diarrhea virus (BVDV). In addition to immunological relationships, the apparent molecular weights of some proteins induced by both viruses were quite similar. HCV genomic RNA was found to be about 12 kb in length, comparable to BVDV RNA. cDNA was synthesized starting from RNA isolated from partially purified virions and cloned in lambda gt11. Screening with the antiserum resulted in identification of several positive clones. Partial sequencing of one HCV-derived cDNA clone revealed a high degree of homology to a portion of the BVDV sequence.

Biosynthesis of Classical Swine Fever Virus Nonstructural Proteins

ABSTRACT Proteolytic processing of polyproteins is considered a crucial step in the life cycle of most positive-strand RNA viruses. An enhancement of NS2-3 processing has been described as a major difference between the noncytopathogenic (non-CP) and the cytopathogenic (CP) biotypes of pestiviruses. The effects of accelerated versus delayed NS2-3 processing on the maturation of the other nonstructural proteins (NSP) have never been compared. In this study, we analyzed the proteolytic processing of NSP in Classical swine fever virus (CSFV). Key to the investigation was a panel of newly developed monoclonal antibodies (MAbs) that facilitated monitoring of all nonstructural proteins involved in virus replication (NS2, NS3, NS4A, NS5A, and NS5B). Applying these MAbs in Western blotting and radioimmunoprecipitation allowed an unambiguous identification of the mature proteins and precursors in non-CP CSFV-infected cells. Furthermore, the kinetics of processing were determined by pulse-chase analyses for non-CP CSFV, CP CSFV, and a CP CSFV replicon. A slow but constant processing of NS4A/B-5A/B occurred in non-CP CSFV-infected cells, leading to balanced low-level concentrations of mature NSP. In contrast, the turnover of the polyprotein precursors was three times faster in CP CSFV-infected cells and in cells transfected with a CP CSFV replicon, causing a substantial increase of mature NSP concentrations. We conclude that a delayed processing not only of NS3 but further of all NSP represents a hallmark of regulation in non-CP pestiviruses.

The Core Protein of Classical Swine Fever Virus Is Dispensable for Virus Propagation In Vitro

Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447Δc), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447Δc. Upon infection of the natural host, Vp447Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.

Viral RNase Involvement in Strategies of Infection

The overwhelming majority of RNase activity is engaged in catabolic processes. Viruses have no metabolism of their own, but rely completely on host cellular energy and substrate provision to support the biochemical processes necessary for virus replication. It is therefore obvious that RNA hydrolysis does not represent an obligate step in the viral life cycle that would have to be governed by viral proteins. Accordingly, RNases are found only rarely in the viral proteomes and serve special functions. In this chapter, several virus-specific RNases will be described and their role in the viral life cycle discussed. The text will concentrate on RNases of members of the nidoviruses, herpesviruses, pestiviruses, and several viruses with segmented negative-strand RNA genome including influenza virus. These enzymes are involved in specific steps of viral gene expression, viral genome replication, shutoff of host cellular gene expression, and interference with the host’s immune response to virus infection.

Molecular Biology of Pestiviruses and Comparison with HCV

Current knowledge on the molecular biology of pestiviruses, which represent a genus of the family Flaviviridae, is briefly summarized and compared with the data available for another genus of the virus family, the hepatitis C virus group. In particular, the genome organization of the two virus groups is compared with respect to the function of the different proteins. Furthermore, a special feature of the pestivirus bovine viral diarrhea virus is presented which is based on RNA recombination and concerns the development of a lethal disease during persistent infection. The recombination frequently involves cellular ubiquitin-coding mRNAs.

9 Cellular Receptors for Alphaviruses

Receptors on host cells that are used by viruses to enter cells and initiate infection have received a great deal of attention of late because of their obvious importance in determining in large part the host range, tissue tropism, and virulence of a virus. The alphaviruses represent an interesting and special situation. The members of this group have an enormous host range that comprises both invertebrate hosts and vertebrate hosts (Chamberlain 1980; Griffin 1986; Niklasson 1988; Peters and Dalrymple 1990). All alphaviruses are transmitted by arthropod vectors, in which the virus replicates. Mosquitoes are used as vectors by most alphaviruses, but Fort Morgan and Bijou Bridge viruses are vectored by swallow bugs, and several alphaviruses, including Sindbis virus (SIN) (Shah et al. 1960) and eastern equine encephalitis virus (EEE) (Scott and Weaver 1989), have been isolated from mites and other hematophagous arthropods as well as from mosquitoes. In the case of EEE, the major vector is the mosquito Culiseta melanura , but other mosquitoes can also transmit the virus and naturally infected chicken mites have been shown to be able to transmit the virus, albeit inefficiently. During the process of mosquito transmission, the virus must productively infect several tissues within the mosquito. The virus is ingested when the insect feeds on a viremic host and first infects cells of the midgut; later the infection must spread to the salivary glands in order for the mosquito to transmit the virus when it next feeds on a vertebrate. A wide variety of vertebrates,...