Identification and characterization of the Remorin gene family in Saccharum and the involvement of ScREM1.5e-1/-2 in SCMV infection on sugarcane.

Introduction: Remorins (REMs) are plant-specific membrane-associated proteins that play important roles in plant-pathogen interactions and environmental adaptations. Group I REMs are extensively involved in virus infection. However, little is known about the REM gene family in sugarcane (Saccharum spp. hyrid), the most important sugar and energy crop around world. Methods: Comparative genomics were employed to analyze the REM gene family in Saccharum spontaneum. Transcriptomics or RT-qPCR were used to analyze their expression files in different development stages or tissues under different treatments. Yeast two hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays were applied to investigate the protein interaction. Results: In this study, 65 REMs were identified from Saccharum spontaneum genome and classified into six groups based on phylogenetic tree analysis. These REMs contain multiple cis-elements associated with growth, development, hormone and stress response. Expression profiling revealed that among different SsREMs with variable expression levels in different developmental stages or different tissues. A pair of alleles, ScREM1.5e-1/-2, were isolated from the sugarcane cultivar ROC22. ScREM1.5e-1/-2 were highly expressed in leaves, with the former expressed at significantly higher levels than the latter. Their expression was induced by treatment with H2O2, ABA, ethylene, brassinosteroid, SA or MeJA, and varied upon Sugarcane mosaic virus (SCMV) infection. ScREM1.5e-1 was localized to the plasma membrane (PM), while ScREM1.5e-2 was localized to the cytoplasm or nucleus. ScREM1.5e-1/-2 can self-interact and interact with each other, and interact with VPgs from SCMV, Sorghum mosaic virus, or Sugarcane streak mosaic virus. The interactions with VPgs relocated ScREM1.5e-1 from the PM to the cytoplasm. Discussion: These results reveal the origin, distribution and evolution of the REM gene family in sugarcane and may shed light on engineering sugarcane resistance against sugarcane mosaic pathogens.

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.

VPg Impact on Ryegrass Mottle Virus Serine-like 3C Protease Proteolysis and Structure.

Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). Nuclear magnetic resonance studies show a Pro-VPg complex interaction and VPg tertiary structure; however, information regarding structural changes of the Pro-VPg complex during interaction is lacking. Here, we solved a full Pro-VPg 3D structure of ryegrass mottle virus (RGMoV) that demonstrates the structural changes in three different conformations due to VPg interaction with Pro. We identified a unique site of VPg interaction with Pro that was not observed in other sobemoviruses, and observed different conformations of the Pro ß2 barrel. This is the first report of a full plant Pro crystal structure with its VPg cofactor. We also confirmed the existence of an unusual previously unmapped cleavage site for sobemovirus Pro in the transmembrane domain: E/A. We demonstrated that RGMoV Pro in cis activity is not regulated by VPg and that in trans, VPg can also mediate Pro in free form. Additionally, we observed Ca2+ and Zn2+ inhibitory effects on the Pro cleavage activity.

Identification of cis-acting replication element in VP2-encoding region of Senecavirus A genome.

Picornavirus possesses one positive-sense, single-stranded RNA genome, in which a cis-acting replication element (cre) is located. The cre is a stem-loop structure that harbors a conserved AAACA motif within its loop region. This motif functions as a template for adding two U residues to the viral VPg, therefore generating a VPg-pUpU that is required for viral RNA synthesis. Senecavirus A (SVA) is an emerging picornavirus. Its cre has not been identified as yet. In the present study, one putative cre containing a typical AAACA motif was computationally predicted to exist within the VP2-encoding sequence of SVA. To test the role of this putative cre, 22 SVA cDNA clones with different point mutations in their cre-formed sequences were constructed in an attempt to rescue replication-competent SVAs. A total of 11 viruses were rescued from their individual cDNA clones, implying that some mutated cres exerted lethal impacts on SVA replication. To eliminate these impacts, an intact cre was artificially inserted into those SVA cDNA clones without ability of recovering virus. The artificial cre was proven to be able of compensating for some, but not all, defects caused by mutated cres, leading to successful recovery of SVAs. These results indicated that the putative cre of SVA was functionally similar to those of other picornaviruses, perhaps involved in the uridylylation of VPg.

All eggs in one basket: How potyvirus infection is controlled at a single cap-independent translation event.

Regulation of protein synthesis is a strong determinant of potyviral pathogenicity. The Potyviridae family is the largest family of plant-infecting positive sense RNA viruses. Similar to the animal-infecting Picornaviridae family, the potyviral RNA genome lacks a 5' cap, and instead has a viral protein (VPg) linked to its 5' end. Potyviral genomes are mainly translated into one large polyprotein relying on a single translation event to express all their protein repertoire. In the absence of the 5' cap, the Potyviridae family depends on cis-acting elements in their 5' untranslated regions (UTR) to recruit the translation machinery. In this review, we summarize the diverse 5'UTR-driven, cap-independent translation mechanisms employed by the Potyviridae family including scanning-dependent mechanism, internal initiation, and the stimulatory role of the VPg. These mechanisms have direct implications on potyviral pathogenicity, including host range specificity and resistance. Finally, we discuss how these viral strategies could not only inform new avenues for engineering and/or breeding for crop resistance but would also provide opportunities for the development of biotechnological tools for large-scale protein production in plant systems.

Corrigendum: CRISPR/Cas9-mediated targeting of susceptibility factor eIF4E-enhanced resistance against Potato virus Y.

[This corrects the article DOI: 10.3389/fgene.2022.922019.].

Expansion of viral genomes with viral protein genome linked copies.

Virus protein-linked genome (VPg) proteins are required for replication. VPgs are duplicated in a subset of RNA viruses however their roles are not fully understood and the extent of viral genomes containing VPg copies has not been investigated in detail. Here, we generated a novel bioinformatics approach to identify VPg sequences in viral genomes using hidden Markov models (HMM) based on alignments of dicistrovirus VPg sequences. From metagenomic datasets of dicistrovirus genomes, we identified 717 dicistrovirus genomes containing VPgs ranging from a single copy to 8 tandem copies. The VPgs are classified into nine distinct types based on their sequence and length. The VPg types but not VPg numbers per viral genome followed specific virus clades, thus suggesting VPgs co-evolved with viral genomes. We also identified VPg duplications in aquamavirus and mosavirus genomes. This study greatly expands the number of viral genomes that contain VPg copies and indicates that duplicated viral sequences are more widespread than anticipated.

The 5'-end motif of Senecavirus A cDNA clone is genetically modified in 36 different ways for uncovering profiles of virus recovery.

Senecavirus A (SVA) is an emerging picornavirus. Its genome is one positive-sense, single-stranded RNA. The viral protein (VPg) is covalently linked to the extreme 5' end of the SVA genome. A complex hairpin-pseudoknot-hairpin (HPH) RNA structure was computationally predicted to form at the 5' end of the SVA genome. A total of three extra "U" residues (UUU) served as a linker between the HPH structure and the VPg, causing putative UUU-HPH formation at the extreme 5' end of the SVA genome. It is unclear how the UUU-HPH structure functions. One SVA cDNA clone (N0) was constructed previously in our laboratory. Here, the N0 was genetically tailored for reconstructing a set of 36 modified cDNA clones (N1 to N36) in an attempt to rescue replication-competent SVAs using reverse genetics. The results showed that a total of nine viruses were successfully recovered. Out of them, five were independently rescued from the N1 to N5, reconstructed by deleting the first five nucleotides (TTTGA) one by one from the extreme 5' end of N0. Interestingly, these five viral progenies reverted to the wild-type or/and wild-type-like genotype, suggesting that SVA with an ability to repair nucleotide defects in its extreme 5' end. The other four were independently rescued from the N26 to N29, containing different loop-modifying motifs in the first hairpin of the HPH structure. These four loop-modifying motifs were genetically stable after serial passages, implying the wild-type loop motif was not a high-fidelity element in the first hairpin during SVA replication. The other genetically modified sequences were demonstrated to be lethal elements in the HPH structure for SVA recovery, suggesting that the putative HPH formation was a crucial cis-acting replication element for SVA propagation.

Large-Scale Mapping of Vocalization-Related Activity in the Functionally Diverse Nuclei in Rat Posterior Brainstem.

The identity and location of vocalization pattern generating (VPG) circuits in mammals is debated. Based on physiological experiments, investigators suggested anterior brainstem circuits in the reticular formation, and anatomic evidence suggested the nucleus retroambiguus (NRA) in the posterior brainstem, or combinations of these sites as the putative mammalian VPG. Additionally, vocalization loudness is a critical factor in acoustic communication. However, many of the underlying neuronal mechanisms are still unknown. Here, we evoked calls by stimulation of the periaqueductal gray in anesthetized male rats, performed a large-scale mapping of vocalization-related activity using the activity marker c-fos, and high-density recordings of brainstem circuits using Neuropixels probes. Both c-fos expression and recording of vocalization-related activity point to a participation of the NRA in vocalization. More important, among our recorded structures, we found that the NRA is the only brainstem area showing a strong correlation between unit activity and call intensity. In addition, we observed functionally diverse patterns of vocalization-related activity in a set of regions around NRA. Dorsal to NRA, we observed activity specific to the beginning and end of vocalizations in the posterior level of the medullary reticular nucleus, dorsal part, whereas medial and lateral to the NRA, we observed activity related to call initiation. No clear vocalization-related activity was observed at anterior brainstem sites. Our findings suggest a set of functionally heterogeneous regions around the NRA contribute to vocal pattern generation in rats.SIGNIFICANCE STATEMENT Vocalization patterns are shaped in the mammalian brainstem, but the identity and location of the circuits involved is debated. Additionally, the neuronal mechanisms of vocal intensity control are still unknown. This study consisted of a large-scale mapping of brainstem vocalization circuits based on the activity marker c-fos and high-density recordings with Neuropixels probes. The results confirm the role of nucleus retroambiguus in call production and point to a key role of neurons in this nucleus in loudness control. Dorsal to the nucleus retroambiguus and in the posterior medulla, the authors identify neurons with activity specific to the beginning and end of vocalizations. The results point to specific neural dials for various aspects of rat vocalization control in the posterior brainstem.

CRISPR/Cas9-Mediated Targeting of Susceptibility Factor eIF4E-Enhanced Resistance Against Potato Virus Y.

Potato (Solanum tuberosum L.) is an important staple food around the world, and potato virus Y (PVY) is a major constraint for potato production. The VPg protein of PVY interacts with the translation initiation factor eIF4E of the host that works as a susceptibility factor during infection. The interaction between eIF4E and VPg was disrupted by CRISPR/Cas9. The homozygous conserved region of eIF4E of the potato variety "Kruda" was mutated by CRISPR/Cas9. Tracking of insertion, deletion, and conversion events was performed by Sanger sequencing with ∼15% editing efficiency. Truncated and mutated eIF4E proteins were unable to interact with VPg, and the virus was not able to exploit the host machinery for replication and systemic spreading. Mutated eIF4E lines showed enhanced resistance to PVYO strain. DAS-ELISA and RT-PCR were used for validation of the observed resistance. PVY resistance in tetraploid lines via CRISPR/Cas9 provides a route to develop novel resistant potato cultivars.

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.

Analysis on Four Derivative Waveforms of Photoplethysmogram (PPG) for Fiducial Point Detection.

Fiducial points of photoplethysmogram (PPG), first derivative PPG (VPG), and second derivative PPG (APG) are essential in extracting numerous parameters to diagnose cardiovascular disease. However, the fiducial points were usually detected using complex mathematical algorithms. Inflection points from derivatives waveforms are not thoroughly studied, whereas they can significantly assist in peak detection. This study is performed to investigate the derivative waveforms of PPG and use them to detect the important peaks of PPG, VPG, and APG. PPGs with different morphologies from 43 ischemic heart disease subjects are analyzed. Inflection points of the derivative waveforms up to the fourth level are observed, and consistent information (derivative markers) is used to detect the fiducial points of PPG, VPG, and APG with proper sequence. Moving average filter and simple thresholding techniques are applied to detect the primary points in VPG and the third derivative waveform. A total of twelve out of twenty derivative markers are found reliable in detecting fiducial points of two common types of PPG. Systolic peaks are accurately detected with 99.64% sensitivity and 99.38% positive predictivity using the 43 IHD dataset and Complex System Laboratory (CSL) Pulse Oximetry Artifact Labels database. The study has introduced the fourth derivative PPG waveform with four main points, which are significantly valuable for detecting the fiducial points of PPG, VPG, and APG.

The RNA-dependent RNA polymerase of the infectious pancreatic necrosis virus is linked to viral mRNA acting as a cap substitute.

The infectious pancreatic necrosis virus (IPNV) is responsible for significant economic losses in the aquaculture industry. It is an unenveloped virus with an icosahedral capsid. Its viral genome comprises two dsRNA segments, A and B. Segment A contains a small ORF, which encodes VP5, and a large ORF, which encodes a polyprotein that generates the structural proteins and the viral protease. Segment B encodes the RNA-dependent RNA polymerase (RdRp), called VP1 in this free form, or Vpg when it covalently attaches to the viral RNA. The viral genome does not have cap or poly(A). Instead, each 5' end is linked to the Vpg. Recently, we demonstrated that mRNA-A contains an internal ribosome entry site (IRES) to command polyprotein synthesis. However, the presence of Vpg on IPNV mRNAs and its impact on cellular translation has not been investigated. This research demonstrates that IPNV mRNAs are linked to Vpg and that this protein inhibits cap-dependent translation on infected cells. Also, it is demonstrated that Vpg interacts with eIF4E and that rapamycin treatment partially diminishes the viral protein synthesis. In addition, we determined that an IRES does not command translation of IPNV mRNA-B. We show that VPg serves as a cap substitute during the initiation of IPNV translation, contributing to understanding the replicative cycle of Birnaviruses. Our results indicate that the viral protein VP1/Vpg is multifunctional, having a significant role during IPNV RNA synthesis as the RdRp and the primer for IPNV RNA synthesis and translation as the viral protein genome, acting as a cap substitute.

Induction of COX-2 by feline calicivirus via activation of the MEK1-ERK1/2 pathway, and attenuation of feline lung inflammation and injury by MEK1 inhibitor AZD6244 (selumetinib).

Feline calicivirus (FCV) is an important and highly prevalent pathogen of cats that causes acute infectious respiratory disease. Here it is shown in vitro that FCV induces the production of cyclooxygenase-2 (COX-2) through the MEK1-ERK1/2 signaling pathway. Screening of FCV proteins revealed that FCV non-structural protein VPg enhanced COX-2 mRNA expression and protein production in CRFK cells in a concentration-dependent manner. Regions 24-54aa and 84-111aa in FCV VPg were essential for up-regulation. In vivo, COX-2 and IL-6 production caused by FCV infection of kittens was significantly suppressed by the MEK1 inhibitor AZD6244 (selumetinib) and lung inflammation and injury were practically eliminated, with body temperature being returned to normal. AZD6244 may therefore find application as an effective therapeutic agent for the treatment of FCV infection.

Specificity of Resistance and Tolerance to Cucumber Vein Yellowing Virus in Melon Accessions and Resistance Breaking with a Single Mutation in VPg.

Cucumber vein yellowing virus (CVYV) is an emerging virus on cucurbits in the Mediterranean Basin, against which few resistance sources are available, particularly in melon. The melon accession PI 164323 displays complete resistance to isolate CVYV-Esp, and accession HSD 2458 presents a tolerance, i.e., very mild symptoms despite virus accumulation in inoculated plants. The resistance is controlled by a dominant allele Cvy-11, while the tolerance is controlled by a recessive allele cvy-2, independent from Cvy-11. Before introducing the resistance or tolerance in commercial cultivars through a long breeding process, it is important to estimate their specificity and durability. Upon inoculation with eight molecularly diverse CVYV isolates, the resistance was found to be isolate-specific because many CVYV isolates induced necrosis on PI 164323, whereas the tolerance presented a broader range. A resistance-breaking isolate inducing severe mosaic on PI 164323 was obtained. This isolate differed from the parental strain by a single amino acid change in the VPg coding region. An infectious CVYV cDNA clone was obtained, and the effect of the mutation in the VPg cistron on resistance to PI 164323 was confirmed by reverse genetics. This represents the first determinant for resistance-breaking in an ipomovirus. Our results indicate that the use of the Cvy-11 allele alone will not provide durable resistance to CVYV and that, if used in the field, it should be combined with other control methods such as cultural practices and pyramiding of resistance genes to achieve long-lasting resistance against CVYV.

Generation of Nucleic Acid Aptamer Candidates against a Novel Calicivirus Protein Target.

Human norovirus is the leading cause of foodborne illness globally. One of the challenges in detecting noroviruses is the identification of a completely broadly reactive ligand; however, all detection ligands generated to date target the viral capsid, the outermost of which is the most variable region of the genome. The VPg is a protein covalently linked to the viral genome that is necessary for replication but hitherto remains underexplored as a target for detection or therapeutics. The purpose of this work was to generate nucleic acid aptamers against human norovirus (Norwalk) and cultivable surrogate (Tulane) VPgs for future use in detection and therapeutics. Eight rounds of positive-SELEX and two rounds of counter-SELEX were performed. Five and eight unique aptamer sequences were identified for Norwalk and Tulane VPg, respectively, all of which were predicted to be stable (∆G < -5.0) and one of which occurred in both pools. All candidates displayed binding to both Tulane and Norwalk VPg (positive:negative > 5.0), and all but two of the candidates displayed very strong binding (positive:negative > 10.0), significantly higher than binding to the negative control protein (p < 0.05). Overall, this work reports a number of aptamer candidates found to be broadly reactive and specific for in vitro-expressed VPgs across genus that could be used for future application in detection or therapeutics. Future work characterizing binding of the aptamer candidates against native VPgs and in therapeutic applications is needed to further evaluate their application.

Comparative Analysis of Novel Strains of Porcine Astrovirus Type 3 in the USA.

Porcine astrovirus type 3 (PoAstV3) has been previously identified as a cause of polioencephalomyelitis in swine and continues to cause disease in the US swine industry. Herein, we describe the characterization of both untranslated regions, frameshifting signal, putative genome-linked virus protein (VPg) and conserved antigenic epitopes of several novel PoAstV3 genomes. Twenty complete coding sequences (CDS) were obtained from 32 diagnostic cases originating from 11 individual farms/systems sharing a nucleotide (amino acid) percent identity of 89.74-100% (94.79-100%), 91.9-100% (96.3-100%) and 90.71-100% (93.51-100%) for ORF1a, ORF1ab and ORF2, respectively. Our results indicate that the 5'UTR of PoAstV3 is highly conserved highlighting the importance of this region in translation initiation while their 3'UTR is moderately conserved among strains, presenting alternative configurations including multiple putative protein binding sites and pseudoknots. Moreover, two predicted conserved antigenic epitopes were identified matching the 3' termini of VP27 of PoAstV3 USA strains. These epitopes may aid in the design and development of vaccine components and diagnostic assays useful to control outbreaks of PoAstV3-associated CNS disease. In conclusion, this is the first analysis predicting the structure of important regulatory motifs of neurotropic mamastroviruses, which differ from those previously described in human astroviruses.

Non-Canonical Translation Initiation Mechanisms Employed by Eukaryotic Viral mRNAs.

Viruses exploit the translation machinery of an infected cell to synthesize their proteins. Therefore, viral mRNAs have to compete for ribosomes and translation factors with cellular mRNAs. To succeed, eukaryotic viruses adopt multiple strategies. One is to circumvent the need for m7G-cap through alternative instruments for ribosome recruitment. These include internal ribosome entry sites (IRESs), which make translation independent of the free 5' end, or cap-independent translational enhancers (CITEs), which promote initiation at the uncapped 5' end, even if located in 3' untranslated regions (3' UTRs). Even if a virus uses the canonical cap-dependent ribosome recruitment, it can still perturb conventional ribosomal scanning and start codon selection. The pressure for genome compression often gives rise to internal and overlapping open reading frames. Their translation is initiated through specific mechanisms, such as leaky scanning, 43S sliding, shunting, or coupled termination-reinitiation. Deviations from the canonical initiation reduce the dependence of viral mRNAs on translation initiation factors, thereby providing resistance to antiviral mechanisms and cellular stress responses. Moreover, viruses can gain advantage in a competition for the translational machinery by inactivating individual translational factors and/or replacing them with viral counterparts. Certain viruses even create specialized intracellular "translation factories", which spatially isolate the sites of their protein synthesis from cellular antiviral systems, and increase availability of translational components. However, these virus-specific mechanisms may become the Achilles' heel of a viral life cycle. Thus, better understanding of the unconventional mechanisms of viral mRNA translation initiation provides valuable insight for developing new approaches to antiviral therapy.

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.