Virus-like particles can be formed by self-assembly of capsid protein (CP) with RNA molecules of increasing length. If the protein "insisted" on a single radius of curvature, the capsids would be identical in size, independent of RNA length. However, there would be a limit to length of the RNA, and one would not expect RNA much shorter than native viral RNA to be packaged unless multiple copies were packaged. On the other hand, if the protein did not favor predetermined capsid size, one would expect the capsid diameter to increase with increase in RNA length. Here we examine the self-assembly of CP from cowpea chlorotic mottle virus with RNA molecules ranging in length from 140 to 12,000 nucleotides (nt). Each of these RNAs is completely packaged if and only if the protein/RNA mass ratio is sufficiently high; this critical value is the same for all of the RNAs and corresponds to equal RNA and N-terminal-protein charges in the assembly mix. For RNAs much shorter in length than the 3,000 nt of the viral RNA, two or more molecules are assembled into 24-and 26-nm-diameter capsids, whereas for much longer RNAs (>4,500 nt), a single RNA molecule is shared/packaged by two or more capsids with diameters as large as 30 nm. For intermediate lengths, a single RNA is assembled into 26-nm-diameter capsids, the size associated with T‫3؍‬ wild-type virus. The significance of these assembly results is discussed in relation to likely factors that maintain T‫3؍‬ symmetry in vivo.

The majority of positive-strand RNA viruses of plants replicate and selectively encapsidate their progeny genomes into stable virions in cytoplasmic compartments of the cell where the opportunity to copackage cellular RNA also exists. Remarkably, highly purified infectious virions contain almost exclusively viral RNA, suggesting that mechanisms exist to regulate preferential packaging of viral genomes. The general principle that governs RNA packaging is an interaction between the structural CP and a specific RNA signal. Mechanisms that enhance selective packaging of viral genomes and formation of infectious virions may involve factors other than CP and nucleic acid sequences. The possible involvement of replicase proteins is an example. Our knowledge concerning genome packaging among spherical plant RNA viruses is still maturing. The main focus of this review is to discuss factors that have limited progress and to evaluate recent technical breakthroughs likely to help unravel the mechanism of RNA packaging among viruses of agronomic importance. A key breakthrough is the development of in vivo systems and comparisons with results obtained in vitro.

To begin elucidation of the relationship between Brome mosaic virus (BMV) replication and encapsidation, we used a T-DNA-based Agrobacterium-mediated transient expression (agroinfiltration) system in Nicotiana benthamiana leaves to express either individual or desired pairs of the three genomic RNAs. The packaging competence of these RNAs into virions formed by the transiently expressed coat protein (CP) was analyzed. We found that in the absence of a functional replicase, assembled virions contained non-replicating viral RNAs (RNA1 or RNA2 or RNA3 or RNA1 + RNA3 or RNA2 + RNA3) as well as cellular RNAs. By contrast, virions assembled in the presence of a functional replicase contained only viral RNAs. To further elucidate the specificity exhibited by the functional viral replicase in RNA packaging, replication-defective RNA1 and RNA2 were constructed by deleting the 3' tRNA-like structure (3' TLS). Co-expression of TLS-less RNA1 and RNA2 with wt RNA3 resulted in efficient synthesis of subgenomic RNA4. Virions recovered from leaves co-expressing TLS-less RNA1 and RNA2 and either CP mRNA or wt RNA3 exclusively contained viral RNAs. These results demonstrated that packaging of BMV genomic RNAs is not replication dependent whereas expression of a functional viral replicase plays an active role in increasing specificity of RNA packaging.

While most T‫3؍‬ single-stranded RNA (ssRNA) viruses package in vivo about 3,000 nucleotides (nt), in vitro experiments have demonstrated that a broad range of RNA lengths can be packaged. Under the right solution conditions, for example, cowpea chlorotic mottle virus (CCMV) capsid protein (CP) has been shown to package RNA molecules whose lengths range from 100 to 10,000 nt. Furthermore, in each case it can package the RNA completely, as long as the mass ratio of CP to nucleic acid in the assembly mixture is 6:1 or higher. Yet the packaging efficiencies of the RNAs can differ widely, as we demonstrate by measurements in which two RNAs compete head-to-head for a limited amount of CP. We show that the relative efficiency depends nonmonotonically on the RNA length, with 3,200 nt being optimum for packaging by the T‫3؍‬ capsids preferred by CCMV CP. When two RNAs of the same length-and hence the same charge-compete for CP, differences in packaging efficiency are necessarily due to differences in their secondary structures and/or three-dimensional (3D) sizes. For example, the heterologous RNA1 of brome mosaic virus (BMV) is packaged three times more efficiently by CCMV CP than is RNA1 of CCMV, even though the two RNAs have virtually identical lengths. Finally, we show that in an assembly mixture at neutral pH, CP binds reversibly to the RNA and there is a reversible equilibrium between all the various RNA/CP complexes. At acidic pH, excess protein unbinds from RNA/CP complexes and nucleocapsids form irreversibly.T he remarkable capacity of the capsid protein (CP) of cowpea chlorotic mottle virus (CCMV) to self-assemble in vitro into nanometer-size capsids around a broad range of anionic materials-its own single-stranded RNA (ssRNA) genome (2, 6), heterologous ssRNAs (3,4,9,24), organic polymers such as polystyrene sulfonate (11,16,23), metal oxide particles (19, 28), functionalized gold nanoparticles (5), and negatively charged nanoemulsion droplets (13)-as well as into a wide range of structures in the absence of any polyanions (1,8,26) has spurred interest in the mechanism of assembly. But a fundamental question has rarely or only inadequately been addressed: how efficient are the assembly processes?To answer this question, it is first necessary to formulate a precise definition of efficiency of assembly, along with a procedure for determining it. In some cases, efficiency has been quantified as the fraction of filled capsids in the mixture of filled and empty capsids observed in electron micrographs of the assembly mixture (14), but this depends on the formation of empty capsids under the conditions of filled-capsid assembly. In other cases, the relative efficiency of packaging an RNA molecule has been defined as the ratio of the number of in vivo capsids containing it to the number formed with the genomic RNA (5), when both are present in the same CP-expressing cell; however, this requires additional knowledge about the relative levels of replication of both RNAs.Several assembly experiments, both in vivo and in...

Four mutant brome mosaic virus (BMV) RNA3 transcripts, bearing single or double base changes in the 3'-CCAOH terminus of the tRNA-like structure, previously characterized as being deficient in vitro with respect to aminoacylation and replication activities, have now been assayed in vivo for their ability to replicate (in the presence of transcripts of wild-type RNA1 and -2) in barley protoplasts and plants. In tests conducted with protoplasts, irrespective of the time postinfection, all four mutants were fully viable, and the relative levels ofboth plus and minus strand replication for each mutant were similar to that of the wild type. Inoculation of barley plants with these mutants resulted in phenotypic symptoms and viral yields that were similar to those from wild-type infections.Analysis of each mutant progeny RNA3 indicated that the altered sequence at the 3' terminus was restored to that of wild type. These observations indicate that there is a rapid turnover and correction of the 3' termini of BMV RNAs in vivo. Such correction is commensurate with the action of tRNA nucleotidyltransferase, but it differs from recombination processes that appear to be relatively infrequent for BMV RNA3. These results support the conclusion that the 3'-CCAoH termini of viral tRNA-like structures function analogously to telomeres of chromosomal DNA.There is currently much interest in the tRNA-like motifs present at the ends of the RNA genomes of several groups of plant viruses. We (1-5) and others (6, 7) have experimentally established in vitro and in vivo that the 3' ends of brome mosaic virus (BMV) and other viruses function as substrates for aminoacyl-tRNA synthetases, tRNA nucleotidyltransferase, and other tRNA-like activities. Studies on the 3' tRNA-like structure of turnip yellow mosaic virus (8, 9) led to discovery of the role of the pseudoknot in forming stable amino acid acceptor stems of viral RNAs. Pseudoknots also appear to participate in forming the structure of biologically active RNAs, such as ribosomal RNAs (10) and the selfsplicing intron in Tetrahymena (11). Weiner and Maizels (12) have postulated a telomeric function for the 3' end ofgenomic RNA molecules and Marsh and Hall (13) have shown that remarkable similarities exist between regions at the 5' ends of BMV RNAs and the internal control regions characteristic of polymerase III promoters of tRNA transcription.tRNA nucleotidyltransferase is the enzyme responsible for the addition to tRNA transcripts ofthe 3'-CCAOH termini that are not encoded in the genome of eukaryotes. It is also important in the maintenance of intact mature tRNA molecules, whose 3' termini are subjected to nuclease attack and undergo constant turnover (14). Host tRNA nucleotidyltransferase has been implicated in the posttranscriptional addition of the 3'-terminal adenosine of BMV RNAs, since this residue has no complement in the minus strand template and is not present in double-stranded replicative form RNAs (15). While the 3'-terminal adenosine is not necessary for initiatio...

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tRNAs, the adapter molecules in protein synthesis, also serve as metabolic cofactors and as primers for viral RNA-directed DNA synthesis. The genomic and subgenomic RNAs of some plant viruses have a 3-terminal tRNA-like structure (TLS) that can accept a specific amino acid and serve as a site for initiation of replication and as a simple telomere. We report a previously undescribed role for the TLS of brome mosaic virus (BMV), and potentially for cellular tRNA, in mediating the assembly of its icosahedral virions. BMV genomic RNAs and subgenomic RNA lacking the TLS failed to assemble into virions when incubated with purified BMV coat protein. Assembly was restored by addition of a 201-nt RNA containing the BMV TLS. TLSs from two other plant viruses as well as tRNAs from wheat germ and yeast were similarly active in the BMV virion assembly reaction, but ribosomal RNA and polyadenylate did not facilitate assembly. Surprisingly, virions assembled from TLS-less BMV RNA in the presence of tRNAs or TLS-containing short RNA did not incorporate the latter molecules. Consistent with a critical role for the BMV TLS in virion assembly, mutations in the BMV genomic RNAs that were designed to disrupt the folding of the TLS also abolished virion assembly. We discuss the likely roles of the TLS in early stages of virion assembly.tRNA-like structure ͉ bromoviruses ͉ virus assembly ͉ RNA packaging T ransfer RNAs (tRNAs) are multifunctional. Their primary role is to be an adapter molecule that translates the codon sequences in mRNA into the amino acid sequence of a protein. tRNAs also participate in specialized functions in cellular metabolism such as biosynthesis of the bacterial cell wall (1), chlorophyll, and heme (2). tRNAs and tRNA-related activities are associated with a variety of RNA viruses, including several plant viruses and the retroviruses (3, 4). Host tRNAs found in the virions of retroviruses function as primers for RNA-directed DNA synthesis (3). The tRNA-like structures (TLSs) found at the 3Ј end of the genomes of some plant viruses serve as efficient origins of replication and as primitive telomeres to ensure that the 3Ј-terminal CCA nucleotides are not lost during replication (4, 5). It has been suggested that these viral TLSs are molecular fossils that may relate to a primordial role for tRNA in RNA replication in the ancient RNA world (6).Brome mosaic virus (BMV) is an example of an RNA virus that has an Ϸ170-nt-long TLS covalently bound to the 3Ј end of its genomic and subgenomic RNAs. BMV is a member of the plant virus family Bromoviridae (7) and the alphavirus-like superfamily of human-, animal-, and plant-infecting positive-strand RNA viruses (8). Mature BMV virions encapsidate three genomic RNAs (B1-B3) and a single subgenomic RNA, B4 (7). Physical and biochemical data suggest that B1-B4 are packaged into three morphologically indistinguishable virus particles: B1 (3.2 kb) and B2 (2.9 kb) are packaged individually into separate particles, whereas the genomic B3 (2.1 kb) and the subgenomic B4 (0.9 kb) are...

We have engineered an optical nanoconstruct composed of genome-depleted brome mosaic virus doped with indocyanine green (ICG), an FDA-approved near-infrared (NIR) chromophore. Constructs are highly monodispersed with standard deviation of ±3.8 nm from a mean diameter of 24.3 nm. They are physically stable and exhibit a high degree of optical stability at physiological temperature (37 °C). Using human bronchial epithelial cells, we demonstrate the effectiveness of the constructs for intracellular optical imaging in vitro, with greater than 90% cell viability after 3 h of incubation. These constructs may serve as a potentially nontoxic and multifunctional nanoplatform for site-specific deep-tissue optical imaging, and therapy of disease.