Coat Protein Binding Research Articles

Using fluorescent proteins to analyze gene expression in real-time

We developed a system using fluorescent proteins to analyze gene expression in real-time, and to follow individual mRNPs. An array of genes coding for a functional mRNA that contains 24 repeats of the MS2 coat protein binding motif combined with the MS2 coat protein fused to GFP or YFP allowed us to analyze the kinetics of transcription in real time and to detect single molecules of RNA in live cells. In these studies we used photo-bleaching of GFP-labeled mRNAs and of a YFP-polII fusion protein and photoactivation of paGFP labeled mRNA. Analysis of the complex process of transcription using fluorescent polymerase as well as fluorescent MS2 proteins provided an opportunity to model the kinetic steps of RNA synthesis. These results yield rate constants for each of the steps of promoter assembly, initiation and elongation. They demonstrate that transcription is inefficient and that polymerases can elongate faster than thought, but can pause stochastically. We have now inserted the stem-loops into the endogenous β-actin gene of mice and can follow transcription and mRNA mobility from this locus. RNAs can then be followed as single molecules in the nucleus and cytoplasm. The analysis demonstrated that subsequent RNA movements were not directed, but governed by rules of simple diffusion. The kinetics of their transport through nuclear pores yielded a biexponential kinetics. Once in the cytoplasm, mRNAs diffuse but also can be directed to their destinations by virtue of a zipcode motif in the RNA. A zipcode binding protein (ZBP1) is essential for this localization and inhibition of translation. β-actin mRNA and newly synthesized protein can thusly be localized to sites of active F-actin polymerization in migrating fibroblasts or growing neurons. Supported by NIH-EB, GM.Key Words: single molecule imaging, RNA, regulation

Combined Use of MS2 and PP7 Coat Fusions Shows that TIA‐1 Dominates hnRNP A1 for K‐SAM Exon Splicing Control

Splicing of the FGFR2 K-SAM exon is repressed by hnRNP A1 bound to the exon and activated by TIA-1 bound to the downstream intron. Both proteins are expressed similarly by cells whether they splice the exon or not, so it is important to know which one is dominant. To answer this question, we used bacteriophage PP7 and bacteriophage MS2 coat fusions to tether hnRNP A1 and TIA-1 to distinct sites on the same pre-mRNA molecule. hnRNP A1 fused to one coat protein was tethered to a K-SAM exon containing the corresponding coat protein's binding site. TIA-1 fused to the other coat protein was tethered to the downstream intron containing that coat protein's binding site. This led to efficient K-SAM exon splicing. Our results show that TIA-1 is dominant for K-SAM exon splicing control and validate the combined use of PP7 and MS2 coat proteins for studying posttranscriptional events.

HRT-Mediated Hypersensitive Response and Resistance to Turnip crinkle virus in Arabidopsis Does Not Require the Function of TIP, the Presumed Guardee Protein

The Arabidopsis resistance protein HRT recognizes the Turnip crinkle virus (TCV) coat protein (CP) to induce a hypersensitive response (HR) in the resistant ecotype Di-17. The CP also interacts with a nuclear-targeted NAC family of host transcription factors, designated TIP (TCV-interacting protein). Because binding of CP to TIP prevents nuclear localization of TIP, it has been proposed that TIP serves as a guardee for HRT. Here, we have tested the requirement for TIP in HRT-mediated HR and resistance by analyzing plants carrying knockout mutation in the TIP gene. Our results show that loss of TIP does not alter HR or resistance to TCV. Furthermore, the mutation in TIP neither impaired the salicylic acid-mediated induction of HRT expression nor the enhanced resistance conferred by overexpression of HRT. Strikingly, the mutation in TIP resulted in increased replication of TCV and Cucumber mosaic virus, suggesting that TIP may play a role in basal resistance but is not required for HRT-mediated signaling.

Single-Molecule Fluorescence Resonance Energy Transfer Assays Reveal Heterogeneous Folding Ensembles in a Simple RNA Stem–Loop

We have examined the folding ensembles present in solution for a series of RNA oligonucleotides that encompass the replicase translational operator stem–loop of the RNA bacteriophage MS2. Single-molecule (SM) fluorescence assays suggest that these RNAs exist in solution as ensembles of differentially base-paired/base-stacked states at equilibrium. There are two distinct ensembles for the wild-type sequence, implying the existence of a significant free energy barrier between “folded” and “unfolded” ensembles. Experiments with sequence variants are consistent with an unfolding mechanism in which interruptions to base-paired duplexes, in this example by the single-stranded loop and a single-base bulge in the base-paired stem, as well as the free ends, act as nucleation points for unfolding. The switch between folded and unfolded ensembles is consistent with a transition that occurs when all base-pairing and/or base-stacking interactions that would orientate the legs of the RNA stem are broken. Strikingly, a U-to-C replacement of a residue in the loop, which creates a high-affinity form of the operator for coat protein binding, results in dramatically different (un)folding behaviour, revealing distinct subpopulations that are either stabilised or destabilised with respect to the wild-type sequence. This result suggests additional reasons for selection against the C-variant stem–loop in vivo and provides an explanation for the increased affinity.

Kinetic Analysis of Arf GAP1 Indicates a Regulatory Role for Coatomer

Arf GAPs are a family of enzymes that catalyze the hydrolysis of GTP bound to Arf. Arf GAP1 is one member of the family that has a critical role in membrane traffic at the Golgi apparatus. Two distinct models for the regulation of Arf GAP1 in membrane traffic have been proposed. In one model, Arf GAP1 functions in a ternary complex with coat proteins and is inhibited by cargo proteins. In another model, Arf GAP1 is recruited to a membrane surface that has defects created by the increased membrane curvature that accompanies transport vesicle formation. Here we have used kinetic and mutational analysis to test predictions of models of regulation of Arf GAP1. We found that Arf GAP1 has a similar affinity for Arf1.GTP as another Arf GAP, ASAP1, but the catalytic rate is approximately 0.5% that of ASAP1. Coatomer stimulated Arf GAP1 activity; however, different from that predicted from the current model, coatomer affected the K(m) and not the k(cat) values. Effects of most mutations in Arf GAP1 paralleled those in ASAP1. Mutation of an arginine that aligned with an arginine presumed to be catalytic in ASAP1 abrogated activity. Peptide from the cytoplasmic tail of cargo proteins inhibited Arf GAP1; however, the unrelated Arf GAP ASAP1 was also inhibited. The curvature of the lipid bilayer had a small effect on activity of Arf GAP1 under the conditions of our experiments. We conclude that coatomer is an allosteric regulator of Arf GAP1. The relevance of the results to the two models of Arf GAP1-mediated regulation of Arf1 is discussed.

Initial Binding Process of the Membrane Insertase YidC with Its Substrate Pf3 Coat Protein Is Reversible

The binding of the inner membrane insertase YidC from Escherichia coli to its substrate, the Pf3 coat protein, was examined in vitro by fluorescence spectroscopy. Purified YidC protein was solubilized with the lipid-like detergent n-dodecylphosphocholine and noncovalently labeled with 1-anilino-naphthalene-8-sulfonate (ANS), whereas the Pf3 coat protein was kept in solution by the addition of 10% (v/v) isopropanol to the buffer. The binding of Pf3 coat protein was analyzed by fluorescence quenching of ANS bound to YidC. All binding curves showed a strict hyperbolic form at pH values between 9.0 and 5.0, indicating a reversible and noncooperative binding between YidC and its substrate. Analysis of the data revealed a dissociation constant K D for the binding process in the range of 1 microM. The pH profile of the K D values suggests that the binding of the Pf3 coat protein is dominated by hydrophobic interactions. The titration experiments provide strong evidence for a conformational change of the insertase upon binding a Pf3 coat protein molecule.

Following the synthesis, transport and translation of mRNA in living cells

In mammalian cells we developed a system using fluorescent proteins to analyze gene expression in real‐time, and to follow individual mRNPs. An array of genes coding for a functional mRNA that contains 24 repeats of the MS2 coat protein binding motif combined with the MS2 coat protein fused to GFP or YFP allowed us to analyze the kinetics of transcription in real time and to detect single molecules of RNA in live cells. In these studies we used photo‐bleaching of GFP‐labeled mRNAs and of a YFP‐polII fusion protein and photoactivation of paGFP labeled mRNA. Analysis of the complex process of transcription using fluorescent polymerase as well as fluorescent MS2 proteins provided an opportunity to model the kinetic steps of RNA synthesis. These results yield rate constants for each of the steps of promoter assembly, initiation and elongation. They demonstrate that transcription is inefficient and that polymerases can elongate each faster than thought, but can pause stochastically. When RNAs leave the transcription site, they can be followed as single molecules in the nucleus. The analysis demonstrated that subsequent RNA mobility was not directed, but governed by rules of simple diffusion. The transport through nuclear pores can be determined as well. Once in the cytoplasm, mRNAs diffuse but also can be directed to their destinations by virtue of a zipcode motif in the RNA. A zipcode binding protein (ZBP1) is essential for this localization and inhibition of translation. Beta‐actin mRNA can be localized to sites of active F‐actin polymerization in migrating fibroblasts or growing neurons. The movement of the RNA follows rules of diffusion, alternating with probabilistic motor‐driven events on microtubles as actin filaments.

The Three-dimensional Structure of Genomic RNA in Bacteriophage MS2: Implications for Assembly

Using cryo-electron microscopy, single particle image processing and three-dimensional reconstruction with icosahedral averaging, we have determined the three-dimensional solution structure of bacteriophage MS2 capsids reassembled from recombinant protein in the presence of short oligonucleotides. We have also significantly extended the resolution of the previously reported structure of the wild-type MS2 virion. The structures of recombinant MS2 capsids reveal clear density for bound RNA beneath the coat protein binding sites on the inner surface of the T = 3 MS2 capsid, and show that a short extension of the minimal assembly initiation sequence that promotes an increase in the efficiency of assembly, interacts with the protein capsid forming a network of bound RNA. The structure of the wild-type MS2 virion at ∼9 Å resolution reveals icosahedrally ordered density encompassing ∼90% of the single-stranded RNA genome. The genome in the wild-type virion is arranged as two concentric shells of density, connected along the 5-fold symmetry axes of the particle. This novel RNA fold provides new constraints for models of viral assembly.

Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro

Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3′-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3′-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA–protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution (“far-Northwestern blotting”). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA–replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3′ transfer RNA signals.

Effect of mutations K97A and E128A on RNA binding and self assembly of papaya mosaic potexvirus coat protein

Papaya mosaic potexvirus (PapMV) coat protein (CP) was expressed (CPdeltaN5) in Escherichia coli and showed to self assemble into nucleocapsid like particles (NLPs). Twenty per cent of the purified protein was found as NLPs of 50 nm in length and 80% was found as a multimer of 450 kDa (20 subunits) arranged in a disk. Two mutants in the RNA binding domain of the PapMV CP, K97A and E128A showed interesting properties. The proteins of both mutants could be easily purified and CD spectra of these proteins showed secondary and tertiary structures similar to the WT protein. The mutant K97A was unable to self assemble and bind RNA. On the contrary, the mutant E128A showed an improved affinity for RNA and self assembled more efficiently in NLPs. E128A NLPs were longer (150 nm) than the recombinant CPdeltaN5 and 100% percent of the protein was found as NLPs in bacteria. E128A NLPs were more resistant to digestion by trypsin than the CPdeltaN5 but were more sensitive to denaturation by heat. We discuss the possible role of K97 and E128 in the assembly of PapMV.

Base‐pairing potential identified by in vitro selection predicts the kinked RNA backbone observed in the crystal structure of the alfalfa mosaic virus RNA‐coat protein complex

The three-dimensional structure of the 3' terminus of alfalfa mosaic virus RNA in complex with an amino-terminal coat protein peptide revealed an unusual RNA fold with inter-AUGC basepairing stabilized by key arginine residues (Guogas, et al., 2004). To probe viral RNA interactions with the full-length coat protein, we have used in vitro genetic selection to characterize potential folding patterns among RNAs isolated from a complex randomized pool. Nitrocellulose filter retention, electrophoretic mobility bandshift analysis, and hydroxyl radical footprinting techniques were used to define binding affinities and to localize the potential RNA-protein interaction sites. Minimized binding sites were identified that included both the randomized domain and a portion of the constant regions of the selected RNAs. The selected RNAs, identified by their ability to bind full-length coat protein, have the potential to form the same unusual inter-AUGC Watson-Crick base pairs observed in the crystal structure, although the primary sequences diverge from the wild-type RNA. A constant feature of both the wild-type RNA and the selected RNAs is a G ribonucleotide in the third position of an AUGC-like repeat. Competitive binding assays showed that substituting adenosine for the constant guanosine in either the wild-type or selected RNAs impaired coat protein binding. These data suggest that the interactions observed in the RNA-peptide structure are likely recapitulated when the full-length protein binds. Further, the results underscore the power of in vitro genetic selection for probing RNA-protein structure and function.

Replication of Alfamo- and Ilarviruses: Role of the Coat Protein

In the family Bromoviridae, a mixture of the three genomic RNAs of bromo-, cucumo-, and oleaviruses is infectious as such, whereas the RNAs of alfamo- and ilarviruses require binding of a few molecules of coat protein (CP) to the 3' end to initiate infection. Most studies on the early function of CP have been done on the alfamovirus Alfalfa mosaic virus (AMV). The 3' 112 nucleotides of AMV RNAs can adopt two different conformations. One conformer consists of a tRNA-like structure that, together with an upstream hairpin, is required for minus-strand promoter activity. The other conformer consists of four hairpins interspersed by AUGC-sequences and represents a strong binding site for CP. Binding of CP to this conformer enhances the translational efficiency of viral RNAs in vivo 40-fold and blocks viral minus-strand RNA synthesis in vitro. AMV CP is proposed to initiate infection by mimicking the function of the poly(A)-binding protein.

Coat protein enhances translational efficiency of Alfalfa mosaic virus RNAs and interacts with the eIF4G component of initiation factor eIF4F

The three plus-strand genomic RNAs of Alfalfa mosaic virus (AMV) and the subgenomic messenger for viral coat protein (CP) contain a 5'-cap structure, but no 3'-poly(A) tail. Binding of CP to the 3' end of AMV RNAs is required for efficient translation of the viral RNAs and to initiate infection in plant cells. To study the role of CP in translation, plant protoplasts were transfected with luciferase (Luc) transcripts with 3'-terminal sequences consisting of the 3' untranslated region of AMV RNA 3 (Luc-AMV), a poly(A) tail of 50 residues [Luc-poly(A)] or a short vector-derived sequence (Luc-control). Pre-incubation of the transcripts with CP had no effect on Luc expression from Luc-poly(A) or Luc-control, but strongly stimulated Luc expression from Luc-AMV. From time-course experiments, it was calculated that CP binding increased the half-life of Luc-AMV by 20 % and enhanced its translational efficiency by about 40-fold. In addition to the 3' AMV sequence, the cap structure was required for CP-mediated stimulation of Luc-AMV translation. Glutathione S-transferase pull-down assays revealed an interaction between AMV CP and initiation factor complexes eIF4F and eIFiso4F from wheatgerm. Far-Western blotting revealed that this binding occurred through an interaction of CP with the eIF4G and eIFiso4G subunits of eIF4F and eIFiso4F, respectively. The results support the hypothesis that the role of CP in translation of viral RNAs mimics the role of the poly(A)-binding protein in translation of cellular mRNAs.

Evaluation of the Conformational Switch Model for Alfalfa Mosaic Virus RNA Replication

Key elements of the conformational switch model describing regulation of alfalfa mosaic virus (AMV) replication (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999) have been tested using biochemical assays and functional studies in nontransgenic protoplasts. Although comparative sequence analysis suggests that the 3' untranslated regions of AMV and ilarvirus RNAs have the potential to fold into pseudoknots, we were unable to confirm that a proposed pseudoknot forms or has a functional role in regulating coat protein-RNA binding or viral RNA replication. Published work has suggested that the pseudoknot is part of a tRNA-like structure (TLS); however, we argue that the canonical sequence and functional features that define the TLS are absent. We suggest here that the absence of the TLS correlates directly with the distinctive requirement for coat protein to activate replication in these viruses. Experimental data are evidence that elevated magnesium concentrations proposed to stabilize the pseudoknot structure do not block coat protein binding. Additionally, covarying nucleotide changes proposed to reestablish pseudoknot pairings do not rescue replication. Furthermore, as described in the accompanying paper (L. M. Guogas, S. M. Laforest, and L. Gehrke, J. Virol. 79:5752-5761, 2005), coat protein is not, by definition, inhibitory to minus-strand RNA synthesis. Rather, the activation of viral RNA replication by coat protein is shown to be concentration dependent. We describe the 3' organization model as an alternate model of AMV replication that offers an improved fit to the available data.

Molecular Genetics of Bacteriophage P22 Scaffolding Protein's Functional Domains

The assembly intermediates of the Salmonella bacteriophage P22 are well defined but the molecular interactions between the subunits that participate in its assembly are not. The first stable intermediate in the assembly of the P22 virion is the procapsid, a preformed protein shell into which the viral genome is packaged. The procapsid consists of an icosahedrally symmetric shell of 415 molecules of coat protein, a dodecameric ring of portal protein at one of the icosahedral vertices through which the DNA enters, and approximately 250 molecules of scaffolding protein in the interior. Scaffolding protein is required for assembly of the procapsid but is not present in the mature virion. In order to define regions of scaffolding protein that contribute to the different aspects of its function, truncation mutants of the scaffolding protein were expressed during infection with scaffolding deficient phage P22, and the products of assembly were analyzed. Scaffolding protein amino acids 1-20 are not essential, since a mutant missing them is able to fully complement scaffolding deficient phage. Mutants lacking 57 N-terminal amino acids support the assembly of DNA containing virion-like particles; however, these particles have at least three differences from wild-type virions: (i) a less than normal complement of the gene 16 protein, which is required for DNA injection from the virion, (ii) a fraction of the truncated scaffolding protein was retained within the virions, and (iii) the encapsidated DNA molecule is shorter than the wild-type genome. Procapsids assembled in the presence of a scaffolding protein mutant consisting of only the C-terminal 75 amino acids contained the portal protein, but procapsids assembled with the C-terminal 66 did not, suggesting portal recruitment function for the region about 75 amino acids from the C terminus. Finally, scaffolding protein amino acids 280 through 294 constitute its minimal coat protein binding site.

Cis-Acting sequences required for coat protein binding and in vitro assembly of Potato virus X

The 5′ region of Potato virus X (PVX) RNA containing an AC-rich single-stranded region and stem–loop 1 (SL1) has been shown to be important for PVX replication (Miller, E.D., Plante, C.A., Kim, K.-H., Brown, J.W., Hemenway, C., 1998. Stem–loop structure in the 5′ region of potato virus X genome required for plus-strand RNA accumulation. J. Mol. Biol. 284, 591–608.). Here, we describe the involvement of SL1 for binding to the PVX coat protein (CP) using an in vitro assembly system and various deletion mutants of the 5′ region of PVX RNA. Internal and 5′ terminal deletions of the 5′-nontranslated region of PVX RNA were assessed for their effects on formation of assembled virus-like particles (VLPs). Mutant RNAs that contain the top region of SL1 or sequences therein bound to CP to form VLPs. In contrast, transcripts of mutants that disrupt SL1 RNA structure were unable to form VLPs. SELEX was used to further confirm the specific RNA recognition of PVX CP using RNA transcripts containing randomized sequences of the upper portion of SL1. Wild-type (wt) sequences along with many other sequences that resemble SL1 structure were selected after fourth and fifth rounds of SELEX (27.0% and 44.4%, respectively). RNA transcripts from several SELEX winners that are predicted to form stable stem–loop structures very closely resembling wt PVX SL1 VLPs. RNA transcripts not predicted to form secondary structures similar to SL1 did not form VLPs in vitro. Taken together, our results suggest that RNA secondary structural elements within SL1 and/or sequences therein are crucial for formation of VLPs and are required for the specific recognition by the CP subunit.

Modeling distinct compartments

Heinrich and Rapoport (page 271) have used mathematical modeling to generate the first explanation of how a bidirectional transport system can generate unique compartments, such as the ER and Golgi, despite constant vesicle movement between them. The model provides testable predictions about the vesicle transport system in cells.In modeling a two-organelle system, the team found that they only needed to include two molecular components of the vesicle transport system: the coat proteins for budding; and the SNARE proteins for fusion. Coat proteins regulate budding from distinct compartments: COPI from the Golgi (to the ER); and COPII from the ER (to the Golgi). Meanwhile, SNARE proteins work in pairs, with a v-SNARE localized in the vesicle membrane and the t-SNARE in the target membrane. Different SNAREs direct vesicle fusion to specific organelles. Figure Differential affinities between coats and SNARE proteins can generate distinct compartments. The new model only worked when it incorporated differential affinity of one coat protein for one set of SNARE proteins versus the other. If both coat proteins bound all SNAREs with equal affinity, then bidirectional vesicle transport would result in two uniform organelles. If the model assumed that one coat protein bound one pair of SNAREs with at least a 10-fold preference over the other SNARE pair, then the model accurately maintained two unique compartments. One nonintuitive aspect of the model is that it predicts that both members of a SNARE pair, the v- and t-SNAREs, accumulate in the target compartment, a prediction borne out by experimental observations from other groups. COPII is known to have high affinity for SNAREs that target the Golgi, and the model predicts that COPI should preferentially bind to ER SNAREs. If, however, the coat protein's binding affinity for a SNARE pair is too strong, then all of those SNARE proteins would accumulate in the target compartment, leaving vesicles to bud from the other compartment without any SNAREs that would allow it to fuse to the target membrane. The model also predicts that if one SNARE pair is overexpressed, then the size of its target compartment would increase.

Template-dependent incorporation of 8-N3AMP into RNA with bacteriophage T7 RNA polymerase.

UV-induced photochemical crosslinking is a powerful approach that can be used for the identification of specific interactions involving nucleic acid-protein and nucleic acid-nucleic acid complexes. 8-AzidoATP (8-N(3)ATP) is a photoaffinity-labeling agent which has been widely used to elucidate the ATP binding site of a variety of proteins. However, its true potential as a photoactivatable nucleotide analog could not be exploited due to the lack of 8-azidoadenosine phosphoramidite, a monomer used in the synthesis of RNA, and the inability of 8-N(3)ATP to serve as an efficient substrate for bacteriophage RNA polymerase. In this study, we explored the ability of SP6, T3, and T7 RNA polymerases and metal ion cofactors to catalyze the incorporation of 8-N(3)AMP into RNA. Whereas transcription buffer containing 2.0-2.5 mM Mn(2+) supports T7 RNA polymerase-mediated insertion of 8-N(3)AMP into RNA, a mixture of 2.5 mM Mn(2+) and 2.5 mM Mg(2+) further improves the yield of 8-N(3)AMP-containing transcript. In addition, both RNA transcription and reverse transcription proceed with high fidelity for the incorporation of 8-N(3)AMP and complementary residue, respectively. Finally, we show that a high-affinity MS2 coat protein binding sequence, in which adenosine residues were replaced by 8-azidoadenosine, crosslinks to the coat protein of the Escherichia coli phage MS2.

Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.

Efficient translation of alfamovirus RNAs requires the binding of coat protein dimers to the 3' termini of the viral RNAs.

The coat protein (CP) of Alfalfa mosaic virus (AMV) is required to initiate infection by the viral tripartite RNA genome whereas infection by the tripartite Brome mosaic virus (BMV) genome is independent of CP. AMV CP stimulates translation of AMV RNA in vivo 50- to 100-fold. The 3' untranslated region (UTR) of the AMV subgenomic CP messenger RNA 4 contains at least two CP binding sites. A CP binding site in the 3'-terminal 112 nucleotides of RNA 4 was found to be required for efficient translation of the RNA whereas an upstream binding site was not. Binding of CP to the AMV 3' UTR induces a conformational change of the RNA but this change alone was not sufficient to stimulate translation. CP mutant R17A is unable to bind to the 3' UTR and translation in vivo of RNA 4 encoding this mutant occurs at undetectable levels. Replacement of the 3' UTR of this mutant RNA 4 by the 3' UTR of BMV RNA 4 restored translation of R17A-CP to wild-type levels. Apparently, the BMV 3' UTR stimulates translation independently of CP. AMV CP mutant N199 is defective in the formation of CP dimers and did not stimulate translation of RNA 4 in vivo although the mutant CP did bind to the 3' UTR. The finding that N199-CP does not promote AMV infection corroborates the notion that the requirement of CP in the inoculum reflects its role in translation of the viral RNAs.