The red clover necrotic mosaic virus genome is composed of two single-stranded RNA components, RNA-1 and RNA-2. The viral capsid protein is translated from a subgenomic RNA (sgRNA) that is transcribed from genomic RNA-1. Here, a 34-nucleotide sequence in RNA-2 is shown to be required for transcription of sgRNA. Mutations that prevent base-pairing between the RNA-1 subgenomic promoter and the 34-nucleotide trans-activator prevent expression of a reporter gene. A model is proposed in which direct binding of RNA-2 to RNA-1 trans-activates sgRNA synthesis. This RNA-mediated regulation of transcription is unusual among RNA viruses, which typically rely on protein regulators.

Amphitrite ornata dehaloperoxidase (DHP) is a heme enzyme with a globin structure, which is capable of oxidizing para-halogenated phenols to the corresponding quinones. Cloning, high-level expression, and purification of recombinant DHP are described. Recombinant DHP was assayed by stopped-flow experiments for its ability to oxidatively debrominate 2,4,6-tribromophenol (TBP). The enzymatic activity of the ferric form of recombinant DHP is intermediate between that of a typical peroxidase (horseradish peroxidase) and a typical globin (horse heart myoglobin). The present study shows that, unlike other known peroxidases, DHP activity requires the addition of substrate, TBP, prior to the cosubstrate, peroxide. The presence of a substrate-binding site in DHP is consistent with a two-electron oxidation mechanism and an obligatory order for activation of the enzyme by addition of the substrate prior to the cosubstrate.

Folded RNA elements that block processive 5' → 3' cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5' end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use "codegradational remodeling," exploiting the exoribonucleases' degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.

This article summarizes studies of viral coat (capsid) proteins (CPs) of RNA plant viruses. In addition, we discuss and seek to interpret the knowledge accumulated to data. CPs are named for their primary function; to encapsidate viral genomic nucleic acids. However, encapsidation is only one feature of an extremely diverse array of structural, functional, and ecological roles played during viral infection and spread. Herein, we consider the evolution of viral CPs and their multitude of interactions with factors encoded by the virus, host plant, or viral vector (biological transmission agent) that influence the infection and epidemiological facets of plant disease. In addition, applications of today's understanding of CPs in the protection of crops from viral infection and use in the manufacture of valuable compounds are considered.

The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 Å by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 ؎ 30 Ca 2؉ ions bound to the capsid and 420 ؎ 25 Mg 2؉ions thought to be in the interior of the capsid. Depletion of both Ca 2؉ and Mg 2؉ ions from RCNMV leads to significant structural changes, including (i) formation of 11-to 13-Å-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca 2؉ and Mg 2؉ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.The Tombusviridae family consists of small, icosahedral plant viruses that are transmitted through the soil and infect their hosts via the root system (34). Given that these viruses must survive harsh environments, it is not surprising that they possess unusually stable and robust capsids. This high degree of virion stability raises the question of how the intracellular environment triggers disassembly or minimally exposes the genome for translation. Structural analyses of several plant viruses have revealed that maintenance of a stable capsid conformation is dependent on the presence of divalent cations bound to the capsid (25,33,36). It has been a longstanding hypothesis that swelling and other conformational changes induced by ion extraction from virions are critical for a productive viral life cycle (13). For Tomato bushy stunt virus (TBSV), the type species of the genus Tombusvirus within the Tombusviridae family, it was hypothesized that Ca 2ϩ ions would be released from viral capsids within infected cells, leading to virion swelling sufficient to expose the viral genome.Crystallographic studies of TBSV (28) revealed a capsid formed by 180 chemically identical capsid protein (CP) subunits in three quasiequivalent conformations (A, B, and C). Each CP subunit is composed of three distinct structural domains, which include the RNA-interacting (R), shell (S), and protruding (P) domains. The conformational differences that distinguish the A, B, and C subunits are localized within the hinge regions between the respective S and P domains. These hinges point either down (in A-B dimers) or up (in C-C dimers). In addition, the loop that ...