Launching of the yeast 20 s RNA narnavirus by expressing the genomic or antigenomic viral RNA in vivo

20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome encodes only its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing, from a vector, genomic strands fused at the 3'-ends to the hepatitis delta virus antigenomic ribozyme. Using this launching system, we analyzed 3'-cis-signals present in the genomic strand for replication. The viral genome has five-nucleotide inverted repeats at both termini (5'-GGGGC... GCCCC-OH). The fifth G from the 3'-end was dispensable for replication, whereas the third and fourth Cs were essential. The 3'-terminal and penultimate Cs could be eliminated or modified to other nucleotides; however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3'-end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the correct size and sequence of the viral 3'-end. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from antigenomic strands provided active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3'-end in the antigenomic strand was essential for replication. In this work, we also present the first conclusive evidence that 20 S and 23 S RNA viruses are independent replicons.     

Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication

Previous studies indicate that the 3' terminal 46 nt of the RNA genome of hepatitis C virus (HCV) are highly conserved among different viral strains and essential for RNA replication. Here, we describe a mutational analysis of the 3' terminal hairpin (stem-loop I) that is putatively formed by this sequence and demonstrate its role in replication of the viral RNA. We show that single base substitutions within the 6-nt loop at positions adjacent to the stem abrogate replication of a subgenomic RNA, whereas substitutions in the three apical nucleotides were well tolerated without loss of replication competence. Single point mutations were also well tolerated within the middle section of the duplex, but not at the penultimate nucleotide positions near either end of the stem. However, complementary substitutions at the -19 and -28 positions (from the 3' end) restored replication competence, providing strong evidence for the existence of the structure and its involvement in RNA replication. This was confirmed by rescue of replicating RNAs from mutants containing complementary 10-nt block substitutions at the base of the stem. Each of these RNAs contained an additional U at the 3' terminus. Further experiments indicated a strong preference for U at the 3' terminal position (followed in order by C, A, and G), and a G at the -2 position. These features of stem-loop I are likely to facilitate recognition of the 3' end of the viral RNA by the viral RNA replicase.     

Structure and function of the 3' terminal six nucleotides of the west nile virus genome in viral replication

Using a self-replicating reporting replicon of West Nile (WN) virus, we performed a mutagenesis analysis to define the structure and function of the 3'-terminal 6 nucleotides (nt) (5'-GGAUCU(OH)-3') of the WN virus genome in viral replication. We show that mutations of nucleotide sequence or base pair structure of any of the 3'-terminal 6 nt do not significantly affect viral translation, but exert discrete effects on RNA replication. (i). The flavivirus-conserved terminal 3' U is optimal for WN virus replication. Replacement of the wild-type 3' U with a purine A or G resulted in a substantial reduction in RNA replication, with a complete reversion to the wild-type sequence. In contrast, replacement with a pyrimidine C resulted in a replication level similar to that of the 3' A or G mutants, with only partial reversion. (ii). The flavivirus-conserved 3' penultimate C and two upstream nucleotides (positions 78 and 79), which potentially base pair with the 3'-terminal CU(OH), are absolutely essential for viral replication. (iii). The base pair structures, but not the nucleotide sequences at the 3rd (U) and the 4th (A) positions, are critical for RNA replication. (iv). The nucleotide sequences of the 5th (G) position and its base pair nucleotide (C) are essential for viral replication. (v). Neither the sequence nor the base pair structure of the 6th nucleotide (G) is critical for WN virus replication. These results provide strong functional evidence for the existence of the 3' flavivirus-conserved RNA structure, which may function as contact sites for specific assembly of the replication complex or for efficient initiation of minus-sense RNA synthesis.     

Repair and polyadenylation of a naturally occurring hepatitis C virus 3' nontranslated region-shorter variant in selectable replicon cell lines

The 3' nontranslated region (NTR) of the hepatitis C virus (HCV) genome is highly conserved and contains specific cis-acting RNA motifs that are essential in directing the viral replication machinery to initiate at the correct 3' end of the viral genome. Since the ends of viral genomes may be damaged by cellular RNases, preventing the initiation of viral RNA replication, stable RNA hairpin structures in the 3' NTR may also be essential in host defense against exoribonucleases. During 3'-terminal sequence analysis of serum samples of a patient with chronic hepatitis related to an HCV1b infection, a number of clones were obtained that were several nucleotides shorter at the extreme 3' end of the genome. These shorter 3' ends were engineered in selectable HCV replicons in order to enable the study of RNA replication in cell culture. When in vitro-transcribed subgenomic RNAs, containing shorter 3' ends, were introduced into Huh-7 cells, a few selectable colonies were obtained, and the 3' terminus of these subgenomic RNAs was sequenced. Interestingly, most genomes recovered from these colonies had regained the wild-type 3' ends, showing that HCV, like several other positive-stranded RNA viruses, has developed a strategy to repair deleted 3' end nucleotides. Furthermore, we found several genomes in these replicon colonies that contained a poly(A) tail and a short linker sequence preceding the poly(A) tail. After recloning and subsequent passage in Huh-7 cells, these poly(A) tails persisted and varied in length. In addition, the connecting linker became highly diverse in sequence and length, suggesting that these tails are actively replicated. The possible terminal repair mechanisms, including roles for the poly(A) tail addition, are discussed.     

Template requirements for de novo RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase on the viral X RNA

The hepatitis C virus (HCV)-encoded NS5B protein is an RNA-dependent RNA polymerase which plays a substantial role in viral replication. We expressed and purified the recombinant NS5B of an HCV genotype 3a from Esherichia coli, and we investigated its ability to bind to the viral RNA and its enzymatic activity. The results presented here demonstrate that NS5B interacts strongly with the coding region of positive-strand RNA, although not in a sequence-specific manner. It was also determined that more than two molecules of polymerase bound sequentially to this region with the direction 3' to 5'. Also, we attempted to determine the initiation site(s) of de novo synthesis by NS5B on X RNA, which contains the last 98 nucleotides of HCV positive-strand RNA. The initiation site(s) on X RNA was localized in the pyrimidine-rich region of stem I. However, when more than five of the nucleotides of stem I in X RNA were deleted from the 3' end, RNA synthesis initiated at another site of the specific ribonucleotide. Our study also showed that the efficiency of RNA synthesis, which was directed by X RNA, was maximized by the GC base pair at the penultimate position from the 3' end of the stem. These results will provide some clues to understanding the mechanism of HCV genomic RNA replication in terms of viral RNA-NS5B interaction and the initiation of de novo RNA synthesis.     

3'-Terminal sequence in poliovirus negative-strand templates is the primary cis-acting element required for VPgpUpU-primed positive-strand initiation

The 5' cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5' cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was not required for positive-strand synthesis. In contrast, altering the primary sequence at the 5'-terminal end of stem a had little or no effect on negative-strand synthesis but dramatically reduced positive-strand initiation and the formation of infectious virus. The inhibition of positive-strand synthesis observed in these reactions was most likely a consequence of nucleotide alterations in the conserved sequence at the 3' ends of negative-strand RNA templates. Previous studies suggested that VPgpUpU synthesized on the cre(2C) hairpin was required for positive-strand synthesis. Therefore, these results are consistent with a model in which preformed VPgpUpU serves as the primer for positive-strand initiation on the 3'AAUUUUGUC5' sequence at the 3' ends of negative-strand templates. Our results suggest that this sequence is the primary cis-acting element that is required for efficient VPgpUpU-primed positive-strand initiation.     

The 5'-terminal region of the Aichi virus genome encodes cis-acting replication elements required for positive- and negative-strand RNA synthesis

Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.     

A long-range pseudoknot in Qbeta RNA is essential for replication

A puzzling aspect of replication of bacteriophage Qbeta RNA has always been that replicase binds at an internal segment, the M-site, some 1450 nt away from the 3' end. Here, we report on the existence of a long-range pseudoknot, base-pairing eight nt in the loop of the 3' terminal hairpin to a single-stranded interdomain sequence located about 1200 nt upstream, close to the internal replicase binding site. Introduction of a single mismatch into this pseudoknot is sufficient to abolish replication, but the inhibition is fully reversed by a second-site substitution that restores the pairing. The pseudoknot is part of an elaborate structure that seems to hold the 3' end in a fixed position vis a vis the replicase binding site. Our results imply that the shape of the RNA confers the functonality. We discuss the possible relevance of our findings for replication of other viral RNAs.     

Sesbania mosaic virus (SeMV) infectious clone: possible mechanism of 3' and 5' end repair and role of polyprotein processing in viral replication

Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.     

Base pairing between hepatitis C virus RNA and microRNA 122 3' of its seed sequence is essential for genome stabilization and production of infectious virus

MicroRNA 122 (miR-122) facilitates hepatitis C virus (HCV) replication by recruiting an RNA-induced silencing complex (RISC)-like complex containing argonaute 2 (Ago2) to the 5' end of the HCV genome, thereby stabilizing the viral RNA. This requires base pairing between the miR-122 "seed sequence" (nucleotides [nt] 2 to 8) and two sequences near the 5' end of the HCV RNA: S1 (nt 22 to 28) and S2 (nt 38 to 43). However, recent reports suggest that additional base pair interactions occur between HCV RNA and miR-122. We searched 606 sequences from a public database (genotypes 1 to 6) and identified two conserved, putatively single-stranded RNA segments, upstream of S1 (nt 2 and 3) and S2 (nt 30 to 34), with potential for base pairing to miR-122 (nt 15 and 16 and nt 13 to 16, respectively). Mutagenesis and genetic complementation experiments confirmed that HCV nt 2 and 3 pair with nt 15 and 16 of miR-122 bound to S1, while HCV nt 30 to 33 pair with nt 13 to 16 of miR-122 at S2. In genotype 1 and 6 HCV, nt 4 also base pairs with nt 14 of miR-122. These 3' supplementary base pair interactions of miR-122 are functionally important and are required for Ago2 recruitment to HCV RNA by miR-122, miR-122-mediated stabilization of HCV RNA, and production of infectious virus. However, while complementary mutations at HCV nt 30 and 31 efficiently rescued the activity of a 15C,16C miR-122 mutant targeting S2, similar mutations at nt 2 and 3 failed to rescue Ago2 recruitment at S1. These data add to the current understanding of miR-122 interactions with HCV RNA but indicate that base pairing between miR-122 and the 5' 43 nt of the HCV genome is more complex than suggested by existing models.     

Unusual loop-sequence flexibility of the proximal RNA replication element in EMCV

Picornaviruses contain stable RNA structures at the 5' and 3' ends of the RNA genome, OriL and OriR involved in viral RNA replication. The OriL RNA element found at the 5' end of the enterovirus genome folds into a cloverleaf-like configuration. In vivo SELEX experiments revealed that functioning of the poliovirus cloverleaf depends on a specific structure in this RNA element. Little is known about the OriL of cardioviruses. Here, we investigated structural aspects and requirements of the apical loop of proximal stem-loop SL-A of mengovirus, a strain of EMCV. Using NMR spectroscopy, we showed that the mengovirus SL-A apical loop consists of an octaloop. In vivo SELEX experiments demonstrated that a large number of random sequences are tolerated in the apical octaloop that support virus replication. Mutants in which the SL-A loop size and the length of the upper part of the stem were varied showed that both stem-length and stability of the octaloop are important determinants for viral RNA replication and virus reproduction. Together, these data show that stem-loop A plays an important role in virus replication. The high degree of sequence flexibility and the lack of selective pressure on the octaloop argue against a role in sequence specific RNA-protein or RNA-RNA interactions in which octaloop nucleotides are involved.     

Generation of a uniform 3'' end RNA of murine leukemia virus.

Using the S1 nuclease mapping technique, we demonstrated that the majority of Moloney murine leukemia RNA molecules, isolated either from the nucleus or cytoplasm of infected mouse cells, share a uniform 3' end located at the border of the R and U-5 regions of the long terminal repeat. When the long terminal repeat sequences were inserted in the pSV plasmid downstream of the simian virus 40 late promoter, the 3' end of the viral RNA was also generated close to the R region of the long terminal repeat. These results demonstrate that the long terminal repeat signals the generation of an authentic 3' end when situated downstream of an actively transcribed region.     

Specific interaction between the classical swine fever virus NS5B protein and the viral genome.

The NS5B protein of the classical swine fever virus (CSFV) is the RNA-dependent RNA polymerase of the virus and is able to catalyze the viral genome replication. The 3' untranslated region is most likely involved in regulation of the Pestivirus genome replication. However, little is known about the interaction between the CSFV NS5B protein and the viral genome. We used different RNA templates derived from the plus-strand viral genome, or the minus-strand viral genome and the CSFV NS5B protein obtained from the Escherichia coli expression system to address this problem. We first showed that the viral NS5B protein formed a complex with the plus-strand genome through the genomic 3' UTR and that the NS5B protein was also able to bind the minus-strand 3' UTR. Moreover, it was found that viral NS5B protein bound the minus-strand 3' UTR more efficiently than the plus-strand 3' UTR. Further, we observed that the plus-strand 3' UTR with deletion of CCCGG or 21 continuous nucleotides at its 3' terminal had no binding activity and also lost the activity for initiation of minus-strand RNA synthesis, which similarly occurred in the minus-strand 3' UTR with CATATGCTC or the 21 nucleotide fragment deleted from the 3' terminal. Therefore, it is indicated that the 3' CCCGG sequence of the plus-strand 3' UTR, and the 3' CATATGCTC fragment of the minus-strand are essential to in vitro synthesis of the minus-strand RNA and the plus-strand RNA, respectively. The same conclusion is also appropriate for the 3' 21 nucleotide terminal site of both the 3' UTRs.