Depurination within the intergenic region of Brome mosaic virus RNA3 inhibits viral replication in vitro and in vivo

Pokeweed antiviral protein (PAP) is a glycosidase of plant origin that has been shown to depurinate some viral RNAs in vitro. We have demonstrated previously that treatment of Brome mosaic virus (BMV) RNAs with PAP inhibited their translation in a cell-free system and decreased their accumulation in barley protoplasts. In the current study, we map the depurination sites on BMV RNA3 and describe the mechanism by which replication of the viral RNA is inhibited by depurination. Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product. This decrease was due to depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3′ terminal core promoter required for initiation of negative-strand RNA synthesis. Depurination within the intergenic region alone inhibited the binding of the replicase to full-length RNA3, whereas depurination outside the intergenic region permitted the replicase to initiate negative-strand synthesis; however, elongation of the RNA product was stalled at the abasic nucleotide. These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of depurination by PAP on BMV replication.     

Cap-independent translation mechanism of red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication

The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5' end and no poly(A) tail at the 3' end. The 3' untranslated region (3' UTR) of RCNMV RNA1 contains an essential RNA element (3'TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3'TE-DR1 of RNA1. However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5' UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.     

Template recognition mechanisms by replicase proteins differ between bipartite positive-strand genomic RNAs of a plant virus

Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3' untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans. The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism.     

Poly(A)-binding protein facilitates translation of an uncapped/nonpolyadenylated viral RNA by binding to the 3' untranslated region

Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.     

In Vitro and In Vivo Studies of the RNA Conformational Switch in Alfalfa Mosaic Virus

The 3′ termini of Alfalfa mosaic virus (AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme) in vitro is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3′ UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the in vitro and in vivo data clearly demonstrate the existence of the conformational switch in the 3′ UTR of AMV RNAs.Alfalfa mosaic virus (AMV) is a plant virus that belongs to one of the five genera in the family Bromoviridae, whose genomes consist of three genomic RNAs (RNAs 1, 2, and 3) and one subgenomic RNA (RNA4) that are capped at the 5′ end and lack polyadenylation at the 3′ terminus (3). RNAs 1 and 2 encode the viral subunits P1 and P2 of the replicase, respectively. RNA3 encodes the movement protein and serves as a template for the synthesis of RNA4, which encodes the coat protein (CP).The role of AMV CP has been the subject of extensive research in the past four decades. Initially, it was found that, in contrast to RNAs of the Bromo-, Cucumo-, and Oleavirus genera, the genomic RNAs of AMV and the closely related genus Ilarvirus were not infectious as such but required the presence of CP in the inoculum (15). This phenomenon was called genome activation and was long considered to compensate for the lack of a tRNA-like structure (TLS) at the 3′ end of their genomic RNAs, a prominent feature of bromo- and cucumovirus RNAs (3). However, in 1999 we demonstrated that the 3′ end of AMV RNAs can adopt an alternative conformation that shows many structural similarities to the TLS of other Bromoviridae, although it could not be charged with an amino acid (20). The tRNA-like (TL) conformation (Fig. ​(Fig.1)1) turned out to be the replicative form of the 3′ termini (19, 20), whereas the other, coat protein binding (CPB), conformer was subsequently shown to be required for translation (16-18). Although other models have been forwarded (9), we have proposed that switching between these two conformations, mediated by CP binding, plays a fundamental role in the life cycle of AMV and ilarviruses by regulating the competing processes of translation and replication of the viral RNAs.Open in a separate windowFIG. 1.The CPB and the TL conformations of the AMV RNA3 3′ terminus. The two conformers of AMV RNA3 3′ 145 nt are shown. (A) CPB conformer. The two major CP binding sites are indicated by brackets. Base pairing between loop D and stem A promotes TL conformation. (B) Secondary structure of the TL conformer.In the present study, the distribution between CPB and TL conformers was further investigated. We addressed how changes in this distribution would affect recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding ezyme) and the viral polymerase in vitro. We also monitored how the binding affinity to CP is affected by this conformational switch in vivo using the yeast three-hybrid (Y3H) system (2, 11, 24). Together, the in vitro and in vivo data clearly demonstrate the existence and function of the conformational switch in the 3′ UTR of AMV RNAs.     

IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3′UTR

The positive-strand RNA genome of the Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) in the 5′untranslated region (5′UTR) and structured sequence elements within the 3′UTR, but no poly(A) tail. Employing a limited set of initiation factors, the HCV IRES coordinates the 5′cap-independent assembly of the 43S pre-initiation complex at an internal initiation codon located in the IRES sequence. We have established a Huh7 cell-derived in vitro translation system that shows a 3′UTR-dependent enhancement of 43S pre-initiation complex formation at the HCV IRES. Through the use of tobramycin (Tob)-aptamer affinity chromatography, we identified the Insulin-like growth factor-II mRNA-binding protein 1 (IGF2BP1) as a factor that interacts with both, the HCV 5′UTR and 3′UTR. We report that IGF2BP1 specifically enhances translation at the HCV IRES, but it does not affect 5′cap-dependent translation. RNA interference against IGF2BP1 in HCV replicon RNA-containing Huh7 cells reduces HCV IRES-mediated translation, whereas replication remains unaffected. Interestingly, we found that endogenous IGF2BP1 specifically co-immunoprecipitates with HCV replicon RNA, the ribosomal 40S subunit, and eIF3. Furthermore eIF3 comigrates with IGF2BP1 in 80S ribosomal complexes when a reporter mRNA bearing both the HCV 5′UTR and HCV 3′UTR is translated. Our data suggest that IGF2BP1, by binding to the HCV 5′UTR and/or HCV 3′UTR, recruits eIF3 and enhances HCV IRES-mediated translation.     

lsm1 mutations impairing the ability of the Lsm1p-7p-Pat1p complex to preferentially bind to oligoadenylated RNA affect mRNA decay in vivo

The poly(A) tail is a crucial determinant in the control of both mRNA translation and decay. Poly(A) tail length dictates the triggering of the degradation of the message body in the major 5′ to 3′ and 3′ to 5′ mRNA decay pathways of eukaryotes. In the 5′ to 3′ pathway oligoadenylated but not polyadenylated mRNAs are selectively decapped in vivo, allowing their subsequent degradation by 5′ to 3′ exonucleolysis. The conserved Lsm1p-7p-Pat1p complex is required for normal rates of decapping in vivo, and the purified complex exhibits strong binding preference for oligoadenylated RNAs over polyadenylated or unadenylated RNAs in vitro. In the present study, we show that two lsm1 mutants produce mutant complexes that fail to exhibit such higher affinity for oligoadenylated RNA in vitro. Interestingly, these mutant complexes are normal with regard to their integrity and retain the characteristic RNA binding properties of the wild-type complex, namely, binding near the 3′-end of the RNA, having higher affinity for unadenylated RNAs that carry U-tracts near the 3′-end over those that do not and exhibiting similar affinities for unadenylated and polyadenylated RNAs. Yet, these lsm1 mutants exhibit a strong mRNA decay defect in vivo. These results underscore the importance of Lsm1p-7p-Pat1p complex–mRNA interaction for mRNA decay in vivo and imply that the oligo(A) tail mediated enhancement of such interaction is crucial in that process.     

The 3′-Untranslated Region of Hepatitis C Virus RNA Enhances Translation from an Internal Ribosomal Entry Site

Translation of most eukaryotic mRNAs and many viral RNAs is enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a positive-stranded RNA genome which does not have a poly(A) tail but has a stretch of 98 nucleotides (X region) at the 3′-untranslated region (UTR), which assumes a highly conserved stem-loop structure. This X region binds a polypyrimidine tract-binding protein (PTB), which also binds to the internal ribosome entry site (IRES) in HCV 5′-UTR. These RNA-protein interactions may regulate its translation. We generated a set of HCV RNAs differing only in their 3′-UTRs and compared their translation efficiencies. HCV RNA containing the X region was translated three- to fivefold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, enhancement in translation. The X region also enhanced translation from another unrelated IRES (from encephalomyocarditis virus RNA), but did not affect the 5′-end-dependent translation of globin mRNA in either monocistronic or bicistronic RNAs. It did not appear to affect RNA stability. The free X region added in trans, however, did not enhance translation, indicating that the translational enhancement by the X region occurs only in cis. These results demonstrate that the highly conserved 3′ end of HCV RNA provides a novel mechanism for enhancement of HCV translation and may offer a target for antiviral agents.     

Conservation of RNA structures enables TNV and BYDV 5' and 3' elements to cooperate synergistically in cap-independent translation

The subgenomic RNA 2 of tobacco necrosis virus A (TNV sgRNA2) encodes the viral coat protein, is unpolyadenylated and presumably uncapped. Here, we show that TNV sgRNA2 is translated cap independently. This cap-independent translation requires the leader and a 140 nt element of the trailer both in wheat germ extract and in tobacco protoplasts. Similar to barley yellow dwarf virus (BYDV), the TNV 5′ and 3′ elements stimulate translation synergistically. Computer-aided phylogenetic analysis of the secondary structure of the TNV trailer revealed that the 3′ translation element is part of a major conserved stem–loop that contains similarities to structures in the BYDV 3′ translation element. These data suggest that the translation mechanisms of TNV sgRNA2 and BYDV RNA are related. To further characterize this relationship, we tested whether cooperativity exists between TNV sgRNA2 and BYDV 5′ and 3′ elements. We found that the TNV sgRNA2 5′ element stimulates translation synergistically with the BYDV 3′ element in vitro. This finding is the first evidence for conservation of structures that enable a 5′–3′ interaction stimulating cap-independent translation.     

Hsp90 Interacts Specifically with Viral RNA and Differentially Regulates Replication Initiation of Bamboo mosaic virus and Associated Satellite RNA

Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3′ untranslated region (3′ UTR) of BaMV genomic RNA, but not with the 3′ UTR of BaMV-associated satellite RNA (satBaMV RNA) or that of genomic RNA of other viruses, such as Potato virus X (PVX) or Cucumber mosaic virus (CMV). Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3′ UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3′ UTR of BaMV RNA during the initiation of BaMV RNA replication.     

Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m⁷GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m⁷GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features.     

Differential Roles of Hsp70 and Hsp90 in the Assembly of the Replicase Complex of a Positive-Strand RNA Plant Virus

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.     

Hepatitis C virus 3′UTR regulates viral translation through direct interactions with the host translation machinery

The 3′ untranslated region (3′UTR) of hepatitis C virus (HCV) messenger RNA stimulates viral translation by an undetermined mechanism. We identified a high affinity interaction, conserved among different HCV genotypes, between the HCV 3′UTR and the host ribosome. The 3′UTR interacts with 40S ribosomal subunit proteins residing primarily in a localized region on the 40S solvent-accessible surface near the messenger RNA entry and exit sites. This region partially overlaps with the site where the HCV internal ribosome entry site was found to bind, with the internal ribosome entry site-40S subunit interaction being dominant. Despite its ability to bind to 40S subunits independently, the HCV 3′UTR only stimulates translation in cis, without affecting the first round translation rate. These observations support a model in which the HCV 3′UTR retains ribosome complexes during translation termination to facilitate efficient initiation of subsequent rounds of translation.     

mRNAs containing the histone 3′ stem–loop are degraded primarily by decapping mediated by oligouridylation of the 3′ end

Metazoan replication-dependent histone mRNAs are only present in S-phase, due partly to changes in their stability. These mRNAs end in a unique stem–loop (SL) that is required for both translation and cell-cycle regulation. Previous studies showed that histone mRNA degradation occurs through both 5′→3′ and 3′→5′ processes, but the relative contributions are not known. The 3′ end of histone mRNA is oligouridylated during its degradation, although it is not known whether this is an essential step. We introduced firefly luciferase reporter mRNAs containing the histone 3′ UTR SL (Luc-SL) and either a normal or hDcp2-resistant cap into S-phase HeLa cells. Both mRNAs were translated, and translation initially protected the mRNAs from degradation, but there was a lag of ∼40 min with the uncleavable cap compared to ∼8 min for the normal cap before rapid decay. Knockdown of hDcp2 resulted in a similar longer lag for Luc-SL containing a normal cap, indicating that 5′→3′ decay is important in this system. Inhibition of DNA replication with hydroxyurea accelerated the degradation of Luc-SL. Knockdown of terminal uridyltransferase (TUTase) 4 but not TUTase 3 slowed the decay process, but TUTase 4 knockdown had no effect on destabilization of the mRNA by hydroxyurea. Both Luc-SL and its 5′ decay intermediates were oligouridylated. Preventing oligouridylation by 3′-deoxyadenosine (cordycepin) addition to the mRNA slowed degradation, in the presence or absence of hydroxyurea, suggesting oligouridylation initiates degradation. The spectrum of oligouridylated fragments suggests the 3′→5′ degradation machinery stalls during initial degradation, whereupon reuridylation occurs.