A Stem-Loop Motif Formed by the Immediate 5′ Terminus of the Bovine Viral Diarrhea Virus Genome Modulates Translation as well as Replication of the Viral RNA

Abstract

Bovine viral diarrhea virus (BVDV), a Pestivirus member of theThe pestivirus genome, a single-stranded, nonsegmented RNA molecule of positive polarity, has a size of approximately 12.5 kilobases and comprises a single open reading frame (ORF) that is flanked by untranslated regions (UTRs) at the 5Ј and 3Ј ends. Upon infection, the viral RNA functions as a messenger in the cytoplasm of the host cell and directs the synthesis of an unstable polyprotein, which is co-and posttranslationally processed to yield a range of different viral proteins. The array of genetic units along the ORF encoding the final products of polyprotein proteolysis has been determined to be 5Ј N pro , C, E rns , E1, E2, p7, NS2-NS3, NS4A, NS4B, NS5A, and NS5B 3Ј (reviewed in reference 30). N pro , a nonstructural autoprotease of unknown function, releases itself from the precursor (49). The activity of host signal peptidases gives rise to C, E rns , E1, and E2 which are destined to form the capsid and the envelope of the virion, respectively (reviewed in reference 44). The nonstructural (NS) proteins being encoded by the C-terminal part of the polyprotein are predominantly generated by the activity of a serine protease domain residing within the N terminus of NS3 (43, 50). Most NS proteins are presumed to act as catalytic components of the viral replication machinery (see below). Along this line, certain enzymatic functions which were demonstrated to be essentially involved in RNA replication, namely, a nucleoside triphosphatase/RNA helicase activity and an RNA-dependent RNA polymerase (RdRp) activity, were found to be associated with the NS3 and the NS5B protein, respectively (36,40,47,52).As with other positive-strand RNA viruses, replication of the pestivirus genome proceeds in an asymmetric manner along a two-step scheme. Supposedly in close functional linkage with the generation or maturation of the polyprotein, the nascent viral proteins and hypothetical cellular components associate with the 3Ј terminus of the genomic RNA to form replication complexes. These catalyze the synthesis of a low copy number of complementary negative-strand RNA intermediates, which subsequently serve as templates for the transcription of an excess of progeny positive-strand RNA molecules (6).cDNA copies of BVDV genomes (24,25,46) that are capable of producing infectious RNA transcripts in vitro permit detailed investigations of the virus life cycle by means of "reverse genetics": i.e., mutagenesis of the viral RNA via the cDNA construct, introduction of the modified RNA into cultured cells, and monitoring of the effects of mutagenesis in vivo. In this way, genetic studies revealed that a subgenomic BVDV RNA (DI9c) consisting mainly of the 5Ј and 3Ј UTRs and the coding region of the nonstructural proteins NS3-NS5B replicates autonomously upon transfection into different host cells. BVDV DI9c RNA was hence verified to encode all fac-* Corresponding author. Mailing address:

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

“…Moderate negative effects on viral replication were also observed when we changed, for example, the spacing between SL stop and SLII (not shown). These findings explained the evolutionary drift of 3ЈV, and they were in striking contrast to observations with cis-acting replication signals in the conserved genomic 5Ј and 3Ј termini, where analogous interventions completely inhibited replication (Yu et al 1999(Yu et al , 2000; for a further Discussion see below).…”

“…Nevertheless, the replication defect of 3ЈV mutants such as mutant 7 could not be explained by the observed reduction of protein synthesis. This becomes apparent in view of the fact that BVDV replicons with mutations in the 5ЈNTR, which reduced the rate of translation by a factor of 2-3, still produced sufficient viral protein to allow RNA replication at the wild-type level (Yu et al 2000; see also Discussion). …”

“…For this purpose, we applied the mutant 7 RNA, that is, the same transcript that was previously tested in the replication assay, to an in vitro translation assay that was based on cytoplasmic extracts of BHK-21 cells. This in vitro translation system was previously shown to reflect the in vivo situation of BVDV protein synthesis rather authentically, because it promotes efficient and accurate translation of the ORF as well as NS3/4A-mediated maturation of the diverse replicon-encoded nonstructural proteins (Grassmann et al , 2001Yu et al 2000; see also Fig. 1A).…”

“…5,6). Considering again that reductions in the rate of translation of the viral proteins by 50%-70% still allowed viral propagation at the wild-type level (Yu et al 2000), an amount of 5% C-terminally extended and potentially inactive NS5B should be irrelevant for the overall viral replication process. Therefore, we favor a second explanation of the replication defect of mutants that revealed a defect in translation termination.…”

A chimeric GB virus B with 5′ nontranslated RNA sequence from hepatitis C virus causes hepatitis in tamarins

“…In addition to domains II-III, some reports suggest that the most 5Ј stem-loop (domain I) within the 5ЈNTR may regulate cap-independent translation of some members of the family Flaviviridae. 24,33,34 However, it made little difference to translation whether domain I was derived from HCV or GBV-B in our chimeric constructs.…”