Biomedical subjects

Sven-Erik Behrens

Publications and source records attributed to Sven-Erik Behrens.

7 recordsLinked to original sources

PubMed2005

Hepatitis C virus and the related bovine viral diarrhea virus considerably differ in the functional organization of the 5' non-translated region: implications for the viral life cycle.

The 5' non-translated regions (5'NTRs) of hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) initiate translation of the viral RNA genome through an internal ribosomal entry site (IRES) and operate as major determinants of the RNA replication cycle. We report on comparative studies with both virus systems demonstrating that the functional organization of the 5'NTRs of HCV and BVDV shows evident differences despite a similar RNA structure. In the BVDV 5'NTR, replication signals are restricted to the 5' terminal domain I. With HCV, we defined specific replication signals in domain I but also in domains II and III that constitute the functional IRES. While the BVDV domain I supports IRES activity, the HCV domain I appears to down-regulate IRES function. These data suggest that HCV and BVDV apply different mechanisms to coordinate viral protein and RNA synthesis, which may explain differences in the replication efficiency of both related viruses.

5' Untranslated Regions↗

PubMed2004

VP1 of infectious bursal disease virus is an RNA-dependent RNA polymerase.

Segment B of the bisegmented, double-stranded RNA genome of infectious bursal disease virus (IBDV) encodes the viral protein VP1. This has been presumed to represent the RNA-dependent RNA polymerase (RdRp) as it contains motifs that are typical for the RdRp of plus-strand RNA viruses. Here it is demonstrated that baculovirus-expressed wild-type but not motif A mutated VP1 acts as an RdRp on IBDV-specific RNA templates. Thus, on a plus-strand IBDV segment A cRNA template, minus-strand synthesis occurred in such a way that a covalently linked double-stranded RNA product was generated (by a 'copy-back' mechanism). Importantly, enzyme activity was observed only with templates that comprised the 3' non-coding region of plus-strand RNAs transcribed from IBDV segments A and B, indicating template specificity. RdRp activity was shown to have a temperature optimum of 37 degrees C and required magnesium ions for enzyme activity. Thus, it has been demonstrated unequivocally that VP1 represents the RdRp of IBDV.

Animals↗

PubMed2004

Complex signals in the genomic 3' nontranslated region of bovine viral diarrhea virus coordinate translation and replication of the viral RNA.

The genomes of positive-strand RNA viruses strongly resemble cellular mRNAs. However, besides operating as a messenger to generate the virus-encoded proteins, the viral RNA serves also as a template during replication. A central issue of the viral life cycle, the coordination of protein and RNA synthesis, is yet poorly understood. Examining bovine viral diarrhea virus (BVDV), we report here on the role of the variable 3'V portion of the viral 3' nontranslated region (3'NTR). Genetic studies and structure probing revealed that 3'V represents a complex RNA motif that is composed of synergistically acting sequence and structure elements. Correct formation of the 3'V motif was shown to be an important determinant of the viral RNA replication process. Most interestingly, we found that a proper conformation of 3'V is required for accurate termination of translation at the stop-codon of the viral open reading frame and that efficient termination of translation is essential for efficient replication of the viral RNA. Within the viral 3'NTR, the complex 3'V motif constitutes also the binding site of recently characterized cellular host factors, the so-called NFAR proteins. Considering that the NFAR proteins associate also with the 5'NTR of the BVDV genome, we propose a model where the viral 3'NTR has a bipartite functional organization: The conserved 3' portion (3'C) is part of the nascent replication complex; the variable 5' portion (3'V) is involved in the coordination of the viral translation and replication. Our data suggest the accuracy of translation termination as a sophisticated device determining viral adaptation to the host.

3' Untranslated Regions↗

PubMed2003

Kinetic profile of a heterocyclic HCV replicon RNA synthesis inhibitor.

Recently, a benzo-1,2,4-thiadiazine was shown to be a potent, specific inhibitor of the hepatitis C virus (HCV) RNA polymerase [J. Biol. Chem. 277 (2002) 32327]. Herein, we present several lines of evidence to demonstrate that thiadiazine compound 4 (C(21)H(21)N(3)O(4)S) is highly synergistic with interferon-alpha (IFN-alpha) and disrupts HCV replicon RNA synthesis with a distinct kinetic profile. A time course analysis after a single treatment with 5 microM compound 4 showed a loss of viral RNA consistent with replicon RNA half-life, suggesting inhibition of 90% of ongoing or newly initiated replicative intermediates. This finding is consistent with the mechanism of action recently reported for compound 4, an RNA synthesis initiation inhibitor [J. Biol. Chem. 278 (2003) 16602]. Further, unlike IFN-alpha, an immediate reduction of HCV replicon RNA synthesis was apparent upon addition of compound 4. Treatment with IFN-alpha showed a delay of approximately 4h prior to inhibition of viral RNA replication, consistent with its signaling kinetics.

Antiviral Agents↗

PubMed2003

Members of the NF90/NFAR protein group are involved in the life cycle of a positive-strand RNA virus.

A major issue of current virology concerns the characterization of cellular proteins that operate as functional components of the viral multiplication process. Here we describe a group of host factors designated as 'NFAR proteins' that are recruited by the replication machinery of bovine viral diarrhea virus, a close relative of the human pathogen hepatitis C virus. The NFAR proteins associate specifically with both the termini of the viral RNA genome involving regulatory elements in the 5' and 3' non-translated regions. Modification of the protein interaction sites in the 3' non-translated region yielded viral RNAs that were replication deficient. Viral replication was also inhibited by RNAi approaches that reduced the concentration of RNA helicase A, a member of the NFAR group, in the host cell's cytoplasm. Further experimental data suggest that NFAR proteins mediate a circular conformation of the viral genome that may be important for the coordination of translation and replication. Because NFAR proteins are presumed components of the antiviral response, we suspect that viral recruitment may also serve to weaken cellular defense mechanisms.

3' Untranslated Regions↗

PubMed2003

Replication studies using genotype 1a subgenomic hepatitis C virus replicons.

Recently, cell-based replicon systems for hepatitis C virus (HCV), in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. To date, one limitation of using these replicon systems to advance drug discovery is the inability of other genotypic derivatives, beyond those of two distinct strains of genotype 1b (HCV-N and Con1), to stably replicate in Huh7 cells. In this report, we evaluated a series of replicon genotype 1a-1b chimeras, as well as a complete genotype 1a replicon clone. A subgenomic replicon construct containing only type 1a sequences failed to generate stable colonies in Huh7 cells even after repeated attempts. Furthermore, addition of an NS5A adaptive mutation (S2204I) which enhances type 1b replicon efficiency was insufficient to confer replication to the wild-type 1a replicon. This subgenomic replicon was subsequently found to be inefficiently translated in Huh7 cells compared to a type 1b replicon, and the attenuation of translation mapped to the N-terminal region of NS3. Therefore, to ensure efficient translation and thereby support replication of the 1a genome, the coding sequence for first 75 residues from type 1a were replaced with the type 1b (strain Con 1) NS3 coding sequence. Although nonstructural proteins were expressed at lower levels with this replicon than with type 1b and although the amount of viral RNA was also severalfold lower (150 copies of positive-strand RNA per cell), the replicon stably replicated in Huh7 cells. Notwithstanding this difference, the ratio of positive- to negative-strand RNA of 26 was similar to that found with the type 1b replicon. Similar results were found for a 1b replicon expressing the type 1a RNA-dependent RNA polymerase. These 1a hybrid replicons maintained sensitivity to alpha interferon (IFN-alpha), albeit with an eightfold-higher 50% inhibitory concentration than type 1b replicons. Evidence is provided herein to confirm that this differential response to IFN-alpha may be attributed directly to the type 1a polymerase.

Adaptation, Physiological↗

PubMed1999

The RNA-dependent RNA polymerases of different members of the family Flaviviridae exhibit similar properties in vitro.

The virus-encoded RNA-dependent RNA polymerase (RdRp), which is required for replication of the positive-strand RNA genome, is a key enzyme of members of the virus family Flaviviridae. By using heterologously expressed proteins, we demonstrate that the 77 kDa NS5B protein of two pestiviruses, bovine viral diarrhoea virus and classical swine fever virus, and the 100 kDa NS5 protein of the West Nile flavivirus possess RdRp activity in vitro. As originally shown for the RdRp of hepatitis C virus, RNA synthesis catalysed by the pestivirus and flavivirus enzymes is strictly primer-dependent in vitro. Accordingly, initiation of RNA polymerization on homopolymeric RNAs and heteropolymeric templates, the latter with a blocked 3'-hydroxyl group, was found to be dependent on the presence of complementary oligonucleotide primer molecules. On unblocked heteropolymeric templates, including authentic viral RNAs, the RdRps were shown to initiate RNA synthesis via intramolecular priming at the 3'-hydroxyl group of the template and 'copy-back' transcription, thus yielding RNase-resistant hairpin molecules. Taken together, the RdRps of different members of the Flaviviridae were demonstrated to exhibit a common reactivity profile in vitro, typical of nucleic acid-polymerizing enzymes.

Animals↗