D'Ann Rochon (1955-2022), a life of passion for plant virology.

D'Ann Rochon passed away on November 29th 2022. She is remembered for her outstanding contributions to the field of plant virology, her strong commitment to high quality science and her dedication to the training and mentorship of the next generation of scientists. She was a research scientist for Agriculture and Agri-Food Canada and an Adjunct Professor for the University of British Columbia. Her research program provided new insights on the infection cycle of tombusviruses and related viruses, including ground-breaking research on the structure of virus particles, the mechanisms of virus transmission by fungal zoospores, and the complexity of plant-virus interactions. She also developed diagnostic antibodies for plum pox virus and little cherry virus 2 that have had a significant impact on the management of these viruses.

Dimerization of an umbravirus RNA genome activates subgenomic mRNA transcription.

Many eukaryotic RNA viruses transcribe subgenomic (sg) mRNAs during infections to control expression of a subset of viral genes. Such transcriptional events are commonly regulated by local or long-range intragenomic interactions that form higher-order RNA structures within these viral genomes. In contrast, here we report that an umbravirus activates sg mRNA transcription via base pair-mediated dimerization of its plus-strand RNA genome. Compelling in vivo and in vitro evidence demonstrate that this viral genome dimerizes via a kissing-loop interaction involving an RNA stem-loop structure located just upstream from its transcriptional initiation site. Both specific and non-specific features of the palindromic kissing-loop complex were found to contribute to transcriptional activation. Structural and mechanistic aspects of the process in umbraviruses are discussed and compared with genome dimerization events in other RNA viruses. Notably, probable dimer-promoting RNA stem-loop structures were also identified in a diverse group of umbra-like viruses, suggesting broader utilization of this unconventional transcriptional strategy.

Structure-Based Regulatory Role for the 5'UTR of RCNMV RNA2.

Red clover necrotic mosaic virus (RCNMV) is a segmented positive-strand RNA virus consisting of RNA1 and RNA2. Previous studies demonstrated that efficient translation of RCNMV RNA2 requires de novo synthesis of RNA2 during infections, suggesting that RNA2 replication is required for its translation. We explored a potential mechanism underlying the regulation of replication-associated translation of RNA2 by examining RNA elements in its 5' untranslated region (5'UTR). Structural analysis of the 5'UTR suggested that it can form two mutually exclusive configurations: a more thermodynamically stable conformation, termed the 5'-basal stem structure (5'BS), in which 5'-terminal sequences are base paired, and an alternative conformation, where the 5'-end segment is single stranded. Functional mutational analysis of the 5'UTR structure indicated that (i) 43S ribosomal subunits enter at the very 5'-end of RNA2; (ii) the alternative conformation, containing unpaired 5'-terminal nucleotides, mediates efficient translation; (iii) the 5'BS conformation, with a paired 5'-end segment, supresses translation; and (iv) the 5'BS conformation confers stability to RNA2 from 5'-to-3' exoribonuclease Xrn1. Based on our results, we suggest that during infections, newly synthesized RNA2s transiently adopt the alternative conformation to allow for efficient translation, then refold into the 5'BS conformation, which supresses translation and promotes efficient RNA2 replication. The potential advantages of this proposed 5'UTR-based regulatory mechanism for coordinating RNA2 translation and replication are discussed.

Complex and simple translational readthrough signals in pea enation mosaic virus 1 and potato leafroll virus, respectively.

Different essential viral proteins are translated via programmed stop codon readthrough. Pea enation mosaic virus 1 (PEMV1) and potato leafroll virus (PLRV) are related positive-sense RNA plant viruses in the family Solemoviridae, and are type members of the Enamovirus and Polerovirus genera, respectively. Both use translational readthrough to express a C-terminally extended minor capsid protein (CP), termed CP-readthrough domain (CP-RTD), from a viral subgenomic mRNA that is transcribed during infections. Limited incorporation of CP-RTD subunits into virus particles is essential for aphid transmission, however the functional readthrough structures that mediate CP-RTD translation have not yet been defined. Through RNA solution structure probing, RNA secondary structure modeling, site-directed mutagenesis, and functional in vitro and in vivo analyses, we have investigated in detail the readthrough elements and complex structure involved in expression of CP-RTD in PEMV1, and assessed and deduced a comparatively simpler readthrough structure for PLRV. Collectively, this study has (i) generated the first higher-order RNA structural models for readthrough elements in an enamovirus and a polerovirus, (ii) revealed a stark contrast in the complexity of readthrough structures in these two related viruses, (iii) provided compelling experimental evidence for the strict requirement for long-distance RNA-RNA interactions in generating the active readthrough signals, (iv) uncovered what could be considered the most complex readthrough structure reported to date, that for PEMV1, and (v) proposed plausible assembly pathways for the formation of the elaborate PEMV1 and simple PLRV readthrough structures. These findings notably advance our understanding of this essential mode of gene expression in these agriculturally important plant viruses.

Trans-Activator Binding Site Context in RCNMV Modulates Subgenomic mRNA Transcription.

Many positive-sense RNA viruses transcribe subgenomic (sg) mRNAs during infections that template the translation of a subset of viral proteins. Red clover necrotic mosaic virus (RCNMV) expresses its capsid protein through the transcription of a sg mRNA from RNA1 genome segment. This transcription event is activated by an RNA structure formed by base pairing between a trans-activator (TA) in RNA2 and a trans-activator binding site (TABS) in RNA1. In this study, the impact of the structural context of the TABS in RNA1 on the TA-TABS interaction and sg mRNA transcription was investigated using in vitro and in vivo approaches. The results (i) generated RNA secondary structure models for the TA and TABS, (ii) revealed that the TABS is partially base paired with proximal upstream sequences, which limits TA access, (iii) demonstrated that the aforementioned intra-RNA1 base pairing involving the TABS modulates the TA-TABS interaction in vitro and sg mRNA levels during infections, and (iv) revealed that the TABS in RNA1 can be modified to mediate sg mRNA transcription in a TA-independent manner. These findings advance our understanding of transcriptional regulation in RCNMV and provide novel insights into the origin of the TA-TABS interaction.

RNA Structure Protects the 5' End of an Uncapped Tombusvirus RNA Genome from Xrn Digestion.

One of the many challenges faced by RNA viruses is the maintenance of their genomes during infections of host cells. Members of the family Tombusviridae are plus-strand RNA viruses with unmodified triphosphorylated genomic 5' termini. The tombusvirus Carnation Italian ringspot virus was used to investigate how it protects its RNA genome from attack by 5'-end-targeting degradation enzymes. In vivo and in vitro assays were employed to determine the role of genomic RNA structure in conferring protection from the 5'-to-3' exoribonuclease Xrn. The results revealed that (i) the CIRV RNA genome is more resistant to Xrn than its sg mRNAs, (ii) the genomic 5'-untranslated region (UTR) folds into a compact RNA structure that effectively and independently prevents Xrn access, (iii) the RNA structure limiting 5' access is formed by secondary and tertiary interactions that function cooperatively, (iv) the structure is also able to block access of RNA pyrophosphohydrolase to the genomic 5' terminus, and (v) the RNA structure does not stall an actively digesting Xrn. Based on its proficiency at impeding Xrn 5' access, we have termed this 5'-terminal structure an Xrn-evading RNA, or xeRNA. These and other findings demonstrate that the 5'UTR of the CIRV RNA genome folds into a complex structural conformation that helps to protect its unmodified 5' terminus from enzymatic decay during infections. IMPORTANCE The plus-strand RNA genomes of plant viruses in the large family Tombusviridae are not 5' capped. Here, we explored how a species in the type genus Tombusvirus protects its genomic 5' end from cellular nuclease attack. Our results revealed that the 5'-terminal sequence of the CIRV genome folds into a complex RNA structure that limits access of the 5'-to-3' exoribonuclease Xrn, thereby protecting it from processive degradation. The RNA conformation also impeded access of RNA pyrophosphohydrolase, which converts 5'-triphosphorylated RNA termini into 5'-monophosphorylated forms, the preferred substrate for Xrn. This study represents the first report of a higher-order RNA structure in an RNA plant virus genome independently conferring resistance to 5'-end-attacking cellular enzymes.

Activation of viral transcription by stepwise largescale folding of an RNA virus genome.

The genomes of RNA viruses contain regulatory elements of varying complexity. Many plus-strand RNA viruses employ largescale intra-genomic RNA-RNA interactions as a means to control viral processes. Here, we describe an elaborate RNA structure formed by multiple distant regions in a tombusvirus genome that activates transcription of a viral subgenomic mRNA. The initial step in assembly of this intramolecular RNA complex involves the folding of a large viral RNA domain, which generates a discontinuous binding pocket. Next, a distally-located protracted stem-loop RNA structure docks, via base-pairing, into the binding site and acts as a linchpin that stabilizes the RNA complex and activates transcription. A multi-step RNA folding pathway is proposed in which rate-limiting steps contribute to a delay in transcription of the capsid protein-encoding viral subgenomic mRNA. This study provides an exceptional example of the complexity of genome-scale viral regulation and offers new insights into the assembly schemes utilized by large intra-genomic RNA structures.

Investigation of Novel RNA Elements in the 3'UTR of Tobacco Necrosis Virus-D.

RNA elements in the untranslated regions of plus-strand RNA viruses can control a variety of viral processes including translation, replication, packaging, and subgenomic mRNA production. The 3' untranslated region (3'UTR) of Tobacco necrosis virus strain D (TNV-D; genus Betanecrovirus, family Tombusviridae) contains several well studied regulatory RNA elements. Here, we explore a previously unexamined region of the viral 3'UTR, the sequence located upstream of the 3'-cap independent translation enhancer (3'CITE). Our results indicate that (i) a long-range RNA-RNA interaction between an internal RNA element and the 3'UTR facilitates translational readthrough, and may also promote viral RNA synthesis; (ii) a conserved RNA hairpin, SLX, is required for efficient genome accumulation; and (iii) an adenine-rich region upstream of the 3'CITE is dispensable, but can modulate genome accumulation. These findings identified novel regulatory RNA elements in the 3'UTR of the TNV-D genome that are important for virus survival.

A trans-activator-like structure in RCNMV RNA1 evokes the origin of the trans-activator in RNA2.

The Red clover necrotic mosaic virus (RCNMV) genome consists of two plus-strand RNA genome segments, RNA1 and RNA2. RNA2 contains a multifunctional RNA structure known as the trans-activator (TA) that (i) promotes subgenomic mRNA transcription from RNA1, (ii) facilitates replication of RNA2, and (iii) mediates particle assembly and copackaging of genome segments. The TA has long been considered a unique RNA element in RCNMV. However, by examining results from RCNMV genome analyses in the ViRAD virus (re-)annotation database, a putative functional RNA element in the polymerase-coding region of RNA1 was identified. Structural and functional analyses revealed that the novel RNA element adopts a TA-like structure (TALS) and, similar to the requirement of the TA for RNA2 replication, the TALS is necessary for the replication of RNA1. Both the TA and TALS possess near-identical asymmetrical internal loops that are critical for efficient replication of their corresponding genome segments, and these structural motifs were found to be functionally interchangeable. Moreover, replacement of the TA in RNA2 with a stabilized form of the TALS directed both RNA2 replication and packaging of both genome segments. Based on their comparable properties and considering evolutionary factors, we propose that the TALS appeared de novo in RNA1 first and, subsequently, the TA arose de novo in RNA2 as a functional mimic of the TALS. This and other related information were used to formulate a plausible evolutionary pathway to describe the genesis of the bi-segmented RCNMV genome. The resulting scenario provides an evolutionary framework to further explore and test possible origins of this segmented RNA plant virus.

A 212-nt long RNA structure in the Tobacco necrosis virus-D RNA genome is resistant to Xrn degradation.

Plus-strand RNA viruses can accumulate viral RNA degradation products during infections. Some of these decay intermediates are generated by the cytosolic 5'-to-3' exoribonuclease Xrn1 (mammals and yeast) or Xrn4 (plants) and are formed when the enzyme stalls on substrate RNAs upon encountering inhibitory RNA structures. Many Xrn-generated RNAs correspond to 3'-terminal segments within the 3'-UTR of viral genomes and perform important functions during infections. Here we have investigated a 3'-terminal small viral RNA (svRNA) generated by Xrn during infections with Tobacco necrosis virus-D (family Tombusviridae). Our results indicate that (i) unlike known stalling RNA structures that are compact and modular, the TNV-D structure encompasses the entire 212 nt of the svRNA and is not functionally transposable, (ii) at least two tertiary interactions within the RNA structure are required for effective Xrn blocking and (iii) most of the svRNA generated in infections is derived from viral polymerase-generated subgenomic mRNA1. In vitro and in vivo analyses allowed for inferences on roles for the svRNA. Our findings provide a new and distinct addition to the growing list of Xrn-resistant viral RNAs and stalling structures found associated with different plant and animal RNA viruses.

Trans-Acting RNA-RNA Interactions in Segmented RNA Viruses.

RNA viruses represent a large and important group of pathogens that infect a broad range of hosts. Segmented RNA viruses are a subclass of this group that encode their genomes in two or more molecules and package all of their RNA segments in a single virus particle. These divided genomes come in different forms, including double-stranded RNA, coding-sense single-stranded RNA, and noncoding single-stranded RNA. Genera that possess these genome types include, respectively, Orbivirus (e.g., Bluetongue virus), Dianthovirus (e.g., Red clover necrotic mosaic virus) and Alphainfluenzavirus (e.g., Influenza A virus). Despite their distinct genomic features and diverse host ranges (i.e., animals, plants, and humans, respectively) each of these viruses uses trans-acting RNA-RNA interactions (tRRIs) to facilitate co-packaging of their segmented genome. The tRRIs occur between different viral genome segments and direct the selective packaging of a complete genome complement. Here we explore the current state of understanding of tRRI-mediated co-packaging in the abovementioned viruses and examine other known and potential functions for this class of RNA-RNA interaction.

Intragenomic Long-Distance RNA-RNA Interactions in Plus-Strand RNA Plant Viruses.

Plant viruses that contain positive-strand RNA genomes represent an important class of pathogen. The genomes of these viruses harbor RNA sequences and higher-order RNA structures that are essential for the regulation of viral processes during infections. In recent years, it has become increasingly evident that, in addition to locally positioned RNA structures, long-distance intragenomic interactions, involving nucleotide base pairing over large distances, also contribute significantly to the control of various viral events. Viral processes that are modulated by such interactions include genome replication, translation initiation, translational recoding, and subgenomic mRNA transcription. Here, we review the structure and function of different types of long-distance RNA-RNA interactions, herein termed LDRIs, present in members of the family Tombusviridae and other plus-strand RNA plant viruses.

Expression of T7-based constructs in tobacco cells.

Bacteriophage T7 promoter and RNA polymerase (T7-Pol) are widely used for recombinant protein expression in bacteria. In plants, there exists conflicting results regarding the efficacy of protein expression from T7-Pol-derived mRNAs. To reconcile these contradictory observations, the expression of green fluorescent protein (GFP) from T7 constructs was evaluated in tobacco protoplasts. T7 constructs transcribed by a nuclearly targeted T7-Pol did not express GFP in plant protoplasts, however T7-Pol lacking a nuclear targeting signal was able to translate cytosolically transcribed mRNAs, but only if the messages contained a viral translation enhancer. GFP expression was further evaluated at the plant level by using agroinfiltration-mediated transient expression system. Unlike for cytosolic expression, nuclear T7 transcripts containing a viral translation enhancer element did not express GFP, and modifications designed to stabilize and facilitate export of T7 transcripts to the cytosol did not improve the expression. We conclude that expression of nuclear T7 constructs is not feasible in tobacco cells, but cytosolic transcription provides an alternative means to over-express RNAs directly in the cytosol.

Atypical RNA Elements Modulate Translational Readthrough in Tobacco Necrosis Virus D.

Tobacco necrosis virus, strain D (TNV-D), is a positive-strand RNA virus in the genus Betanecrovirus and family Tombusviridae The production of its RNA-dependent RNA polymerase, p82, is achieved by translational readthrough. This process is stimulated by an RNA structure that is positioned immediately downstream of the recoding site, termed the readthrough stem-loop (RTSL), and a sequence in the 3' untranslated region of the TNV-D genome, called the distal readthrough element (DRTE). Notably, a base pairing interaction between the RTSL and the DRTE, spanning ∼3,000 nucleotides, is required for enhancement of readthrough. Here, some of the structural features of the RTSL, as well as RNA sequences and structures that flank either the RTSL or DRTE, were investigated for their involvement in translational readthrough and virus infectivity. The results revealed that (i) the RTSL-DRTE interaction cannot be functionally replaced by stabilizing the RTSL structure, (ii) a novel tertiary RNA structure positioned just 3' to the RTSL is required for optimal translational readthrough and virus infectivity, and (iii) these same activities also rely on an RNA stem-loop located immediately upstream of the DRTE. Functional counterparts for the RTSL-proximal structure may also be present in other tombusvirids. The identification of additional distinct RNA structures that modulate readthrough suggests that regulation of this process by genomic features may be more complex than previously appreciated. Possible roles for these novel RNA elements are discussed.IMPORTANCE The analysis of factors that affect recoding events in viruses is leading to an ever more complex picture of this important process. In this study, two new atypical RNA elements were shown to contribute to efficient translational readthrough of the TNV-D polymerase and to mediate robust viral genome accumulation in infections. One of the structures, located close to the recoding site, could have functional equivalents in related genera, while the other structure, positioned 3' proximally in the viral genome, is likely limited to betanecroviruses. Irrespective of their prevalence, the identification of these novel RNA elements adds to the current repertoire of viral genome-based modulators of translational readthrough and provides a notable example of the complexity of regulation of this process.

Tombusvirus polymerase: Structure and function.

Tombusviruses are small icosahedral viruses that possess plus-sense RNA genomes ∼4.8kb in length. The type member of the genus, tomato bushy stunt virus (TBSV), encodes a 92kDa (p92) RNA-dependent RNA polymerase (RdRp) that is responsible for viral genome replication and subgenomic (sg) mRNA transcription. Several functionally relevant regions in p92 have been identified and characterized, including transmembrane domains, RNA-binding segments, membrane targeting signals, and oligomerization domains. Moreover, conserved tombusvirus-specific motifs in the C-proximal region of the RdRp have been shown to modulate viral genome replication, sg mRNA transcription, and trans-replication of subviral replicons. Interestingly, p92 is initially non-functional, and requires an accessory viral protein, p33, as well as viral RNA, host proteins, and intracellular membranes to become active. These and other host factors, through a well-orchestrated process guided by the viral replication proteins, mediate the assembly of membrane-associated virus replicase complexes (VRCs). Here, we describe what is currently known about the structure and function of the tombusvirus RdRp and how it utilizes host components to build VRCs that synthesize viral RNAs.

Protein expression strategies in Tobacco necrosis virus-D.

Tobacco necrosis virus (TNV-D) has a plus-strand RNA genome that is neither 5' capped nor 3' poly-adenylated. Instead, it utilizes a 3' cap-independent translational enhancer (3'CITE) located in its 3' untranslated region (UTR) for translation of its proteins. We have examined the protein expression strategies used by TNV-D and our results indicate that: (i) a base pairing interaction between conserved ACCA and UGGU motifs in the genomic 5'UTR and 3'CITE, respectively, is not required for efficient plant cell infection, (ii) similar potential 5'UTR-3'CITE interactions in the two viral subgenomic mRNAs are not needed for efficient translation of viral proteins in vitro, (iii) a small amount of capsid protein is translated from the viral genome by a largely 3'CITE-independent mechanism, (iv) the larger of two possible forms of capsid protein is efficiently translated, and (v) p7b is translated from subgenomic mRNA1 by a leaky scanning mechanism.

Biologically-supported structural model for a viral satellite RNA.

Satellite RNAs (satRNAs) are a class of small parasitic RNA replicon that associate with different viruses, including plus-strand RNA viruses. Because satRNAs do not encode a polymerase or capsid subunit, they rely on a companion virus to provide these proteins for their RNA replication and packaging. SatRNAs recruit these and other required factors via their RNA sequences and structures. Here, through a combination of chemical probing analysis of RNA structure, phylogenetic structural comparisons, and viability assays of satRNA mutants in infected cells, the biological importance of a deduced higher-order structure for a 619 nt long tombusvirus satRNA was assessed. Functionally-relevant secondary and tertiary RNA structures were identified throughout the length of the satRNA. Notably, a 3'-terminal segment was found to adopt two mutually-exclusive RNA secondary structures, both of which were required for efficient satRNA accumulation. Accordingly, these alternative conformations likely function as a type of RNA switch. The RNA switch was also found to engage in a required long-range kissing-loop interaction with an upstream sequence. Collectively, these results establish a high level of conformational complexity within this small parasitic RNA and provide a valuable structural framework for detailed mechanistic studies.

Pelarspovirus, a proposed new genus in the family Tombusviridae.

Currently, the family Tombusviridae encompasses thirteen viral genera that contain single-stranded, positive-sense RNA genomes and isometric virions; the exception being the genus Umbravirus, whose members do not encode a coat protein (CP). A new genus, tentatively named Pelarspovirus, is proposed to be added to this family and would include five members, with Pelargonium line pattern virus recommended as the type species. Viruses assigned to this proposed genus have monopartite genomes encoding five open reading frames (ORFs) that include two 5'-proximal replication proteins, two centrally located movement proteins (MP1 and MP2) and a 3'-proximal CP that, at least for pelargonium line pattern virus (PLPV), has been shown to act also as suppressor of RNA silencing. Distinguishing characteristics of these viruses include i) production of a single, tricistronic subgenomic RNA for expression of MP and CP genes, ii) presence of a non-AUG start codon (CUG or GUG) initiating the MP2 ORF, iii) absence of AUG codons in any frame between the AUG initiation codons of MP1 and CP genes, and iv) sequence-based phylogenetic clustering of all encoded proteins in separate clades from those of other family members.

Exploring the architecture of viral RNA genomes.

The genomes of RNA viruses contain local structural elements and long-range interactions that control various steps in virus replication. While many individual RNA elements have been characterized, it remains less clear how the structure and activity of such elements are integrated and regulated within the complex context of complete viral genomes. Recent technical advances, particularly the development of high-throughput solution structure mapping methods, have made secondary structural analysis of entire viral RNA genomes feasible. As a consequence, whole-genome structural models have been deduced for a number of plus-strand RNA viruses and retroviruses and these structures have provided intriguing functional and evolutionary insights into global genome architecture.