A novel in vitro replication system for Dengue virus. Initiation of RNA synthesis at the 3'-end of exogenous viral RNA templates requires 5'- and 3'-terminal complementary sequence motifs of the viral RNA

Positive strand viral replicases are membrane-bound complexes of viral and host proteins. The mechanism of viral replication and the role of host proteins are not well understood. To understand this mechanism, a viral replicase assay that utilizes extracts from dengue virus-infected mosquito (C6/36) cells and exogenous viral RNA templates is reported in this study. The 5'- and 3'-terminal regions (TR) of the template RNAs contain the conserved elements including the complementary (cyclization) motifs and stem-loop structures. RNA synthesis in vitro requires both 5'- and 3'-TR present in the same template molecule or when the 5'-TR RNA was added in trans to the 3'-untranslated region (UTR) RNA. However, the 3'-UTR RNA alone is not active. RNA synthesis occurs by elongation of the 3'-end of the template RNA to yield predominantly a double-stranded hairpin-like RNA product, twice the size of the template RNA. These results suggest that an interaction between 5'- and 3'-TR of the viral RNA that modulates the 3'-UTR RNA structure is required for RNA synthesis by the viral replicase. The complementary cyclization motifs of the viral genome also seem to play an important role in this interaction.     

Similarity between 5'- and 3'-terminal nucleotide sequences and double-stranded RNA-derived sequences of eukaryotic mRNA

It has been reported recently that parts of the nucleotide sequences present in the 5′- and 3′-terminal regions of cytoplasmic mRNA are derived from double-stranded hairpin structures of heterogeneous nuclear RNA—a putative mRNA precursor (Naora, 1979). In order to explore the nature of double-stranded hairpin structures, using the sequencing data of human and rabbit globin mRNA and hen ovalbumin mRNA, we examined the following possibility: that certain regions of both the 5′- and 3′-terminal nucleotide sequences of mature mRNA were present in double-stranded hairpin structures covalently linked to both sides of the message sequence in the precursor mRNA molecule and that these double-stranded hairpin structures are similar to each other. The results support the above possibility by showing substantial similarity of nucleotide sequences between the 5′- and 3′-terminal regions of these mRNAs in terms of the formation of similar double-stranded hairpin structures.     

Interaction between the cellular protein eEF1A and the 3'-terminal stem-loop of West Nile virus genomic RNA facilitates viral minus-strand RNA synthesis

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.     

5'-terminal phosphorylation and secondary structure of double-stranded RNA from a fungal virus.

Different double-stranded RNA species from Penicillium stoloniferum virus have been phosphorylated at the 5′ termini with the aid of polynucleotide kinase. A very low phosphate uptake has been observed which, especially in the case of a relatively small molecular component, was increased several times by pretreatment with RNAase t₁. Adenosine and uridine have been detected at the 5′-termini of this RNA component. Digestion with RNAase T₁, an enzyme which does not cut across the two strands of a double-stranded RNA molecule, produced a new uridine terminus and increased the efficiency of phosphorylation. It is concluded that this double-stranded RNA molecule contains single-stranded stretches at or near the 5′-termini. The possibility of a circular structure being formed by the annealing of single-stranded tails is discussed.     

In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.     

In vitro selection of novel RNA ligands that bind human cytomegalovirus and block viral infection

Ribonuclease-resistant RNA molecules that bind to infectious human cytomegalovirus (HCMV) were isolated in vitro from a pool of randomized sequences after 16 cycles of selection and amplification. The two ligands (L13 and L19) characterized exhibited high HCMV-binding affinity in vitro and effectively inhibited viral infection in tissue culture. Their antiviral activity was also specific as they only reacted with two different strains of HCMV but not with the related herpes simplex virus 1 and human cells. These two ligands appeared to function as antivirals by blocking viral entry. Ultraviolet (UV) crosslinking studies suggested that L13 and L19 bind to HCMV essential glycoproteins B and H, respectively. Thus, RNA ligands that bind to different surface antigens of HCMV can be simultaneously isolated by the selection procedure. Our study demonstrates the feasibility of using these RNA ligands as a research tool to identify viral proteins required for infectivity and as an antiviral agent to block viral infection.     

In vitro and in vivo mutational analysis of the 3'-terminal regions of hepatitis e virus genomes and replicons

Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3'-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3'-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3'-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3' termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3' terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3'-terminal mutations in vivo. Incorporation of the 3'-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.     

Interaction between polypeptide 3ABC and the 5'-terminal structural elements of the genome of Aichi virus: implication for negative-strand RNA synthesis

Secondary structural elements at the 5' end of picornavirus genomic RNA function as cis-acting replication elements and are known to interact specifically with viral P3 proteins in several picornaviruses. In poliovirus, ribonucleoprotein complex formation at the 5' end of the genome is required for negative-strand synthesis. We have previously shown that the 5'-end 115 nucleotides of the Aichi virus genome, which are predicted to fold into two stem-loops (SL-A and SL-C) and one pseudoknot (PK-B), act as a cis-acting replication element and that correct folding of these structures is required for negative-strand synthesis. In this study, we investigated the interaction between the 5'-terminal 120 nucleotides of the genome and the P3 proteins, 3AB, 3ABC, 3C, and 3CD, by gel shift assay and Northwestern analysis. The results showed that 3ABC and 3CD bound to the 5'-terminal region specifically. The binding of 3ABC was observed on both assays, while that of 3CD was detected only on Northwestern analysis. No binding of 3AB or 3C was observed. Binding assays using mutant RNAs demonstrated that disruption of the base pairings of the stem of SL-A and one of the two stem segments of PK-B (stem-B1) abolished the 3ABC binding. In addition, the specific nucleotide sequence of stem-B1 was responsible for the efficient 3ABC binding. These results suggest that the interaction of 3ABC with the 5'-terminal region of the genome is involved in negative-strand synthesis. On the other hand, the ability of 3CD to interact with the 5'-terminal region did not correlate with the RNA replication ability.     

Selection of DNA aptamers that bind the RNA-dependent RNA polymerase of hepatitis C virus and inhibit viral RNA synthesis in vitro

The RNA-dependent RNA polymerase (NS5B) of the hepatitis C virus (HCV) plays a key role in the life cycle of the virus. In order to find inhibitors of the HCV polymerase, we screened a library of 81 nucleotide (nt)-long synthetic DNA containing 35 random nucleotides by the Systematic Evolution of Ligands by Exponential enrichment (SELEX) approach. Thirty ligands selected for their binding affinity to the NS5B were classified into four groups on the basis of their sequence homologies. Among the selected molecules, two were able to inhibit in vitro the polymerase activity of the HCV NS5B. These aptamers appeared to be specific for HCV polymerase, as no inhibition of poliovirus 3D polymerase activity was observed. The binding and inhibitory potential of one aptamer (27v) was associated with the 35 nt-long variable region. This oligonucleotide displayed an apparent dissociation constant (K(d)) in the nanomolar range. Our results showed that it was able to compete with RNA templates corresponding to the 3'-ends of the (+) and the (-) HCV RNA for binding to the polymerase. The fact that a DNA aptamer could interfere with the binding of natural templates of the enzyme could help in performing structure-function analysis of the NS5B and might constitute a basis for further structure-based drug design of this crucial enzyme of HCV replication.     

A relationship between adenosine 3'-5'-cyclic monophosphate levels and deoxyribonucleic acid synthesis in Euglena

Using the binding protein method we found that cAMP levels in normal, exponentially growing Euglena stay constant on per cell and protein basis. The level rises slightly when cells enter the stationary stage. Cells growing in low vitamin B12 medium show the same pattern during predeficiency growth. Upon becoming vitamin B12 deficient, the cAMP level decreases. Replenishment of these cells with the vitamin causes an immediate drop, followed by a sharp rise in cAMP. This is followed by resumption of DNA synthesis. The cAMP level drops and rises again when DNA duplication is completed and during the G2 period. The level of the cAMP drops again followed by resumption of cell division. the data suggest a relation exists between cAMP level, resumption and completion of DNA synthesis, and cell division.     

In vitro synthesis and stability of RNA in isolated nuclei from bovine lymphocytes

Nuclei from Concanavalin A-stimulated lymphocytes (30 hr after Con A addition) incorporate up to 5 times more (3-H)UTP into RNA than nuclei from resting lymphocytes. The incorporation kinetics is linear for almost 60 min. 14–20% of the $\text{in vitro}$ labeled RNA is polyadenylated. Poly(A) (?)RNA from both types of nuclei sediments from 4–5S up to more than 30S on sucrose gradients. Nuclei from stimulated cells synthesize about double the amount of RNA larger than 18S than nuclei from resting cells. The same holds for poly(A) (+)RNA. Poly(A) (?) RNA labeled during 10 min in both types of nuclei is stable during a 30 min chase. Under the same conditions poly(A) (+)RNA in nuclei from resting cells is degraded to about 50% during the chase whereas it is stable in nuclei from stimulated cells.     

Interactions of the RNA polymerase with the viral genome at the 5'- and 3'-ends contribute to 20S RNA narnavirus persistence in yeast

20S RNA narnavirus is a positive strand RNA virus found in the yeast Saccharomyces cerevisiae. The viral genome (2514 nucleotides) only encodes a single protein (p91), the RNA-dependent RNA polymerase and does not have capsid proteins to form intracellular virions. The genomic RNA has no 3' poly(A) tail and perhaps no cap structure at the 5'-end; thus resembling an intermediate of mRNA degradation. The virus, however, escapes the host surveillance and replicates in the yeast cytoplasm persistently. The viral genome is not naked but exists in the form of a ribonucleoprotein complex with p91 in a 1:1 stoichiometry. Here we investigated interactions between p91 and the viral genome. Our results indicate that p91 directly or indirectly interacts with the RNA at the 5'- and 3'-end regions and to a lesser extent at a central part. The 3'-end site is identical to or overlaps with the 3' cis signal for replication identified previously. The 5'-site is at the second stem loop structure from the 5'-end (nucleotides 72-104), and this structure also contains a cis signal for replication. Analysis of mutants in the structure revealed a tight correlation between replication and formation of complexes. These results highlight the importance of ribonucleoprotein complexes for the viral life cycle. We will discuss implications of these findings especially on how the virus escapes from mRNA degradation pathways and resides in the cytoplasm persistently despite the lack of a protective capsid.