Unraveling the molecular basis of the life cycle of hepatitis C virus (HCV), a prevalent agent of human liver disease, entails the identification of cell-encoded factors that participate in the replication of the viral RNA genome. This study provides evidence that the so-called NF/NFAR proteins, namely, NF90/NFAR-1, NF110/NFAR-2, NF45, and RNA helicase A (RHA), which mostly belong to the dsRBM protein family, are involved in the HCV RNA replication process. NF/NFAR proteins were shown to specifically bind to replication signals in the HCV genomic 59 and 39 termini and to promote the formation of a looplike structure of the viral RNA. In cells containing replicating HCV RNA, the generally nuclear NF/NFAR proteins accumulate in the cytoplasmic viral replication complexes, and the prototype NFAR protein, NF90/NFAR-1, stably interacts with a viral protein.HCV replication was inhibited in cells where RNAi depleted RHA from the cytoplasm. Likewise, HCV replication was hindered in cells that contained another NF/NFAR protein recruiting virus. The recruitment of NF/NFAR proteins by HCV is assumed to serve two major purposes: to support 59-39 interactions of the viral RNA for the coordination of viral protein and RNA synthesis and to weaken host-defense mechanisms.
The complete Npro coding sequences were determined for 16 pestiviruses isolated from cattle, pig, and several wild ruminant species including reindeer, bison, deer, and bongo. Phylogenetic analysis enabled the segregation of pestiviruses into the established species bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, border disease virus (BDV), and classical swine fever virus (CSFV). For BVDV-1 five distinct subgroups were identified, while BVDV-2, BDV, and CSFV were each subdivided into two subgroups. The virus isolates from bongo and deer as well as one porcine virus isolate belong to BVDV-1. Interestingly, the isolates from reindeer and bison are distinct from the established pestivirus species. The Npro sequences from these two viruses are more similar to BDV than to the other pestivirus species. Calculation of the pairwise evolutionary distances allowed a clear separation of the categories species, subgroup, and isolate only when the reindeer/bison viruses were considered as members of an additional pestivirus species. Furthermore, the entire E2 coding sequences of a representative set of virus isolates covering all recognized species and subgroups were studied. Segregation of pestiviruses based on the E2 region was identical with that obtained with the N(pro) sequences.
For the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP 821, a duplication of the genomic region encoding part of NS2, NS3, NS4A, and part of NS4B together with a nonviral insertion was detected. Further analyses including molecular cloning and sequencing of the putative cellular recombination partner showed that the insertion in CP 821 originated from a bovine mRNA encoding the cellular protein NEDD8, which is 58% identical to ubiquitin. To our knowledge the genome of CP 821 represents the first viral RNA with a NEDD8 coding insertion. Remarkably, the insertion site differs from that described for insertions of ubiquitin. The NEDD8 sequence allows an additional cleavage of the viral polyprotein, whereby an NS3 with an unusual N-terminus is generated. Furthermore, the CP 821-specific genomic alterations were introduced into an infectious noncytopathogenic (noncp) BVDV cDNA clone. After transfection of bovine cells with the respective RNA, a cp virus was recovered. This showed that the NEDD8 coding insertion together with the duplicated viral sequences represents the genetic basis for cytopathogenicity of CP 821. In addition to the recovered cp virus, noncp BVDV rapidly evolved after transfection. This is the first time that a change from the cp to the noncp phenotype was demonstrated in the course of replication in tissue culture cells.
Here we report on a segment in the genomic 39 non-translated region (39NTR) of bovine viral diarrhea virus (BVDV) that is accessible for the insertion of foreign sequence elements such as the 59NTR of hepatitis C virus. Recombinant viruses exhibited replication kinetics similar to those of the parental strain, and characterization of RNA species after several passages revealed that foreign inserts had the same genetic stability as the BVDV 39NTR. The generation of such BVDV recombinants is relevant for several applications.Infections with bovine viral diarrhea virus (BVDV) elicit various diseases in cattle. For example, vertical infections may lead to persistently infected (PI) animals, which may die of mucosal disease (MD) when cytopathic (cp) BVDV biotypes emerge by mutation during virus replication .Within the family Flaviviridae, BVDV belongs to the genus Pestivirus, together with Classical swine fever virus (CSFV). The approximately 12 kb positive-strand RNA genome consists of a long open reading frame (ORF) and 59 and 39 non-translated regions (NTRs). Translation initiates at an internal ribosomal entry site (IRES) involving the 59NTR and the 59 portion of the ORF. The resulting polyprotein is processed by cellular and viral proteases to yield the viral structural and non-structural (NS) proteins (Lindenbach et al., 2007). In concert with cellular factors, the proteins NS3, NS4A, NS4B, NS5A and NS5B form viral replication complexes (RC) that catalyse the synthesis of progeny positive-strand viral RNAs via negative-strand RNA intermediates (Behrens & Isken, 2006).Studies on the molecular mechanisms of BVDV RNA replication were facilitated significantly by functional DNA copies of the viral genome. One of the first cDNAs that enabled in vitro transcription of infectious viral RNA was generated with BVDV CP7 (Meyers et al., 1996), a cp virus isolate from a PI animal that died of MD (Tautz et al., 1994). Considering its key role during viral RNA replication, the viral 39NTR has been particularly wellstudied. As with all pestiviruses, the BVDV 39NTR consists of a variable region (39V) downstream of the translational stop codon, and a conserved region (39C) at the 39 end (Deng & Brock, 1993) (Fig. 1). 39C contains inalterable elements that are assumed to compose the negative-strand promoter of the initial RC (Yu et al., 1999). Conversely, 39V displays a remarkable heterogeneity in size and nucleotide composition between different virus subtypes, and certain mutations within 39V are well-tolerated (Isken et al., 2004;Pankraz et al., 2005). Still, 39V also encodes conserved features, such as the so-called 'pseudo-stop codons', i.e. nucleotide triplets within the 39NTR that resemble translational stop codons and that are present in translational frame with the viral ORF. The presence of these 'pseudo-stops', as well as proper folding of the SL stop stem-loop structure in 39V and association of the cellular NFAR proteins with SL stop , were shown to be important for accurate termination of translation at the stop co...
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