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.

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 speci®cally with both the termini of the viral RNA genome involving regulatory elements in the 5¢ and 3¢ non-translated regions. Modi®cation of the protein interaction sites in the 3¢ non-translated region yielded viral RNAs that were replication de®cient. 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.

As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of theFlaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized defective interfering particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5′ and 3′ untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3′ end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5′ third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5′ end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.

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:

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 3V portion of the viral 3 nontranslated region (3NTR). Genetic studies and structure probing revealed that 3V represents a complex RNA motif that is composed of synergistically acting sequence and structure elements. Correct formation of the 3V motif was shown to be an important determinant of the viral RNA replication process. Most interestingly, we found that a proper conformation of 3V 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 3NTR, the complex 3V 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 5NTR of the BVDV genome, we propose a model where the viral 3NTR has a bipartite functional organization: The conserved 3 portion (3C) is part of the nascent replication complex; the variable 5 portion (3V) 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.

The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5 and 3 untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cis or in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to be cis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.Bovine viral diarrhea virus (BVDV) types I and II, border disease virus of sheep, and classical swine fever virus constitute the genus Pestivirus, comprising widely distributed pathogens of ruminants and pigs (reviewed in reference 31). Together with the genera Flavivirus and Hepacivirus (hepatitis C viruses [HCVs]), the pestiviruses are classified in the family Flaviviridae (reviewed in reference 24), all members of which are characterized by an enveloped virion that harbors a singlestranded, linear RNA genome of positive polarity. The genomic RNA, which in the case of pestiviruses has a length of approximately 12.5 kb, consists of a single open reading frame (ORF) and untranslated regions (UTRs) at the 5Ј and 3Ј ends, respectively. Following infection, it operates initially as a messenger in the cytoplasm. Translation is mediated by an internal ribosomal entry site (IRES) within the 5Ј UTR (22) and leads to the synthesis of a polyprotein that is co-and posttranslationally processed into a range of viral proteins. The ord...

Bovine viral diarrhea virus (BVDV), a member of the genusPestivirus in the family Flaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5′ and 3′ ends. Computer modeling suggested the 3′ UTR comprised single-stranded regions as well as stem-loop structures—features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364–2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3′ UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3′C) of the DI9c 3′ UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3′ terminus and a considerably less stable stem-loop, SL II, that forms the 5′ portion of 3′C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3′-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3′C region of the BVDV 3′ UTR create a common cis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors.

Studies on the replication of the pestivirus bovine viral diarrhea virus (BVDV) were considerably facilitated by the recent discovery of an autonomous subgenomic BVDV RNA replicon (DI9c). DI9c comprises mainly the untranslated regions of the viral genome and the coding region of the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To assess the significance of the NS3-associated nucleoside triphosphatase/helicase activity during RNA replication and to explore other functional features of NS3, we generated a repertoire of DI9c derivatives bearing in-frame mutations in different parts of the NS3 coding unit. Most alterations resulted in deficient replicons, several of which encoded an NS3 protein with an inhibited protease function. Three lesions permitted replication, though at a lower level than that of the wild-type RNA, i.e., replacement of the third position of the DEYH helicase motif II by either T or F and an insertion of four amino acid residues in the C-terminal part of NS3. While polyprotein proteolysis was found to be almost unaffected in these latter replicons, in vitro studies with the purified mutant NS3 proteins revealed a significantly impaired helicase activity for the motif II substitutions. NS3 with a DEFH motif, moreover, showed a significantly lower ATPase activity. In contrast, the C-terminal insertion had no negative impact on the ATPase/RNA helicase activity of NS3. All three mutations affected the synthesis of both replication products—negative-strand intermediate and progeny positive-strand RNA—in a symmetric manner. Unexpectedly, various attempts to rescue or enhance the replication capability of nonfunctional or less functional DI9c NS3 derivatives, respectively, by providing intact NS3 intrans failed. Our experimental data thus demonstrate that the diverse enzymatic activities of the NS3 protein—in particular the ATPase/RNA helicase—play a pivotal role even during early steps of the viral replication pathway. They may further indicate the C-terminal part of NS3 to be an important functional determinant of the RNA replication process.