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We experimentally register stimulated low-frequency Raman scattering (SLFRS) in the suspension of brome mosaic virus (BMV) in phosphate buffer with very high conversion efficiency. We identify two components of the SLFRS spectrum as the breathing and quadrupole modes of BMV and determine damping characteristics and gain factors for these modes. We show that, using the core–shell model for BMV and taking into account the influence of the environment, the acoustic properties of individual components of such a composite nanosystem can be determined. Thus, we define the sound velocity in the RNA core of BMV, in view of spectral characteristics of SLFRS.

Plus-stranded RNA viruses, the largest group among eukaryotic viruses, are capable of reprogramming host cells by subverting host proteins and membranes, by co-opting and modulating protein and ribonucleoprotein complexes, and by altering cellular pathways during infection. To achieve robust replication, plus-stranded RNA viruses interact with numerous cellular molecules via protein–protein, RNA–protein, and protein–lipid interactions using molecular mimicry and other means. These interactions lead to the transformation of the host cells into viral “factories" that can produce 10,000–1,000,000 progeny RNAs per infected cell. This chapter presents the progress that was made largely in the last 15 years in understanding virus–host interactions during RNA virus replication. The most commonly employed approaches to identify host factors that affect plus-stranded RNA virus replication are described. In addition, we discuss many of the identified host factors and their proposed roles in RNA virus replication. Altogether, host factors are key determinants of the host range of a given virus and affect virus pathology, host–virus interactions, as well as virus evolution. Studies on host factors also contribute insights into their normal cellular functions, thus promoting understanding of the basic biology of the host cell. The knowledge obtained in this fast-progressing area will likely stimulate the development of new antiviral methods as well as novel strategies that could make plus-stranded RNA viruses useful in bio- and nanotechnology.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the Arteriviridae family, which includes enveloped and positive-sense single-stranded RNA viruses (Meulenberg et al., 1993). The size of the viral genome is approximately 15 kb, and eight functional genes have been identified, five of which encode viral structural proteins (Meulenberg et al., 1997). The organization, structure, and replication strategies of the viral genome are similar to those of coronaviruses (Snijder and Spaan, 1995). Interaction of the N protein with the 3’-end of the viral genome suggests that the N protein may regulate viral RNA synthesis. Several cellular proteins have been reported to bind to the 3’-untranslated region (UTR) of RNA viruses (Nakhasi et al., 1991; Pardigon and Strauss, 1992).

A potential pseudoknot was found in the 3′ untranslated region of the bovine coronavirus genome beginning 63 nt downstream from the stop codon of the N gene. Mutation analysis of the pseudoknot in a cloned defective interfering RNA indicated that this structural element is necessary for defective interfering RNA replication.

Positive strand RNA viruses, i.e., viruses that encapsidate single stranded messenger-sense RNA, are a numerically and practically important class of viruses in eukaryotic hosts. Such viruses are particularly significant and well diversified in plants, comprising by far the largest single class of known plant viruses (Matthews, 1982). Research interest in positive strand RNA viruses derives from many perspectives, including their role as highly successful pathogens, their facility at high level gene transmission and expression, and the novelty of their RNA-dependent replication, gene expression, and recombination pathways. The value of understanding RNA-based genetic processes is underscored by the growing suggestion that RNA genomes may have preceded DNA genomes as one stage in the early evolution of life (Joyce, 1989). Thus, as well as illuminating their own unusual biochemistry and genetics, RNA virus studies may provide general insights into replication, survival, and evolution in the putative primordial RNA world. In addition, since many of its features are widely conserved among otherwise diverse viruses of both plants and animals (see below), the viral RNA replication apparatus is a potential target for the development of broad spectrum antiviral agents, which would have great practical benefits. The genetic pathways of RNA viruses have also provided and should continue to provide further new ways to amplify and regulate gene expression in applied genetic engineering (French et al., 1986; Xiong et al., 1989). Finally, viruses possess a number of attractive features as general models for plant-microbe interactions, including uniquely small genomes, ready manipulation by recombinant DNA techniques, and a growing understanding of their life cycle. This chapter discusses some aspects of replication, recombination, and host specificity in one representative group of positive strand RNA plant viruses, the bromoviruses.

Antisense oligodeoxyribonucleotides and nuclease resistant, phosphate-modified oligonucleotide analogues can be used to inhibit virus gene expression both in vitro and in cell culture. Oligomers targeted to bind to functional regions of viral mRNAs such as the initiation codon region or splice junction regions of pre-mRNA, can interfere with mRNA expression in a sequence specific manner. Antisense oligonucleotides and oligonucleotide analogues have been used to inhibit virus protein synthesis and replication in cells infected with vesicular stomatitis virus, herpes simplex virus and human immunodeficiency virus. Because the inhibitory effects of the oligomers depend upon the oligomer sequence, it appears that antisense oligomers could be used as research tools to study and manipulate virus gene expression in cell culture and could be developed into highly specific and effective antiviral agents.

Recent developments have brought together viruses which were not previously supposed to bear any relationship except that they were simple RNA-containing plant viruses. These viruses are the spherical bromoviruses, once thought to have a very small genome (Bockstahler and Kaesberg, 1961); the small spherical cucumber mosaic virus and its relatives, which, in contrast to bromoviruses, are transmitted by aphids; the aphid-transmitted alfalfa mosaic virus, which is one of the very few small viruses with bacilliform particles; and a number of structurally not very well studied viruses among which are tobacco streak virus and citrus leaf rugose virus, which seem to have in common the possession of spherical virions of different size.

Brome Mosaic Virus (BMV; MW 4.6 106) is a small spherical plant virus of 133 A radius which possesses a divided genome of single-stranded RNA (MW about 106) contained in a protein capsid which is composed of 180 copies of a single polypeptide of MW 20,300. The proteins are located at quasiequivalent crystallographic sites on an icosahedron of triangulation number T = 3: twelve pentamers form the vertices and twenty hexamers the faces of the icosahedron. The surface structure is occasionally visible on electron micrographs of negatively stained virus, and has been analyzed to 25 A by image reconstruction of two-dimensional arrays1 of the closely related Cowpea Chlorotic Mottle Virus (Figure 1; Figure 2 shows a schematic view of the central protein arrangement visible in Figure 1).

The 3’-ends of several plant viral RNAs show a number of functional characteristics of tRNAs1,2; they are recognized by a set of tRNA-specific proteins, including aminoacyl-tRNA synthetases. So, the RNA of turnip yellow mosaic virus can be valylated by yeast valyl-tRNA synthetase3,4 with kinetic constants very close to those of the aminoacylation of yeast tRNAVal by this enzyme5. The 3′-ends of these viral RNAs however, are lacking several characteristic primary structural features of tRNAs such as strategic D- or T-loop sequences and modified bases. Moreover, they cannot be folded a priori into a canonical tRNA cloverleaf. In the case of TYMV RNA, the question arose as to “how do two structures as different as tRNAVal and the 3′-region of the viral RNA behave in such a similar fashion in the presence of valyl-tRNA synthetase?”. This question might be answered if one supposes that similar structural domains are recognized by the synthetase and exist at the level of the three dimensional structures and even at the level of their secondary structures. In this view, the secondary structure of the 3′-end of TYMV RNA was established by enzymatic footprinting methodologies6. A three-dimensional L-shaped conformation mimicking tRNA, but involving a new RNA folding principle, the pseudoknot, was proposed by the Leiden group7,8 for the 86 last nucleotides of this RNA. A rigourous graphical modelling allowed to assess the reality of this pseudoknotted folding9. To understand the involvement of the 3′-end of the TYMV RNA in aminoacylation, the direct contact points between this part of the RNA and yeast valyl-tRNA synthetase were determined10. Moreover, the study of the valylation of tRNA-like transcripts from cloned cDNA of TYMV RNA permitted to determine the minimal length of the RNA necessary for optimal valylation11.

Tobacco mosaic was first characterized as an infectious disease in 1886 by Mayer and the virus has been the subject of intensive investigation since the 1930s. Early studies focused on chemistry and structure, and much of our fundamental knowledge of the chemical and physical properties of simple RNA-containing viruses derives from TMV. While the large amounts of easily extracted, easily purified virus available in high yield facilitated these studies, the complex nature of the virus-host system has slowed our understanding of the details of TMV replication (and of all other plant viruses, for that matter); for instance, when a leaf is inoculated with virus, initially only a very small proportion of the cells become infected. These initial centers serve as foci for the subsequent rounds of replication in adjacent cells. Thus, early events in the few initially infected cells must be detected against an overwhelming background of uninfected cells. Further difficulties arise from the relatively slow rate of virus replication when compared with animal or bacterial viruses and, most troublesome, the fact that plant virus infection does not shut down host protein and nucleic acid synthesis. Thus, it is sometimes not easy to distinguish virus-directed proteins and nucleic acids from those of the host. Fortunately, recently developed technologies such as molecular hybridization and Western blotting having solved some of these problems.

The following overview of plant viral translation will center on the viruses with large, presumably multigenic RNAs (molecular weight over 106). This includes both the classical homodisperse viruses like TMV, and the large components of the multicomponent viruses like the bromoviridae.* Such viruses have lent themselves well to biochemical analyses and in vitro studies, but the mode of gene expression in vivo has proven difficult to elucidate.

The protein subunits of brome mosaic virus can self-assemble in vitro, in the absence of RNA, to form capsidlike particles that are similar in size to the original virus capsid. The protein exists essentially as dimers at neutral pH. The assembly process is triggered by a decrease in pH. The kinetics of assembly were followed by using stopped-flow techniques coupled with neutron or x-ray small-angle scattering. Neutron scattering experiments were performed at the I.L.L, Grenoble, and x-ray studies at L.U.R.E., Orsay. Both the x-ray and neutron investigations showed that the assembly process starts rapidly: the intensity at the origin, I(O), achieves 60% of its maximal value in a few hundred milliseconds and 80% in a few seconds. Furthermore, analysis of the scattering curves suggests that aggregates similar in size to the native viral capsid are present after only 300 msec. Then, the process gradually slows down, as indicated by I(O), which continues to increase very slowly over a period of hours. Finally, the possible existence of intermediate species is discussed on the basis of our experimental data.

The hordeivirus group includes three rod-shaped viruses with multipartite genomes: barley stripe mosaic virus (BSMV), Poa semilatent virus, and Lychnis ringspot virus, which are distantly related serologically and partly differ in their host ranges.

Unlike higher organisms, viruses utilize a wide range of strategies in the replication of their genome. This is clearly a consequence of their need to deal with genomes that may be single-stranded (ss), double-stranded (ds), or partially ds; with genomes that may be composed of DNA, RNA, or both (at different stages in their life cycle); and with genomic structures which may be simple circles, simple linear molecules, single-stranded linear structures with terminal hairpin folds, ds linear structures in which the complementary strands are covalently linked at the ends, multipartite structures, and complex linear structures. In some cases the genome may also include covalently-linked proteins.

In vitro translation of plant virus RNAs not only yields valuable information about the proteins encoded by these RNAs, but it also gives information about the protein synthesizing machinery of the host. In the present paper we only discuss the results obtained with plant viruses with a genome consisting of single stranded RNA(s) with the plus polarity (genomic RNA has the same polarity as the messenger RNA). The viruses are discussed according to the type of structure present at the 5′ terminus, the 3′ terminus and the strategy employed to express the genetic information. In the last paragraph the possible functions of the virus coded proteins are discussed.

All characterized members of the Bromoviruses, Cucumoviruses, Ilarviruses, and also alfalfa mosaic virus (AMV) contain a tripartite genome in that the three largest of the four major encapsidated RNAs are required for infectivity. The Ilarviruses and AMV are distinguished from the two other virus groups by the additional requirement for infection of either a small amount of coat protein or its subgenomic mRNA, RNA 4 (reviewed in van Vloten-Doting and Jaspars, 1977). A further distinguishing feature is the inability of the RNAs of Ilarviruses and AMV to be aminoa-cylated in the presence of plant aminoacyl tRNA synthetases, whereas the RNAs of the Bromoviruses and Cucumoviruses that have been tested accept tyrosine (Hall, 1979; Koper-Zwarthoff and Bol, 1980). This division of the viruses into two groups on the basis of the ability or inability of their RNAs to be aminoacylated indicates significant differences in the biological and structural properties of the RNAs.

The genome of plant viruses is either carried by large RNA molecules containing the whole genetic information (tobacco mosaic virus for example) or by several pieces of nucleic acid each containing a part of the total genetic information (alfalfa mosaic virus, brome mosaic virus). But whatever the type of virus the shortest RNA piece it contains is on the order of 1 000 nucleotides. Under these circumstances the sequence work on plant viral RNA’s has been aimed at solving relatively limited sequences of strategic importance rather than at trying to sequence the entire molecule. The sequence problems of interest belong to three main categories.