Viral Genome-linked Protein Research Articles

The Molecular Maze of Potyviral and Host Protein Interactions.

The negative effects of potyvirus diseases on the agricultural industry are extensive and global. Understanding how protein-protein interactions contribute to potyviral infections is imperative to developing resistant varieties that help counter the threat potyviruses pose. While many protein-protein interactions have been reported, only a fraction are essential for potyviral infection. Accumulating evidence demonstrates that potyviral infection processes are interconnected. For instance, the interaction between the eukaryotic initiation factor 4E (eIF4E) and viral protein genome-linked (VPg) is crucial for both viral translation and protecting viral RNA (vRNA). Additionally, recent evidence for open reading frames on the reverse-sense vRNA and for nonequimolar expression of viral proteins has challenged the previous polyprotein expression model. These discoveries will surely reveal more about the potyviral protein interactome. In this review, we present a synthesis of the potyviral infection cycle and discuss influential past discoveries and recent work on protein-protein interactions in various infection processes.

Recessive resistance against beet chlorosis virus is conferred by the eukaryotic translation initiation factor (iso)4E in Beta vulgaris.

Eukaryotic translation initiation factors (eIFs) are important for mRNA translation but also pivotal for plant-virus interaction. Most of these plant-virus interactions were found between plant eIFs and the viral protein genome-linked (VPg) of potyviruses. In case of lost interaction due to mutation or deletion of eIFs, the viral translation and subsequent replication within its host is negatively affected, resulting in a recessive resistance. Here we report the identification of the Beta vulgaris Bv-eIF(iso)4E as a susceptibility factor towards the VPg-carrying beet chlorosis virus (genus Polerovirus). Using yeast two-hybrid and bimolecular fluorescence complementation assays, the physical interaction between Bv-eIF(iso)4E and the putative BChV-VPg was detected, while the VPg of the closely related beet mild yellowing virus (BMYV) was found to interact with the two isoforms Bv-eIF4E and Bv-eIF(iso)4E. These VPg-eIF interactions within the polerovirus-beet pathosystem were demonstrated to be highly specific, as single mutations within the predicted cap-binding pocket of Bv-eIF(iso)4E resulted in a loss of interaction. To investigate the suitability of eIFs as a resistance resource against beet infecting poleroviruses, B.vulgaris plants were genome edited by CRISPR/Cas9 resulting in knockouts of different eIFs. Asimultaneous knockout of the identified BMYV-interaction partners Bv-eIF4E and Bv-eIF(iso)4E was not achieved, but Bv-eIF(iso)4EKO plants showed a significantly lowered BChV accumulation and decrease in infection rate from 100% to 28.86%, while no influence on BMYV accumulation was observed. Still, these observations support that eIFs are promising candidate genes for polerovirus resistance breeding in sugar beet.

Tryptanthrin Derivative B1 Binds Viral Genome-Linked Protein (VPg) of Potato Virus Y.

Potato virus Y (PVY) is a plant virus that is known to be responsible for substantial economic losses in agriculture. Within the PVY genome, viral genome-linked protein (VPg) plays a pivotal role in the viral translation process. In this study, VPg was used as a potential target for analyzing the antiviral activity of tryptanthrin derivatives. In vitro, the dissociation constants of B1 with PVY VPg were 0.69 μmol/L (measured by microscale thermophoresis) and 4.01 μmol/L (measured via isothermal titration calorimetry). B1 also strongly bound to VPg proteins from three other Potyviruses. Moreover, in vivo experiments demonstrated that B1 effectively suppressed the expression of the PVY gene. Molecular docking experiments revealed that B1 formed a hydrogen bond with N121 and that no specific binding occurred between B1 and the PVY VPgN121A mutant. Therefore, N121 is a key amino acid residue in PVY VPg involved in B1 binding. These results highlight the potential of PVY VPg as a potential target for the development of antiviral agents.

Predictive Modeling of Proteins Encoded by a Plant Virus Sheds a New Light on Their Structure and Inherent Multifunctionality.

Plant virus genomes encode proteins that are involved in replication, encapsidation, cell-to-cell, and long-distance movement, avoidance of host detection, counter-defense, and transmission from host to host, among other functions. Even though the multifunctionality of plant viral proteins is well documented, contemporary functional repertoires of individual proteins are incomplete. However, these can be enhanced by modeling tools. Here, predictive modeling of proteins encoded by the two genomic RNAs, i.e., RNA1 and RNA2, of grapevine fanleaf virus (GFLV) and their satellite RNAs by a suite of protein prediction software confirmed not only previously validated functions (suppressor of RNA silencing [VSR], viral genome-linked protein [VPg], protease [Pro], symptom determinant [Sd], homing protein [HP], movement protein [MP], coat protein [CP], and transmission determinant [Td]) and previously identified putative functions (helicase [Hel] and RNA-dependent RNA polymerase [Pol]), but also predicted novel functions with varying levels of confidence. These include a T3/T7-like RNA polymerase domain for protein 1AVSR, a short-chain reductase for protein 1BHel/VSR, a parathyroid hormone family domain for protein 1EPol/Sd, overlapping domains of unknown function and an ABC transporter domain for protein 2BMP, and DNA topoisomerase domains, transcription factor FBXO25 domain, or DNA Pol subunit cdc27 domain for the satellite RNA protein. Structural predictions for proteins 2AHP/Sd, 2BMP, and 3A? had low confidence, while predictions for proteins 1AVSR, 1BHel*/VSR, 1CVPg, 1DPro, 1EPol*/Sd, and 2CCP/Td retained higher confidence in at least one prediction. This research provided new insights into the structure and functions of GFLV proteins and their satellite protein. Future work is needed to validate these findings.

Ryegrass mottle virus complete genome determination and development of infectious cDNA by combining two methods- 3' RACE and RNA-Seq.

Ryegrass mottle virus (RGMoV; genus: Sobemovirus) is a single-stranded positive RNA virus with a 30 nm viral particle size. It exhibits T = 3 symmetry with 180 coat protein (CP) subunits forming a viral structure. The RGMoV genome comprises five open reading frames that encode P1, Px, a membrane-anchored 3C-like serine protease, a viral genome-linked protein, P16, an RNA-dependent RNA polymerase, and CP. The RGMoV genome size varies, ranging from 4175 nt (MW411579.1) to 4253 nt (MW411579.1) in the deposited sequences. An earlier deposited RGMoV complete genome sequence of 4212 nt length (EF091714.1) was used to develop an infectious complementary DNA (icDNA) construct for in vitro gRNA transcription from the T7 promoter. However, viral infection was not induced when the transcribed gRNA was introduced into oat plants, indicating the potential absence of certain sequences in either the 5' or 3' untranslated regions (UTR) or both. The complete sequence of the 3' UTR was determined through 3' end RACE, while the 5' UTR was identified using high-throughput sequencing (HTS)-RNA-Seq to resolve the potential absences. Only the icDNA vector containing the newly identified UTR sequences proved infectious, resulting in typical viral infection symptoms and subsequent propagation of progeny viruses, exhibiting the ability to cause repeated infections in oat plants after at least one passage. The successful generation of icDNA highlighted the synergistic potential of utilizing both methods when a single approach failed. Furthermore, this study demonstrated the reliability of HTS as a method for determining the complete genome sequence of viral genomes.

Evaluation of a Series of Turnip Mosaic Virus Chimeric Clones Reveals Two Amino Acid Sites Critical for Systemic Infection in Chinese Cabbage.

Two infectious clones of turnip mosaic virus (TuMV), pKBC-1 and pKBC-8, with differential infectivity in Chinese cabbage (Brassica rapa subsp. pekinensis), were obtained. Both infected Nicotiana benthamiana systemically, inducing similar symptoms, whereas only virus KBC-8 infected Chinese cabbage systemically. To identify the determinants affecting infectivity on Chinese cabbage, chimeric clones were constructed by restriction fragment exchange between the parental clones and tested on several Chinese cabbage cultivars. Chimeric clones p1N8C and p8N1C demonstrated that the C-terminal portion of the polyprotein determines systemic infection of Chinese cabbage despite only three amino acid differences in this region, in the cylindrical inclusion (CI), viral protein genome-linked (VPg), and coat protein (CP). A second pair of hybrid constructs, pHindIII-1N8C and pHindIII-8N1C, failed to infect cultivars CR Victory and Jinseonnorang systemically, yet pHindIII-1N8C caused hypersensitive response-like lesions on inoculated leaves of these cultivars, and could systemically infect cultivars CR Chusarang and Jeongsang; this suggests that R genes effective against TuMV may exist in the first two cultivars but not the latter two. Constructs with single amino acid changes in both VPg (K2045E) and CP (Y3095H) failed to infect Chinese cabbage, implying that at least one of these two amino acid substitutions is essential for successful infection on Chinese cabbage. Successful infection by mutant KBC-8-CP-H and delayed infection with mutant HJY1-VPg-E following mutation or reversion suggested that VPg (2045K) is the residue required for infection of Chinese cabbage and involved in the interaction between VPg and eukaryotic initiation factor eIF(iso)4E, confirmed by yeast two-hybrid assay.

Synthesis and Screening of Chemical Agents Targeting Viral Protein Genome-Linked Protein of Telosma Mosaic Virus.

The viral protein genome-linked protein (VPg) of telosma mosaic virus (TeMV) plays an important role in viral reproduction. In this study, the expression conditions of TeMV VPg were explored. A series of novel benzenesulfonamide derivatives were synthesized. The binding sites of the target compounds and TeMV VPg were studied by molecular docking, and the interaction was verified by microscale thermophoresis. The study revealed that the optimal expression conditions for TeMV VPg were in Escherichia coli Rosetta with IPTG concentration of 0.8 mM and induction temperature of 25 °C. Compounds A4, A6, A9, A16, and A17 exhibited excellent binding affinity to TeMV VPg, with Kd values of 0.23, 0.034, 0.19, 0.086, and 0.22 μM, respectively. LYS 121 is the key amino acid site. Compounds A9 inhibited the expression of TeMV VPg in Nicotiana benthamiana. The results suggested that TeMV VPg is a potential antiviral target to screen anti-TeMV compounds.

Identification of a novel dicistro-like virus associated with the roots of tomato plants.

Viruses belonging to the family Dicistroviridae have a monopartite positive-sense single-stranded RNA genome and infect a variety of arthropods. Using high-throughput sequencing, we detected a novel dicistro-like virus, tentatively named "tomato root-associated dicistro-like virus" (TRaDLV), in the roots of tomato plants showing yellow mosaic symptoms on the leaves. The diseased tomato plants were coinfected with multiple plant viruses, and TRaDLV was present in the roots but not in the leaves. The genome of TRaDLV is 8726 nucleotides in length, excluding the poly(A) tail, and contains two open reading frames (ORFs) separated by an intergenic region (IGR). The TRaDLV genome showed characteristics similar to those of dicistroviruses, including the presence of a 3C-like protease domain, repeated amino acid sequences representing multiple copies of viral genome-linked protein (VPg)-like sequences in the ORF1 polyprotein, and a series of stem-loop structures resembling an internal ribosome entry site in the IGR. Phylogenetic analysis revealed that TRaDLV clustered with unclassified dicistro-like viruses from invertebrates or identified in samples of plant-derived material. These findings indicate the existence of a novel dicistro-like virus that may associate with plant roots or a root-inhabiting organism.

Molecular Tools to Infer Resistance-Breaking Abilities of Rice Yellow Mottle Virus Isolates.

Rice yellow mottle virus (RYMV) is a major biotic constraint to rice cultivation in Africa. RYMV shows a high genetic diversity. Viral lineages were defined according to the coat protein (CP) phylogeny. Varietal selection is considered as the most efficient way to manage RYMV. Sources of high resistance were identified mostly in accessions of the African rice species, Oryza glaberrima. Emergence of resistance-breaking (RB) genotypes was observed in controlled conditions. The RB ability was highly contrasted, depending on the resistance sources and on the RYMV lineages. A molecular marker linked to the adaptation to susceptible and resistant O. glaberrima was identified in the viral protein genome-linked (VPg). By contrast, as no molecular method was available to identify the hypervirulent lineage able to overcome all known resistance sources, plant inoculation assays were still required. Here, we designed specific RT-PCR primers to infer the RB abilities of RYMV isolates without greenhouse experiments or sequencing steps. These primers were tested and validated on 52 isolates, representative of RYMV genetic diversity. The molecular tools described in this study will contribute to optimizing the deployment strategy of resistant lines, considering the RYMV lineages identified in fields and their potential adaptability.

Multiple Viral Protein Genome-Linked Proteins Compensate for Viral Translation in a Positive-Sense Single-Stranded RNA Virus Infection.

Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of plus-stranded RNA viruses. VPg is covalently linked to the 5' end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses have multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use cricket paralysis virus (CrPV), a dicistrovirus that has four nonidentical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses contain two main open reading frames (ORFs) that are driven by distinct internal ribosome entry sites (IRESs). We systematically generated single and combinatorial deletions and mutations of VPg1 to VPg4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, mutations of which in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant negative effect. Using viral minigenome reporters that monitor dicistrovirus 5' untranslated (UTR) and IRES translation revealed a relationship between VPg copy number and the ratio of distinct IRES translation activities. We uncovered a novel viral strategy whereby VPg copies in dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote infection. IMPORTANCE Genetic duplication is exceedingly rare in small RNA viral genomes, as there is selective pressure to prevent RNA genomes from expanding. However, some small RNA viruses encode multiple copies of a viral protein, most notably an unusual viral protein that is linked to the viral RNA genome. Here, we investigate a family of viruses that contains multiple viral protein genome-linked proteins and reveal a novel viral strategy whereby viral protein copy number counterbalances differences in viral protein synthesis mechanisms.

Genomic and Pathogenic Diversity of Barley Yellow Mosaic Virus and Barley Mild Mosaic Virus Isolates in Fields of China and Their Compatibility with Resistance Genes of Cultivated Barley.

Plant viruses transmitted by the soilborne plasmodiophorid Polymyxa graminis constantly threaten global production of cereal crops. Although the yellow mosaic virus disease of barley has been known to be present for a long time in China, the understanding of the diversity of the viral pathogens and their interactions with host resistance remains limited. In this study, we conducted a nationwide survey of P. graminis and the barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) it transmits, followed by genomic and pathogenic diversity analyses of both viruses. BaYMV and BaMMV were found exclusively in the region downstream of the Yangtze River, despite the national distribution of its transmission vector P. graminis. Analysis of the genomic variations of BaYMV and BaMMV revealed an elevated rate of nonsynonymous substitutions in the viral genome-linked protein (VPg), in which most substitutions were located in its interaction surface with the host eukaryotic translation initiation factor 4E (eIF4E). VPg sequence diversity was associated with the divergence in virus pathogenicity that was identified through multiple field trials. The majority of the resistance genes, including the widely applied rym4 and rym5 (alleles of eIF4E), as well as the combination of rym1/11 and rym5, are not sufficient to protect cultivated barley against viruses in China. Collectively, these results provide insights into virulence specificity and interaction mode with host resistance in cultivated barley, which has significant implications in breeding for the broad-spectrum resistance barley varieties.

Cassava Brown Streak Viruses express second 6-kilodalton (6K2) protein with varied polarity and three dimensional (3D) structures: Basis for trait discrepancy between the virus species.

Cassava Brown Streak Virus (CBSV) and Ugandan Cassava Brown Streak Virus (UCBSV) are the two among six virus species speculated to cause the most catastrophic Brown Streak Disease of Cassava (CBSD) in Africa and Asia. Cassava Brown Streak Virus (CBSV) is hard to breed resistance for compared to Ugandan Cassava Brown Streak Virus (UCBSV) species. This is exemplified by incidences of CBSV species rather than UCBSV species in elite breeding line, KBH 2006/0026 at Bagamoyo, Tanzania. It is not yet understood as to why CBSV species could breakdown CBSD-resistance in the KBH 2006/0026 unlike the UCBSV species. This marks the first in silico study conducted to understand molecular basis for the trait discrepancy between CBSV and UCBSV species from structural biology view point. Following ab initio modelling and analysis of physical-chemical properties of second 6-kilodalton (6K2) protein encoded by CBSV and UCBSV species, using ROBETTA server and Protein Parameters tool, respectively we report that; three dimensional (3D) structures and polarity of the protein differs significantly between the two virus species. (95% and 5%) and (85% and 15%) strains of 20 CBSV and 20 UCBSV species respectively, expressed the protein in homo-trimeric and homo-tetrameric forms, correspondingly. 95% and 85% of studied strain population of the two virus species expressed hydrophilic and hydrophobic 6K2, respectively. Based on findings of the curent study, we hypothesize that; (i) The hydrophilic 6K2 expressed by the CBSV species, favour its faster systemic movement via vascular tissues of cassava host and hence result into higher tissue titres than the UCBSV species encoding hydrophobic form of the protein. t and (ii) The hydrophilic 6K2 expressed byCBSV species have additional interaction advantage with Nuclear Inclusion b protease domain (NIb) and Viral genome-linked protein (VPg), components of Virus Replication Complex (VRC) and hence contributing to faster replication of viral genome than the hydrophobic 6K2 expressed by the UCBSV species. Experimental studies are needed to resolve the 3D structures of the 6K2, VPg and NIb and comprehend complex molecular interactions between them. We suggest that, the 6K2 gene should be targeted for improvement of RNA interference (RNAi)-directed transgenesis of virus-resistant cassava as a more effective way to control the CBSD besides breeding.

Phosphorylation of eukaryotic initiation factor eIFiso4E enhances the binding rates to VPg of turnip mosaic virus.

Binding of phosphorylated eIFiso4E with viral genome-linked protein (VPg) of turnip mosaic virus was examined by stopped-flow, fluorescence, circular dichroism (CD) spectroscopy, and molecular docking analysis. Phosphorylation of eIFiso4E increased (4-fold) the binding rates as compared to unphosphorylated eIFiso4E with VPg. Stopped-flow kinetic studies of phosphorylated eIFiso4E with VPg showed a concentration-independent conformational change. The dissociation rate was about 3-fold slower for eIFiso4E∙VPg complex upon phosphorylation. Phosphorylation enhanced the association rates and lowered the dissociation rates for the eIFiso4E∙VPg binding, with having higher preferential binding to eIFiso4Ep. Binding rates for the interaction of eIFiso4Ep with VPg increased (6-fold) with an increase in temperature, 278 K to 298 K. The activation energies for binding of eIFiso4Ep and eIFiso4E with VPg were 37.2 ± 2.8 and 52.6 ± 3.6 kJ/mol, respectively. Phosphorylation decreased the activation energy for the binding of eIFiso4E to VPg. The reduced energy barrier suggests more stable platform for eIFiso4Ep∙VPg initiation complex formation, which was further supported by molecular docking analysis. Moreover, far-UV CD studies revealed that VPg formed complex with eIFiso4Ep with substantial change in the secondary structure. These results suggested that phosphorylation, not only reduced the energy barrier and dissociation rate but also enhanced binding rate, and an overall conformational change, which provides a more stable platform for efficient viral translation.

VPg of Potato Virus Y and Potato Cap-Binding eIF4E Factors: Selective Interaction and Its Supposed Mechanism.

Potato virus Y (PVY) is one of the most common and harmful plant viruses. Translation of viral RNA starts with the interaction between the plant cap-binding translation initiation factors eIF4E and viral genome-linked protein (VPg) covalently attached to the viral RNA. Disruption of this interaction is one of the natural mechanisms of plant resistance to PVY. The multigene eIF4E family in the potato (Solanum tuberosum L.) genome contains genes for the translation initiation factors eIF4E1, eIF4E2, and eIF(iso)4E. However, which of these factors can be recruited by the PVY, as well as the mechanism of this interaction, remain obscure. Here, we showed that the most common VPg variant from the PVY strain NTN interacts with eIF4E1 and eIF4E2, but not with eIF(iso)4E. Based on the VPg, eIF4E1, and eIF4E2 models and data on the natural polymorphism of VPg amino acid sequence, we suggested that the key role in the recognition of potato cap-binding factors belongs to the R104 residue of VPg. To verify this hypothesis, we created VPg mutants with substitutions at position 104 and examined their ability to interact with potato eIF4E factors. The obtained data were used to build the theoretical model of the VPg-eIF4E2 complex that differs significantly from the earlier models of VPg complexes with eIF4E proteins, but is in a good agreement with the current biochemical data.

Protein Nucleotidylylation in +ssRNA Viruses.

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5′ end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.

Calicivirus Non-structural Proteins: Potential Functions in Replication and Host Cell Manipulation.

The Caliciviridae are a family of viruses with a single-stranded, non-segmented RNA genome of positive polarity. The ongoing discovery of caliciviruses has increased the number of genera in this family to 11 (Norovirus, Nebovirus, Sapovirus, Lagovirus, Vesivirus, Nacovirus, Bavovirus, Recovirus, Salovirus, Minovirus, and Valovirus). Caliciviruses infect a wide range of hosts that include fishes, amphibians, reptiles, birds, and marine and land mammals. All caliciviruses have a genome that encodes a major and a minor capsid protein, a genome-linked viral protein, and several non-structural proteins. Of these non-structural proteins, only the helicase, protease, and RNA-dependent RNA polymerase share clear sequence and structural similarities with proteins from other virus families. In addition, all caliciviruses express two or three non-structural proteins for which functions have not been clearly defined. The sequence diversity of these non-structural proteins and a multitude of processing strategies suggest that at least some have evolved independently, possibly to counteract innate and adaptive immune responses in a host-specific manner. Studying these proteins is often difficult as many caliciviruses cannot be grown in cell culture. Nevertheless, the study of recombinant proteins has revealed many of their properties, such as intracellular localization, capacity to oligomerize, and ability to interact with viral and/or cellular proteins; the release of non-structural proteins from transfected cells has also been investigated. Here, we will summarize these findings and discuss recent in silico studies that identified previously overlooked putative functional domains and structural features, including transmembrane domains that suggest the presence of viroporins.

Norovirus VPg Binds RNA through a Conserved N-Terminal K/R Basic Patch.

The viral protein genome-linked (VPg) of noroviruses is a multi-functional protein that participates in essential roles during the viral replication cycle. Predictive analyses indicate that murine norovirus (MNV) VPg contains a disordered N-terminal region with RNA binding potential. VPg proteins were expressed with an N-terminal spidroin fusion protein in insect cells and the interaction with RNA investigated by electrophoretic mobility shift assays (EMSA) against a series of RNA probes (pentaprobes) representing all possible five nucleotide combinations. MNV VPg and human norovirus (HuNV) VPg proteins were directly bound to RNA in a non-specific manner. To identify amino acids involved in binding to RNA, all basic (K/R) residues in the first 12 amino acids of MNV VPg were mutated to alanine. Removal of the K/R amino acids eliminated RNA binding and is consistent with a K/R basic patch RNA binding motif within the disordered N-terminal region of norovirus VPgs. Finally, we show that mutation of the K/R basic patch required for RNA binding eliminates the ability of MNV VPg to induce a G0/G1 cell cycle arrest.

Insights Into Natural Genetic Resistance to Rice Yellow Mottle Virus and Implications on Breeding for Durable Resistance.

Rice is the main food crop for people in low- and lower-middle-income countries in Asia and sub-Saharan Africa (SSA). Since 1982, there has been a significant increase in the demand for rice in SSA, and its growing importance is reflected in the national strategic food security plans of several countries in the region. However, several abiotic and biotic factors undermine efforts to meet this demand. Rice yellow mottle virus (RYMV) caused by Solemoviridae is a major biotic factor affecting rice production and continues to be an important pathogen in SSA. To date, six pathogenic strains have been reported. RYMV infects rice plants through wounds and rice feeding vectors. Once inside the plant cells, viral genome-linked protein is required to bind to the rice translation initiation factor [eIF(iso)4G1] for a compatible interaction. The development of resistant cultivars that can interrupt this interaction is the most effective method to manage this disease. Three resistance genes are recognized to limit RYMV virulence in rice, some of which have nonsynonymous single mutations or short deletions in the core domain of eIF(iso)4G1 that impair viral host interaction. However, deployment of these resistance genes using conventional methods has proved slow and tedious. Molecular approaches are expected to be an alternative to facilitate gene introgression and/or pyramiding and rapid deployment of these resistance genes into elite cultivars. In this review, we summarize the knowledge on molecular genetics of RYMV-rice interaction, with emphasis on host plant resistance. In addition, we provide strategies for sustainable utilization of the novel resistant sources. This knowledge is expected to guide breeding programs in the development and deployment of RYMV resistant rice varieties.

Mapping of sequences in the 5' region and 3' UTR of tomato ringspot virus RNA2 that facilitate cap-independent translation of reporter transcripts in vitro.

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5'-viral genome-linked protein (VPg) and have a 3' polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3' untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5' regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5' region and 3' UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5' region and 3' UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5' proximal coding region of the RNA2 polyprotein and in the RNA2 3' UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3' UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3' UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5' region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773-972) of the 1547 nt long 3' UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.

Selective Interaction of Sugarcane eIF4E with VPgs from Sugarcane Mosaic Pathogens.

Eukaryotic translation initiation factor 4E (eIF4E) plays a key role in the infection of potyviruses in susceptible plants by interacting with viral genome-linked protein (VPg). Sugarcane (Saccharum spp.) production is threatened by mosaic disease caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and Sugarcane streak mosaic virus (SCSMV). In this study, two eIF4Es and their isoform eIF(iso)4E and 4E-binding protein coding genes were cloned from sugarcane cultivar ROC22 and designated SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP, respectively. Real-time quantitative PCR analysis showed different expression profiles of these four genes upon SCMV challenge. A subcellular localization assay showed that SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP were distributed in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SceIF4Ea/b and SceIF(iso)4E were selectively employed by different sugarcane mosaic pathogens, i.e., SCMV-VPg interacted with SceIF4Ea/b and SceIF(iso)4E, SrMV-VPg interacted with both SceIF4Eb and SceIF(iso)4E, and SCSMV-VPg interacted only with SceIF(iso)4E. Intriguingly, the BiFC assays, but not the Y2H assays, showed that ScnCBP interacted with the VPgs of SCMV, SrMV, and SCSMV. Competitive interaction assays showed that SCMV-VPg, SrMV-VPg, and SCMV-VPg did not compete with each other to interact with SceIF(iso)4E, and SceIF(iso)4E competed with SceIF4Eb to interact with SrMV-VPg but not SCMV-VPg. This study sheds light on the molecular mechanism of sugarcane mosaic pathogen infection of sugarcane plants and benefits sugarcane breeding against the sugarcane mosaic disease.