An RNA Element at the 5′-End of the Poliovirus Genome Functions as a General Promoter for RNA Synthesis

RNA structures present throughout RNA virus genomes serve as scaffolds to organize multiple factors involved in the initiation of RNA synthesis. Several of these RNA elements play multiple roles in the RNA replication pathway. An RNA structure formed around the 5′- end of the poliovirus genomic RNA has been implicated in the initiation of both negative- and positive-strand RNA synthesis. Dissecting the roles of these multifunctional elements is usually hindered by the interdependent nature of the viral replication processes and often pleiotropic effects of mutations. Here, we describe a novel approach to examine RNA elements with multiple roles. Our approach relies on the duplication of the RNA structure so that one copy is dedicated to the initiation of negative-strand RNA synthesis, while the other mediates positive-strand synthesis. This allows us to study the function of the element in promoting positive-strand RNA synthesis, independently of its function in negative-strand initiation. Using this approach, we demonstrate that the entire 5′-end RNA structure that forms on the positive-strand is required for initiation of new positive-strand RNAs. Also required to initiate positive-strand RNA synthesis are the binding sites for the viral polymerase precursor, 3CD, and the host factor, PCBP. Furthermore, we identify specific nucleotide sequences within “stem a” that are essential for the initiation of positive-strand RNA synthesis. These findings provide direct evidence for a trans-initiation model, in which binding of proteins to internal sequences of a pre-existing positive-strand RNA affects the synthesis of subsequent copies of that RNA, most likely by organizing replication factors around the initiation site.     

Interplay of RNA Elements in the Dengue Virus 5′ and 3′ Ends Required for Viral RNA Replication

Dengue virus (DENV) is a member of the Flavivirus genus of positive-sense RNA viruses. DENV RNA replication requires cyclization of the viral genome mediated by two pairs of complementary sequences in the 5′ and 3′ ends, designated 5′ and 3′ cyclization sequences (5′-3′ CS) and the 5′ and 3′ upstream of AUG region (5′-3′ UAR). Here, we demonstrate that another stretch of six nucleotides in the 5′ end is involved in DENV replication and possibly genome cyclization. This new sequence is located downstream of the AUG, designated the 5′ downstream AUG region (5′ DAR); the motif predicted to be complementary in the 3′ end is termed the 3′ DAR. In addition to the UAR, CS and DAR motifs, two other RNA elements are located at the 5′ end of the viral RNA: the 5′ stem-loop A (5′ SLA) interacts with the viral RNA-dependent RNA polymerase and promotes RNA synthesis, and a stem-loop in the coding region named cHP is involved in translation start site selection as well as RNA replication. We analyzed the interplay of these 5′ RNA elements in relation to RNA replication, and our data indicate that two separate functional units are formed; one consists of the SLA, and the other includes the UAR, DAR, cHP, and CS elements. The SLA must be located at the 5′ end of the genome, whereas the position of the second unit is more flexible. We also show that the UAR, DAR, cHP, and CS must act in concert and therefore likely function together to form the tertiary RNA structure of the circularized DENV genome.Dengue virus (DENV), a member of the Flaviviridae family, is a human pathogen causing dengue fever, the most common mosquito-borne viral disease in humans. The virus has become a major international public health concern, with 3 billion people at risk for infection and an estimated 50 million dengue cases worldwide every year (28). Neither specific antiviral therapies nor licensed vaccines are available, and the biology of the virus is poorly understood.DENV is a small enveloped virus containing a positive-stranded RNA genome with a length of approximately 10.7 kb. The virus encodes one large polyprotein that is co- and posttranslationally cleaved into 10 viral proteins. The structural proteins C, prM/M, and E are located in the N terminus, followed by the nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (6, 10). NS5, the largest of the viral proteins, functions as an RNA-dependent RNA polymerase (RdRP) (29). The coding region is flanked at both ends by untranslated regions (UTR). The 5′ end has a type I cap structure (m⁷GpppAmp) mediating cap-dependent translation, but the virus can switch to a noncanonical translation mechanism under conditions in which translation factors are limiting (13). Cellular mRNAs are known to circularize via a protein-protein bridge between eIF4G and eIF4E (the cap binding complex) at the 5′ end and the poly(A) binding protein (PABP) at the 3′ end, enhancing translation efficiency. Despite the fact that the DENV 3′ UTR lacks a poly(A) tail, recent findings demonstrated binding of PABP to the 3′ UTR and an effect on RNA translation, suggesting a similar mechanism (12, 26).In addition to a presumed protein-mediated genome circularization regulating viral translation, an RNA-RNA-based 5′ and 3′ (5′-3′) end interaction, which can occur in the absence of proteins, leads to circularization of the viral genome (1, 3, 4, 18, 20, 30, 33, 34). This cyclization of the genome is necessary for viral RNA replication, and thus far, two complementary sequences at the 5′ and 3′ ends have been identified (3). The first are the cyclization sequences (CS) present in the capsid-coding region at the 5′ end (5′ CS) and upstream of the 3′ stem-loop (3′ SL) in the 3′ UTR (3′ CS) (2, 4, 18, 20, 30). A second sequence, known as the 5′ upstream AUG region (5′ UAR) element in the 5′ UTR, base pairs with its complementary 3′ UAR counterpart, which is located at the bottom part of 3′ SL (1, 4, 30). Recently, the structure of the 5′ end of the DENV genome hybridized to the 3′ end was determined in solution (25), confirming previous computer-predicted structures for genome cyclization (4, 20, 30). Besides the base pairing between 5′-3′ UAR and 5′-3′ CS sequences, a third stretch of nucleotides was identified to form a double-stranded (ds) region between the 5′ and 3′ ends.In addition to RNA sequences involved in 5′-3′-end interactions that are necessary for cyclization, the 5′ end of the viral genome harbors at least two more functional RNA elements, the stem-loop A (SLA) and capsid-coding region hairpin (cHP). The SLA consists of the first 70 nucleotides (nt) of the genome, forming a stable stem-loop structure. This structure has been confirmed in several studies and identified as a promoter element for RNA synthesis that recruits the viral RdRp NS5 (16, 22). Once NS5 is bound to the SLA at the 5′ end, it is believed to be delivered to the initiation site of minus-strand RNA synthesis at the 3′ end via 5′-3′ RNA-RNA circularization. In addition, a short poly(U) tract located immediately downstream of SLA has been shown to be necessary for RNA synthesis, although it is not involved in genome circularization (22). Finally, the cHP element resides within the capsid-coding region; it directs start codon selection and is essential for RNA replication (8, 9). The cHP structure is more important than its primary sequence. For start codon selection, it is believed that the cHP stalls the scanning initiation complex over the first AUG, favoring its recognition (9). In the case of RNA replication, the cHP likely stabilizes the overall 5′-3′ panhandle structure or participates in recruitment of factors associated with the replicase machinery (8).In this study, we demonstrate that in addition to the 5′ CS and 5′ UAR sequences, a third stretch of nucleotides in the 5′ end is required for RNA replication and appears to be involved in genome circularization. This new motif is located downstream of the AUG and was therefore designated the downstream AUG region (5′ DAR) element, with the predicted counterpart in the 3′ end designated the 3′ DAR. Our results indicate that the 5′ DAR modulates RNA-RNA interaction and RNA replication, and restoring complementarity between the 5′ DAR and 3′ DAR rescues detrimental effects caused by mutations in the 5′ DAR on genome circularization and RNA replication. Although the role of the predicted 3′ DAR counterpart is less conclusive, it may serve to make other structures and sequences in the 3′ end available for 5′-3′ RNA-RNA interaction to facilitate the replication-competent conformation of the DENV genome.Furthermore, we analyzed the functional interplay of RNA elements in the viral 5′ end, showing that two separate units are formed during replication. The first consists of the SLA, and it must be located at the very 5′ end of the genome. The second unit includes UAR, DAR, cHP, and CS elements, and the positional requirements are more flexible within the DENV RNA 5′ terminus. However, all four elements in the second unit must act in concert, forming a functional tertiary RNA structure of the circularized viral genome.     

Functional Analysis of RNA Structures Present at the 3′ Extremity of the Murine Norovirus Genome: the Variable Polypyrimidine Tract Plays a Role in Viral Virulence

Interactions of host cell factors with RNA sequences and structures in the genomes of positive-strand RNA viruses play various roles in the life cycles of these viruses. Our understanding of the functional RNA elements present in norovirus genomes to date has been limited largely to in vitro analysis. However, we recently used reverse genetics to identify evolutionarily conserved RNA structures and sequences required for norovirus replication. We have now undertaken a more detailed analysis of RNA structures present at the 3′ extremity of the murine norovirus (MNV) genome. Biochemical data indicate the presence of three stable stem-loops, including two in the untranslated region, and a single-stranded polypyrimidine tract [p(Y)] of variable length between MNV isolates, within the terminal stem-loop structure. The well-characterized host cell pyrimidine binding proteins PTB and PCBP bound the 3′-untranslated region via an interaction with this variable sequence. Viruses lacking the p(Y) tract were viable both in cell culture and upon mouse infection, demonstrating that this interaction was not essential for virus replication. However, competition analysis with wild-type MNV in cell culture indicated that the loss of the p(Y) tract was associated with a fitness cost. Furthermore, a p(Y)-deleted mutant showed a reduction in virulence in the STAT1^−/− mouse model, highlighting the role of RNA structures in norovirus pathogenesis. This work highlights how, like with other positive-strand RNA viruses, RNA structures present at the termini of the norovirus genome play important roles in virus replication and virulence.Noroviruses are now well established as the leading cause of nonbacterial gastroenteritis in the developed world (reviewed in references 31 and 34), but recent studies have also demonstrated links with more significant clinical disease, e.g., exacerbation of inflammatory bowel disease (20), an outbreak of necrotizing enterocolitis (41), and the induction of seizures in neonates (8). The understanding of norovirus translation, replication, and virulence has lagged behind that for other viruses due to the inability to propagate human noroviruses efficiently in tissue culture. Recent advances in the field have led to the generation of norovirus replicons (5, 6), the demonstration that human norovirus RNA is infectious in tissue culture (13), and an unconfirmed report that a highly differentiated cell culture system can be infected with human noroviruses (38). Although many caliciviruses have been used as models for the study of human noroviruses (reviewed in reference 42), the identification of murine norovirus (MNV) in 2003 (19) led to unprecedented advances in the analysis of many aspects of norovirus biology, as to date this is the only norovirus which replicates efficiently in tissue culture. In addition, the recent development of reverse genetic systems for murine norovirus (7, 44) has allowed the identification of a virulence determinant in the major capsid protein (1) and the first identification of functional RNA sequences/structures in the norovirus genome (35).The murine norovirus genome carries four potential open reading frames (ORFs) (Fig. ​(Fig.1A).1A). ORF1 encodes a large polyprotein which is posttranslationally cleaved by the virus-encoded protease (NS6) into several proteins which are involved in various aspects of genome replication (37). ORF2 and ORF3 code for the major and minor capsid proteins VP1 and VP2, respectively. In addition, the MNV genome is also known to contain a fourth potential ORF (Fig. ​(Fig.1A)1A) (ORF4), which is highly conserved between numerous strains (40) and whose expression and function have yet to be examined.Open in a separate windowFIG. 1.The murine norovirus genome contains three 3′-terminal stem-loop RNA structures. (A) Schematic representation of murine norovirus genome, highlighting the four predicted open reading frames and the mature replicase proteins produced from ORF1. (B) Predicted RNA secondary structure of the 3′ end of the MNV genome. The positions of the RNase cleavage sites, as determined by limited RNase digestion followed by primer extension, are highlighted on the bioinformatically predicted structure for the MNV 3′ end. The genetic sequence variation of 38 published murine norovirus 3′-end sequences is also provided, highlighting those bases which either are invariant, show signs of covariation, vary but maintain base pairing, or vary without maintaining base pairing. ClustalW analysis was performed using all available murine norovirus sequences in the NCBI database (details available on request). The stop codon of the VP2 coding sequence in SL1 is also highlighted. (C to F) In vitro-transcribed RNA encompassing SL1, -2, and -3, with a poly(A) tail of 27 nucleotides in length, was subjected to limited RNase digestion with dilutions of RNases and to subsequent primer extension analysis. A sequencing ladder obtained using the same primer allows the identification of the RNase cleavage sites. Analysis was performed a minimum of three times, and one representative gel is shown. Nucleotide positions are numbered according to their positions in the murine norovirus genome. Note that data are shown for regions containing RNase cleavage sites only.Due to the limited coding capacity of their genomes, small positive-strand RNA viruses rely heavily on host cell nucleic acid binding proteins for efficient genome translation and replication (reviewed in reference 22). These host cell factors often play many roles in the virus life cycle by interacting with specific RNA sequences and/or structures present within the viral genome. Such factors are also a major determinant of viral tropism, as their relative expression levels may determine the efficiency with which a virus can replicate in a particular tissue and, as a result, cause disease. Previous work with human norovirus has identified several host factors interacting with the 5′ and 3′ ends of the viral genome (15, 16). Although a recent study clearly indicated that some cellular factors are potentially involved in norovirus genome circularization (32), the identities of these and the functions of the known binding proteins have yet to be determined.Our previous studies using computational analysis resulted in the identification of several highly conserved RNA structures at various positions in the MNV genome (35). Furthermore, preliminary analysis using reverse genetics demonstrated that these RNA structures played an important role in unknown aspects of the MNV life cycle. We have now extended this analysis to perform a more detailed characterization of the RNA structures present at the 3′ end of the MNV genome, identifying sequences and structures important for replication in tissue culture. We have also demonstrated that a polypyrimidine-rich [p(Y)] tract, present as a single-stranded terminal loop, is nonessential for virus replication in tissue culture but contributes to virulence in the STAT1^−/− mouse model of MNV pathogenesis. This is the first report of RNA structures playing a role in the virulence of any member of the Caliciviridae family.     

Synthesis and Activity of Oligonucleotides Containing a Biologically Active Nucleoside at the 2′End

Abstract

The antiviral activity of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdUrd), acyclovir and other antiherpetic nucleosides depends on a selective phosphorylation by the herpesvirus-induced thymidine kinase in the infected cells. Viruses not encoding a specific thymidine kinase (TK) activity are resistant to the action of these nucleoside analogues. The nucleoside monophosphates are as such poorly taken up by the cells. In order to circumvent the necessity of intracellular phosphorylation, we synthesized four core oligonucleotides bearing a biological active nucleoside at the 2′end. It was hypothesized that these core oligonucleotides, like core 2–5A itself, would be taken up within the cell and that, following intracellular 2′-5′ phosphodiesterase cleavage, the 5′-monophosphate of the active product would be formed. In these circumstances, activity against TK- strains could be expected.     

Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3′-End

Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus Enterovirus, which includes more than 150 human rhinovirus (HRV) serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3′-end. This suggests that packaging also occurs in an ordered manner resulting in the 3′-poly-(A) tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses.     

A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

The 5′ leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.     

The Structure of Bacteriophage φCb5 Reveals a Role of the RNA Genome and Metal Ions in Particle Stability and Assembly

The structure of the Leviviridae bacteriophage φCb5 virus-like particle has been determined at 2.9 Å resolution and the structure of the native bacteriophage φCb5 at 3.6 Å. The structures of the coat protein shell appear to be identical, while differences are found in the organization of the density corresponding to the RNA. The capsid is built of coat protein dimers and in shape corresponds to a truncated icosahedron with T = 3 quasi-symmetry. The capsid is stabilized by four calcium ions per icosahedral asymmetric unit. One is located at the symmetry axis relating the quasi-3-fold related subunits and is part of an elaborate network of hydrogen bonds stabilizing the interface. The remaining calcium ions stabilize the contacts within the coat protein dimer. The stability of the φCb5 particles decreases when calcium ions are chelated with EDTA. In contrast to other leviviruses, φCb5 particles are destabilized in solution with elevated salt concentration. The model of the φCb5 capsid provides an explanation of the salt-induced destabilization of φCb5, since hydrogen bonds, salt bridges and calcium ions have important roles in the intersubunit interactions.Electron density of three putative RNA nucleotides per icosahedral asymmetric unit has been observed in the φCb5 structure. The nucleotides mediate contacts between the two subunits forming a dimer and a third subunit in another dimer. We suggest a model for φCb5 capsid assembly in which addition of coat protein dimers to the forming capsid is facilitated by interaction with the RNA genome. The φCb5 structure is the first example in the levivirus family that provides insight into the mechanism by which the genome-coat protein interaction may accelerate the capsid assembly and increase capsid stability.     

Incoming RNA Virus Nucleocapsids Containing a 5′-Triphosphorylated Genome Activate RIG-I and Antiviral Signaling

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Highlights? RIG-I is activated by the incoming RNA virus nucleocapsids during infection ? RIG-I activation requires a 5′triphosphate dsRNA structure on the nucleocapsids ? Viral nucleocapsids trigger conformational switching and oligomerization of RIG-I ? RIG-I directly binds to viral nucleocapsids containing a 5′triphosphate dsRNA structure     

Common Sequence at the 5′ Ends of the Segmented RNA Genomes of Influenza A and B Viruses

Guanylyl- and methyltransferases, isolated from purified vaccinia virus, were used to specifically label the 5′ ends of the genome RNAs of influenza A and B viruses. All eight segments were labeled with [α-³²P]guanosine 5′-triphosphate or S-adenosyl[methyl-³H]methionine to form “cap” structures of the type m⁷G(5′)pppN^m-, of which unmethylated (p)ppN- represents the original 5′ end. Further analyses indicated that m⁷G(5′)pppA^m, m⁷G(5′)pppA^mpGp, and m⁷G(5′)pppA^mpGpUp were released from total and individual labeled RNA segments by digestion with nuclease P1, RNase T1, and RNase A, respectively. Consequently, the 5′-terminal sequences of most or all individual genome RNAs of influenza A and B viruses were deduced to be (p)ppApGpUp. The presence of identical sequences at the ends of RNA segments of both types of influenza viruses indicates that they have been specifically conserved during evolution.     

Recombination in Enteroviruses Is a Biphasic Replicative Process Involving the Generation of Greater-than Genome Length ‘Imprecise’ Intermediates

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of ‘evolution by duplication’ in the positive-strand RNA viruses.     

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 5′ UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 3′ UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 3′ end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 3′ end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA–RNA interaction between the 5′ and 3′ ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 5′–3′ end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.     

Deuteration and Tritiation of 2′-Deoxyribonucleosides in the 5′ and 4′ Positions

Abstract

The deuterations of 2′-deoxyguanosine in the 4′ and 5′ positions have been described elsewhere (1). The starting material is the 5′-aldehyde formed by mild oxidation with N,N-dicyclohexyl carbodiimide in dimethyl sulphoxide of the fully protected nucleoside with free 5′-alcoholic function. The 5′4euteration was achieved by reduction with deuterated sodium borohydride. Incorporation of deuterium in the 4′-position was achieved v i a an enhanced keto-enol tautomerim by heating the aldehyde in 50/50 D20/pyridine, with subsequent reduction of the aldehyde with NaBH4. The 6-furanoid form was isolated from the I-lyxo by-product by reverse phase HPLC. Applied to pyrimidine 2′-deoxyribonucleosides, this method was shown to give deuterated 2′-deoxycytidine and thymidine in good yield.