Structures required for poly(A) tail-independent translation overlap with, but are distinct from, cap-independent translation and RNA replication signals at the 3' end of Tobacco necrosis virus RNA

Tobacco necrosis necrovirus (TNV) RNA lacks both a 5' cap and a poly(A) tail but is translated efficiently, owing in part to a Barley yellow dwarf virus (BYDV)-like cap-independent translation element (BTE) in its 3' untranslated region (UTR). Here, we identify sequence downstream of the BTE that is necessary for poly(A) tail-independent translation in vivo by using RNA encoding a luciferase reporter gene flanked by viral UTRs. Deletions and point mutations caused loss of translation that was restored by adding a poly(A) tail, and not by adding a 5' cap. The two 3'-proximal stem-loops in the viral genome contribute to poly(A) tail-independent translation, as well as RNA replication. For all necroviruses, we predict a conserved 3' UTR secondary structure that includes the BTE at one end of a long helical axis and the stem-loops required for poly(A) tail-independent translation and RNA replication at the other end. This work shows that a viral genome can harbor distinct cap- and poly(A) tail-mimic sequences in the 3' UTR.     

Inhibition of dengue virus translation and RNA synthesis by a morpholino oligomer targeted to the top of the terminal 3' stem-loop structure

Dengue virus (DEN) is a major public health problem worldwide and causes a spectrum of diseases, for which no antiviral treatments exist. Peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs) complementary to the DEN 5' stem-loop (5'SL) and to the DEN 3' cyclization sequence (3'CS) inhibit DEN replication, presumably by blocking critical RNA-RNA or RNA-protein interactions involved in viral translation and/or RNA synthesis. Here, a third P-PMO, complementary to the top of the 3' stem-loop (3'SLT), inhibited DEN replication in BHK cells. Using a novel DEN2 reporter replicon and a DEN2 reporter mRNA, we determined that the 5'SL P-PMO inhibited viral translation, the 3'CS P-PMO blocked viral RNA synthesis but not viral translation, and the 3'SLT P-PMO inhibited both viral translation and RNA synthesis. These results show that the 3'CS and the 3'SL domains regulate DEN translation and RNA synthesis and further demonstrate that P-PMOs are potentially useful as antiviral agents.     

Role of RNA structures present at the 3'UTR of dengue virus on translation, RNA synthesis, and viral replication

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.     

An infectious clone of the West Nile flavivirus

West Nile (WN) virus is the most widespread among flaviviruses, but until recently it was not known on the American continent. We describe here design of a subgenomic replicon, as well as a full-length infectious clone of the lineage II WN strain, which appeared surprisingly stable compared to other flavivirus infectious clones. This infectious clone was used to investigate effects of 5'- and 3'-nonrelated sequences on virus replication and infectivity of synthetic RNA. While a long nonrelated sequence at the 3'-end delayed but did not prevent establishment of the productive infectious cycle, a much shorter extra sequence at the 5'-end completely abrogated virus replication. Replacement of the conserved 5'-adenosine residue substantially delayed, but did not prevent, establishment of virus infection. In all cases, the recovered virus had restored its authentic 5'- and 3'-end genome sequences. However, the presence of extensive nonrelated sequences at both 5'- and 3'-ends could not be repaired.     

West Nile virus genome cyclization and RNA replication require two pairs of long-distance RNA interactions

West Nile virus (WNV) genome cyclization and replication require two pairs of long-distance RNA interactions. Besides the previously reported 5'CS/3'CSI (conserved sequence) interaction, a 5'UAR/3'UAR (upstream AUG region) interaction also contributes to genome cyclization and replication. WNVs containing mutant 5'UARs capable of forming the 5'/3' viral RNA interaction were replicative. In contrast, WNV containing a 5'UAR mutation that abolished the 5'/3' viral RNA interaction was non-replicative; however, the replication defect could be rescued by a single-nucleotide adaptation that restored the 5'/3' RNA interaction. The 5'UAR/3'UAR interaction is critical for RNA synthesis, but not for viral translation. Antisense oligomers targeting the 5'UAR/3'UAR interaction effectively inhibited WNV replication. Phylogenic analysis showed that the 3'UAR could alternate between pairing with the 5'UAR or with the 3' end of the flaviviral genome. Therefore, the 5'UAR/3'UAR pairing may release the 3' end of viral genome (as a template) during the initiation of minus-strand RNA synthesis.     

Cellular proteins bind to the poly(U) tract of the 3' untranslated region of hepatitis C virus RNA genome

UV cross-linking analyses were performed in an attempt to determine cellular protein-viral RNA interactions with the 3' untranslated region (3' UTR) of the hepatitis C virus RNA genome. Two cellular proteins, with estimated molecular masses of 58 kDa (p58) and 35 kDa (p35), respectively, were found to specifically bind to the 3' UTR. The p58 protein was determined to be the polypyrimidine tract-binding protein. In addition to binding to the conserved 98 nucleotides (nt) of the 3' UTR, p58 also binds to the poly(U) tract of the 3' UTR. The p35 protein was found to interact only with the poly(U) tract of the 3' UTR. These conclusions are supported by the following findings: (1) p58, and not p35, binds to the 3' end conserved 98 nt, (2) both p58 and p35 bind to a 3' UTR RNA with a deletion of the conserved 98 nt, (3) the 98-nt deletion mutant 3' UTR competed out both p58 and p35 binding, (4) a poly(U) homopolymer competed out both p58 and p35 binding, (5) a 3' UTR RNA with deletion of the poly(U) tract competed out only p58 binding but not p35 binding, and (6) an RNA containing the variable region of the 3' UTR with a deletion of both poly(U) tract and 98 nt failed to compete for binding of either p58 or p35. Interaction of these cellular proteins with the HCV 3' UTR is probably involved in regulation of translation and/or replication of the HCV RNA genome.     

Structure-function relationship in viral RNA genomes: The case of hepatitis C virus

The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus(HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5'-untranslatable regions(5'UTRs) and 3'UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5' terminus of the viral genome and regulated by distal functional RNA domains placed at the 3' end. Subsequent RNA replication strongly depends on the 3'UTR folding and is also influenced by the 5' end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNARNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.     

Functional analysis of the stem-loop structures at the 5' end of the Aichi virus genome

Aichi virus is a member of the family Picornaviridae. Computer-assisted secondary structure prediction suggested the formation of three stem-loop structures (SL-A, SL-B, and SL-C from the 5' end) within the 5'-end 120 nucleotides of the genome. We have already shown that the most 5'-end stem-loop, SL-A, is critical for viral RNA replication. Here, using an infectious cDNA clone and a replicon harboring a luciferase gene, we revealed that formation of SL-B and SL-C on the positive strand is essential for viral RNA replication. In addition, the specific nucleotide sequence of the loop segment of SL-B was also shown to be critical for viral RNA replication. Mutations of the upper and lower stems of SL-C that do not disrupt the base-pairings hardly affected RNA replication, but decreased the yields of viable viruses significantly compared with for the wild-type. This suggests that SL-C plays a role at some step besides RNA replication during virus infection.     

5' end-dependent translation initiation of hepatitis C viral RNA and the presence of putative positive and negative translational control elements within the 5' untranslated region.

Hepatitis C virus (HCV) is a distant relative of pestiviruses and flaviviruses, but it has a 5' untranslated region (UTR) with some features structurally similar to that of picornaviruses. In order to test the role of the 5' UTR in controlling the expression of the HCV polyprotein, we fused full-length or deleted versions of the 5' UTR of HCV-1 RNA to chloramphenicol acetyl transferase (CAT) mRNA to monitor CAT activity in vivo. We found: (1) the full-length 5' UTR of HCV-1 RNA is translationally inactive while 5' deletions which mimic a 5' subgenomic RNA detected in vivo are active, (2) an efficient cis-acting element which represses translation is found at the 5' terminus, (3) a putative element which enhances translation is found near the 3' terminus of the 5' UTR, (4) additional cis-acting elements including small open reading frames (ORFs) upstream from the putative enhancer element downregulate translation. We did not find evidence supporting the existence of an internal ribosome entry site in the 5' UTR of HCV-1 RNA. These data suggest that HCV may employ a distinctive translation control strategy such as the generation of subgenomic viral mRNA in infected cells. Translational control of HCV might be responsible for some of the characteristic pathobiology seen in viral infection.     

Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique beta-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and beta-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the beta-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR(345)), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416-8428).     

Core protein-mediated 5'-3' annealing of the West Nile virus genomic RNA in vitro

Genome cyclization through conserved RNA sequences located in the 5' and 3' terminal regions of flavivirus genomic RNA is essential for virus replication. Although the role of various cis-acting RNA elements in panhandle formation is well characterized, almost nothing is known about the potential contribution of protein cofactors to viral RNA cyclization. Proteins with nucleic acid chaperone activities are encoded by many viruses (e.g., retroviruses, coronaviruses) to facilitate RNA structural rearrangements and RNA-RNA interactions during the viral replicative cycle. Since the core protein of flaviviruses is also endowed with potent RNA chaperone activities, we decided to examine the effect of West Nile virus (WNV) core on 5'-3' genomic RNA annealing in vitro. Core protein binding resulted in a dramatic, dose-dependent increase in 5'-3' complex formation. Mutations introduced in either the UAR (upstream AUG region) or CS (conserved sequence) elements of the viral RNA diminished core protein-dependent annealing, while compensatory mutations restored the 5'-3' RNA interaction. The activity responsible for stimulating RNA annealing was mapped to the C-terminal RNA-binding region of WNV core protein. These results indicate that core protein - besides its function in viral particle formation - might be involved in the regulation of flavivirus genomic RNA cyclization, and thus virus replication.     

Classical swine fever virus NS5A regulates viral RNA replication through binding to NS5B and 3'UTR

In this report, classical swine fever virus (CSFV) NS5A inhibit viral RNA replication when its concentration reached and surpassed the level of NS5B. Three amino acid fragments of CSFV NS5A, 137-172, 224-268 and 390-414 individually were shown to be essential to NS5B binding. The former two fragments were independently necessary for regulation of viral RNA replication and correlated with NS5B and 3′UTR binding activity. We also found that amino acids W143, V145, P227, T246, P257, K399, T401, E406 and L413 of CSFV NS5A were essential to NS5B binding activity. Furthermore, these amino acids were shown to be necessary for viral RNA replication and infection and conserved in NS5A proteins of CSFV, BDV, BVDV and HCV. These results indicated that NS5A may regulate viral RNA replication by binding to NS5B and 3′UTR. NS5A can still regulate viral RNA synthesis through binding to 3′UTR when binding to NS5B is not available.     

Enhancement of dengue virus translation: role of the 3' untranslated region and the terminal 3' stem-loop domain

An essential step for a productive infection by the dengue flavivirus (DEN) is translation of the m(7)G-capped, nonpolyadenylated positive-sense RNA genome. We have recently identified sequences within the DEN 3' untranslated region (UTR) that modulate viral translation. Here, we show that the DEN type 2 (DEN2) 3'UTR stimulated translation of m(7)G-capped DEN2 5'UTR-containing reporter mRNAs in baby hamster kidney (BHK) cells compared to a 3' vector sequence. Analogous to the 3' poly(A) tail, the DEN2 3'UTR also enhanced translation of reporter mRNAs containing (i) a nonfunctional A cap, (ii) the 5'UTR of human beta-globin, or (iii) a viral internal ribosome entry site (IRES). In all cases, approximately half of the translation efficiency was due to the terminal 3' stem-loop (3'SL) domain. In addition, the 3'SL domain increased the association of mRNAs with polysomes. Together, these results indicate that the DEN2 3'UTR, mediated in part by the 3'SL domain, enhances translation initiation, possibly after recognition of the 5' cap structure.     

Conserved nucleotides in the terminus of the 3′ UTR region are important for the replication and infectivity of porcine reproductive and respiratory syndrome virus

The 3′ untranslated region (3′ UTR), including the poly (A) tail, reportedly plays an important role in arterivirus replication, but the roles of the cis-acting elements present in the 3′ UTR of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unknown. In the present study, PCR-based mutagenic analysis was conducted on the 3′ UTR of PRRSV infectious full-length cDNA clone pAPRRS to investigate the structure and function of the conserved terminal nucleotides between the poly (A) tail and the 3′ UTR region. Our findings indicated that the conservation of the primary sequence of the 3′ terminal nucleotides, rather than the surrounding secondary structure, was vital for viral replication and infectivity. Four nucleotides (nt) (5′-¹⁵⁵¹⁷AAUU¹⁵⁵²⁰-3′) at the 3′ proximal end of the 3′ UTR and the dinucleotide 5′-AU-3′ exerted an important regulatory effect on viral viability. Of the five 3′-terminal nucleotides of the 3′ UTR (5′-¹⁵⁵⁰³AACCA¹⁵⁵⁰⁷-3′), at least three, including the last dinucleotide (5′-CA-3′), were essential for maintaining viral infectivity. Taken together, the 3′-terminal conserved sequence plays a critical role in PRRSV replication and may function as a contact site for specific assembly of the replication complex.     

The 3' stem loop of the West Nile virus genomic RNA can suppress translation of chimeric mRNAs

Cis-acting elements that regulate translation have been identified in the 3' noncoding regions (NCRs) of cellular and viral mRNAs. As one means of analyzing the effect on translation of the conserved 3' terminal RNA structure of the West Nile virus (WNV) genome, the translation efficiencies of chimeric mRNAs composed of a CAT reporter gene flanked by viral or nonviral 5' and 3' terminal sequences were compared. In vitro, the WNV 3'(+) stem loop (SL) RNA reduced the translation efficiencies of chimeric mRNAs with either viral or nonviral 5' NCRs, suggesting that a specific 3'-5' RNA-RNA interaction was not involved. In contrast, the 3' terminal sequence of a togavirus, rubella virus, enhanced translation efficiency. The WNV 3'(+)SL reduced translation efficiency both in cis and in trans and of both capped and uncapped chimeric mRNAs. We have previously reported that three cellular proteins bind specifically to the WNV 3'(+)SL RNA. Competition between the WNV 3'(+)SL and the 5' terminus of the chimeric mRNAs for proteins involved in translation initiation could explain the translation inhibition observed.     

The conserved structures of the 5' nontranslated region of Citrus tristeza virus are involved in replication and virion assembly

The genomic RNA of different isolates of Citrus tristeza virus (CTV) reveals an unusual pattern of sequence diversity: the 3' halves are highly conserved (homology >90%), while the 5' halves show much more dissimilarity, with the 5' nontranslated region (NTR) containing the highest diversity (homology as low as 42%). Yet, positive-sense sequences of the 5' NTR were predicted to fold into nearly identical structures consisting of two stem-loops (SL1 and SL2) separated by a short spacer region. The predicted most stable secondary structures of the negative-sense sequences were more variable. We introduced mutations into the 5' NTR of a CTV replicon to alter the sequence and/or the predicted secondary structures with or without additional compensatory changes designed to restore predicted secondary structures, and examined their effect on replication in transfected protoplasts. The results suggested that the predicted secondary structures of the 5' NTR were more important for replication than the primary structure. Most mutations that were predicted to disrupt the secondary structures fail to replicate, while compensatory mutations were allowed replication to resume. The 5' NTR mutations that were tolerated by the CTV replicon were examined in the full-length virus for effects on replication and production of the multiple subgenomic RNAs. Additionally, the ability of these mutants to produce virions was monitored by electron microscopy and by passaging the progeny nucleocapsids to another batch of protoplasts. Some of the mutants with compensatory sequence alterations predicted to rebuild similar secondary structures allowed replication at near wild-type levels but failed to passage, suggesting that the 5' NTR contains sequences required for both replication and virion assembly.