The 6-kilodalton peptide 1 in plant viruses of the family Potyviridae is a viroporin.

Potyviridae, the largest family of plant RNA viruses, includes many important pathogens that significantly reduce the yields of many crops worldwide. In this study, we report that the 6-kilodalton peptide 1 (6K1), one of the least characterized potyviral proteins, is an endoplasmic reticulum-localized protein. AI-assisted structure modeling and biochemical assays suggest that 6K1 forms pentamers with a central hydrophobic tunnel, can increase the cell membrane permeability of Escherichia coli and Nicotiana benthamiana, and can conduct potassium in Saccharomyces cerevisiae. An infectivity assay showed that viral proliferation is inhibited by mutations that affect 6K1 multimerization. Moreover, the 6K1 or its homologous 7K proteins from other viruses of the Potyviridae family also have the ability to increase cell membrane permeability and transmembrane potassium conductance. Taken together, these data reveal that 6K1 and its homologous 7K proteins function as viroporins in viral infected cells.

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.

A Zinc Finger Motif in the P1 N Terminus, Highly Conserved in a Subset of Potyviruses, Is Associated with the Host Range and Fitness of Telosma Mosaic Virus.

P1 is the first protein translated from the genomes of most viruses in the family Potyviridae, and it contains a C-terminal serine-protease domain that cis-cleaves the junction between P1 and HCPro in most cases. Intriguingly, P1 is the most divergent among all mature viral factors, and its roles during viral infection are still far from understood. In this study, we found that telosma mosaic virus (TelMV, genus Potyvirus) in passion fruit, unlike TelMV isolates present in other hosts, has two stretches at the P1 N terminus, named N1 and N2, with N1 harboring a Zn finger motif. Further analysis revealed that at least 14 different potyviruses, mostly belonging to the bean common mosaic virus subgroup, encode a domain equivalent to N1. Using the newly developed TelMV infectious cDNA clones from passion fruit, we demonstrated that N1, but not N2, is crucial for viral infection in both Nicotiana benthamiana and passion fruit. The regulatory effects of N1 domain on P1 cis cleavage, as well as the accumulation and RNA silencing suppression (RSS) activity of its cognate HCPro, were comprehensively investigated. We found that N1 deletion decreases HCPro abundance at the posttranslational level, likely by impairing P1 cis cleavage, thus reducing HCPro-mediated RSS activity. Remarkably, disruption of the Zn finger motif in N1 did not impair P1 cis cleavage and HCPro accumulation but severely debilitated TelMV fitness. Therefore, our results suggest that the Zn finger motif in P1s plays a critical role in viral infection that is independent of P1 protease activity and self-release, as well as HCPro accumulation and silencing suppression. IMPORTANCE Viruses belonging to the family Potyviridae represent the largest group of plant-infecting RNA viruses, including a variety of agriculturally and economically important viral pathogens. Like all picorna-like viruses, potyvirids employ polyprotein processing as the gene expression strategy. P1, the first protein translated from most potyvirid genomes, is the most variable viral factor and has attracted great scientific interest. Here, we defined a Zn finger motif-encompassing domain (N1) at the N terminus of P1 among diverse potyviruses phylogenetically related to bean common mosaic virus. Using TelMV as a model virus, we demonstrated that the N1 domain is key for viral infection, as it is involved both in regulating the abundance of its cognate HCPro and in an as-yet-undefined key function unrelated to protease processing and RNA silencing suppression. These results advance our knowledge of the hypervariable potyvirid P1s and highlight the importance for infection of a previously unstudied Zn finger domain at the P1 N terminus.

N6-methyladenosine RNA modification promotes viral genomic RNA stability and infection.

Molecular manipulation of susceptibility (S) genes that are antipodes to resistance (R) genes has been adopted as an alternative strategy for controlling crop diseases. Here, we show the S gene encoding Triticum aestivum m6A methyltransferase B (TaMTB) is identified by a genome-wide association study and subsequently shown to be a positive regulator for wheat yellow mosaic virus (WYMV) infection. TaMTB is localized in the nucleus, is translocated into the cytoplasmic aggregates by binding to WYMV NIb to upregulate the m6A level of WYMV RNA1 and stabilize the viral RNA, thus promoting viral infection. A natural mutant allele TaMTB-SNP176C is found to confer an enhanced susceptibility to WYMV infection through genetic variation analysis on 243 wheat varieties. Our discovery highlights this allele can be a useful target for the molecular wheat breeding in the future.

Proteome expansion in the Potyviridae evolutionary radiation.

Potyviridae, the largest family of known RNA viruses (realm Riboviria), belongs to the picorna-like supergroup and has important agricultural and ecological impacts. Potyvirid genomes are translated into polyproteins, which are in turn hydrolyzed to release mature products. Recent sequencing efforts revealed an unprecedented number of potyvirids with a rich variability in gene content and genomic layouts. Here, we review the heterogeneity of non-core modules that expand the structural and functional diversity of the potyvirid proteomes. We provide a family-wide classification of P1 proteinases into the functional Types A and B, and discuss pretty interesting sweet potato potyviral ORF (PISPO), putative zinc fingers, and alkylation B (AlkB)-non-core modules found within P1 cistrons. The atypical inosine triphosphate pyrophosphatase (ITPase/HAM1), as well as the pseudo tobacco mosaic virus-like coat protein (TMV-like CP) are discussed alongside homologs of unrelated virus taxa. Family-wide abundance of the multitasking helper component proteinase (HC-pro) is revised. Functional connections between non-core modules are highlighted to support host niche adaptation and immune evasion as main drivers of the Potyviridae evolutionary radiation. Potential biotechnological and synthetic biology applications of potyvirid leader proteinases and non-core modules are finally explored.

An Importin-ß-like Protein from Nicotiana benthamiana Interacts with the RNA Silencing Suppressor P1b of the Cucumber Vein Yellowing Virus, Modulating Its Activity.

During a plant viral infection, host-pathogen interactions are critical for successful replication and propagation of the virus through the plant. RNA silencing suppressors (RSSs) are key players of this interplay, and they often interact with different host proteins, developing multiple functions. In the Potyviridae family, viruses produce two main RSSs, HCPro and type B P1 proteins. We focused our efforts on the less known P1b of cucumber vein yellowing virus (CVYV), a type B P1 protein, to try to identify possible factors that could play a relevant role during viral infection. We used a chimeric expression system based on plum pox virus (PPV) encoding a tagged CVYV P1b in place of the canonical HCPro. We used that tag to purify P1b in Nicotiana-benthamiana-infected plants and identified by mass spectrometry an importin-ß-like protein similar to importin 7 of Arabidopsis thaliana. We further confirmed the interaction by bimolecular fluorescence complementation assays and defined its nuclear localization in the cell. Further analyses showed a possible role of this N. benthamiana homolog of Importin 7 as a modulator of the RNA silencing suppression activity of P1b.

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.

A unique internal ribosome entry site representing a dynamic equilibrium state of RNA tertiary structure in the 5'-UTR of Wheat yellow mosaic virus RNA1.

Internal ribosome entry sites (IRESes) were first reported in RNA viruses and subsequently identified in cellular mRNAs. In this study, IRES activity of the 5'-UTR in Wheat yellow mosaic virus (WYMV) RNA1 was identified, and the 3'-UTR synergistically enhanced this IRES activity via long-distance RNA-RNA interaction between C80U81and A7574G7575. Within the 5'-UTR, the hairpin 1(H1), flexible hairpin 2 (H2) and linker region (LR1) between H1 and H2 played an essential role in cap-independent translation, which is associated with the structural stability of H1, length of discontinuous stems and nucleotide specificity of the H2 upper loop and the long-distance RNA-RNA interaction sites in LR1. The H2 upper loop is a target region of the eIF4E. Cytosines (C55, C66, C105 and C108) in H1 and H2 and guanines (G73, G79 and G85) in LR1 form discontinuous and alternative base pairing to maintain the dynamic equilibrium state, which is used to elaborately regulate translation at a suitable level. The WYMV RNA1 5'-UTR contains a novel IRES, which is different from reported IRESes because of the dynamic equilibrium state. It is also suggested that robustness not at the maximum level of translation is the selection target during evolution of WYMV RNA1.

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant.

Cassava brown streak disease (CBSD) is a leading cause of cassava losses in East and Central Africa, and is currently having a severe impact on food security. The disease is caused by two viruses within the Potyviridae family: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), which both encode atypical Ham1 proteins with highly conserved inosine triphosphate (ITP) pyrophosphohydrolase (ITPase) domains. ITPase proteins are widely encoded by plant, animal, and archaea. They selectively hydrolyse mutagenic nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby function to reduce mutation rates. It has previously been hypothesized that U/CBSVs encode Ham1 proteins with ITPase activity to reduce viral mutation rates during infection. In this study, we investigate the potential roles of U/CBSV Ham1 proteins. We show that both CBSV and UCBSV Ham1 proteins have ITPase activities through in vitro enzyme assays. Deep-sequencing experiments found no evidence of the U/CBSV Ham1 proteins providing mutagenic protection during infections of Nicotiana hosts. Manipulations of the CBSV_Tanza infectious clone were performed, including a Ham1 deletion, ITPase point mutations, and UCBSV Ham1 chimera. Unlike severely necrotic wild-type CBSV_Tanza infections, infections of Nicotiana benthamiana with the manipulated CBSV infectious clones do not develop necrosis, indicating that that the CBSV Ham1 is a necrosis determinant. We propose that the presence of U/CBSV Ham1 proteins with highly conserved ITPase motifs indicates that they serve highly selectable functions during infections of cassava and may represent a euphorbia host adaptation that could be targeted in antiviral strategies.

The Triticum Mosaic Virus Internal Ribosome Entry Site Relies on a Picornavirus-Like YX-AUG Motif To Designate the Preferred Translation Initiation Site and To Likely Target the 18S rRNA.

Several viruses encode an internal ribosome entry site (IRES) at the 5' end of their RNA, which, unlike most cellular mRNAs, initiates translation in the absence of a 5' m7GpppG cap. Here, we report a uniquely regulated translation enhancer found in the 739-nucelotide (nt) sequence of the Triticum mosaic virus (TriMV) leader sequence that distinguishes the preferred initiation site from a plethora of IRES-encoded AUG triplets. Through deletion mutations of the TriMV 5' untranslated region (UTR), we show that the TriMV 5' UTR encodes a cis-acting picornaviral Y16-X11-AUG-like motif with a 16-nt polypyrimidine CU-tract (Y16), at a precise, 11-nt distance (X11) from the preferred 13th AUG. Phylogenetic analyses indicate that this motif is conserved among potyviral leader sequences with multiple AUGs. Consistent with a broadly conserved mechanism, the motif could be functionally replaced with known picornavirus YX-AUG motifs and is predicted to function as target sites for the 18S rRNA by direct base pairing. Accordingly, mutations that disrupted overall complementarity to the 18S rRNA markedly reduced TriMV IRES activity, as did the delivery of antisense oligonucleotides designed to block YX-AUG accessibility. To our knowledge, this is the first report of a plant viral IRES YX-AUG motif, and our findings suggest that a conserved mechanism regulates translation for multiple economically important plant and animal positive single-stranded RNA viruses.IMPORTANCE Uncapped viral RNAs often rely on their 5' leader sequences to initiate translation, and the Triticum mosaic virus (TriMV) devotes an astonishing 7% of its genome to directing ribosomes to the correct AUG. Here we uncover a novel mechanism by which a TriMV cis-regulatory element controls cap-independent translation. The upstream region of the functional AUG contains a 16-nt polypyrimidine tract located 11 nt from the initiation site. Based on functional redundancy with similar motifs derived from human picornaviruses, the motif is likely to operate by directing ribosome targeting through base pairing with 18S rRNA. Our results provide the first report of a broad-spectrum mechanism regulating translation initiation for both plant- and animal-hosted picornaviruses.

Wheat streak mosaic virus coat protein is a determinant for vector transmission by the wheat curl mite.

Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae), is transmitted by the wheat curl mite (Aceria tosichella Keifer). The requirement of coat protein (CP) for WSMV transmission by the wheat curl mite was examined using a series of viable deletion and point mutations. Mite transmission of WSMV was completely abolished with deletions comprising CP amino acids 58-100. In contrast, the amino-proximal (amino acids 6-27 and 36-57) and carboxy-terminal (14 amino acids) regions of CP were expendable for mite transmission. Mutation of aspartic acid residues at amino acid positions 289 or 326 (D289A or D326A) at the carboxy-proximal region of CP significantly reduced mite transmission. Remarkably, every wheat plant infected by mutants D289A or D326A through mite transmission but not with in vitro transcripts contained a second-site mutation of R131C and N275H, respectively. Collectively, these data demonstrate for the first time that CP is a determinant for an eriophyid-transmitted plant virus.

Wheat streak mosaic virus Coat Protein Deletion Mutants Elicit More Severe Symptoms Than Wild-Type Virus in Multiple Cereal Hosts.

Previously, we reported that coat protein (CP) of Wheat streak mosaic virus (WSMV) (genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. In this study, we demonstrated that WSMV mutants with deletion of CP amino acids 58 to 84 but not of 36 to 57 induced severe chlorotic streaks and spots, followed by acute chlorosis in wheat, maize, barley, and rye compared with mild to moderate chlorotic streaks and mosaic symptoms by wild-type virus. Deletion of CP amino acids 58 to 84 from the WSMV genome accelerated cell-to-cell movement, with increased accumulation of genomic RNAs and CP, compared with the wild-type virus. Microscopic examination of wheat tissues infected by green fluorescent protein-tagged mutants revealed that infection by mutants lacking CP amino acids 58 to 84 caused degradation of chloroplasts, resulting in acute macroscopic chlorosis. The profile of CP-specific proteins was altered in wheat infected by mutants causing acute chlorosis, compared with mutants eliciting wild-type symptoms. All deletion mutants accumulated CP-specific major protein similarly to that in wild-type virus; however, mutants that elicit acute chlorosis failed to accumulate a 31-kDa minor protein compared with wild-type virus or mutants lacking amino acids 36 to 57. Taken together, these data suggest that deletion of CP amino acids 58 to 84 from the WSMV genome enhanced accumulation of CP and genomic RNA, altered CP-specific protein profiles, and caused severe symptom phenotypes in multiple cereal hosts.

A viral suppressor of RNA silencing inhibits ARGONAUTE 1 function by precluding target RNA binding to pre-assembled RISC.

In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine.

Formation of Potato Virus A-Induced RNA Granules and Viral Translation Are Interrelated Processes Required for Optimal Virus Accumulation.

RNA granules are cellular structures, which play an important role in mRNA translation, storage, and degradation. Animal (+)RNA viruses often co-opt RNA granule proteins for viral reproduction. However, the role of RNA granules in plant viral infections is poorly understood. Here we use Potato virus A (PVA) as a model potyvirus and demonstrate that the helper component-proteinase (HCpro), the potyviral suppressor of RNA silencing, induces the formation of RNA granules. We used confocal microscopy to demonstrate the presence of host RNA binding proteins including acidic ribosomal protein P0, argonaute 1 (AGO1), oligouridylate-binding protein 1 (UBP1), varicose (VCS) and eukaryotic initiation factor iso4E (eIF(iso)4E) in these potyvirus-induced RNA granules. We show that the number of potyviral RNA granules is down-regulated by the genome-linked viral protein (VPg). We demonstrated previously that VPg is a virus-specific translational regulator that co-operates with potyviral RNA granule components P0 and eIF(iso)4E in PVA translation. In this study we show that HCpro and varicose, components of potyviral RNA granules, stimulate VPg-promoted translation of the PVA, whereas UBP1 inhibits this process. Hence, we propose that PVA translation operates via a pathway that is interrelated with potyviral RNA granules in PVA infection. The importance of these granules is evident from the strong reduction in viral RNA and coat protein amounts that follows knock down of potyviral RNA granule components. HCpro suppresses antiviral RNA silencing during infection, and our results allow us to propose that this is also the functional context of the potyviral RNA granules we describe in this study.

A Unique 5' Translation Element Discovered in Triticum Mosaic Virus.

UNLABELLED: Several plant viruses encode elements at the 5' end of their RNAs, which, unlike most cellular mRNAs, can initiate translation in the absence of a 5' m7GpppG cap. Here, we describe an exceptionally long (739-nucleotide [nt]) leader sequence in triticum mosaic virus (TriMV), a recently emerged wheat pathogen that belongs to the Potyviridae family of positive-strand RNA viruses. We demonstrate that the TriMV 5' leader drives strong cap-independent translation in both wheat germ extract and oat protoplasts through a novel, noncanonical translation mechanism. Translation preferentially initiates at the 13th start codon within the leader sequence independently of eIF4E but involves eIF4G. We truncated the 5' leader to a 300-nucleotide sequence that drives cap-independent translation from the 5' end. We show that within this sequence, translation activity relies on a stem-loop structure identified at nucleotide positions 469 to 490. The disruption of the stem significantly impairs the function of the 5' untranslated region (UTR) in driving translation and competing against a capped RNA. Additionally, the TriMV 5' UTR can direct translation from an internal position of a bicistronic mRNA, and unlike cap-driven translation, it is unimpaired when the 5' end is blocked by a strong hairpin in a monocistronic reporter. However, the disruption of the identified stem structure eliminates such a translational advantage. Our results reveal a potent and uniquely controlled translation enhancer that may provide new insights into mechanisms of plant virus translational regulation. IMPORTANCE: Many members of the Potyviridae family rely on their 5' end for translation. Here, we show that the 739-nucleotide-long triticum mosaic virus 5' leader bears a powerful translation element with features distinct from those described for other plant viruses. Despite the presence of 12 AUG start codons within the TriMV 5' UTR, translation initiates primarily at the 13th AUG codon. The TriMV 5' UTR is capable of driving cap-independent translation in vitro and in vivo, is independent of eIF4E, and can drive internal translation initiation. A hairpin structure at nucleotide positions 469 to 490 is required for the cap-independent translation and internal translation initiation abilities of the element and plays a role in the ability of the TriMV UTR to compete against a capped RNA in vitro. Our results reveal a novel translation enhancer that may provide new insights into the large diversity of plant virus translation mechanisms.

Cotranslational coat protein-mediated inhibition of potyviral RNA translation.

UNLABELLED: Potato virus A (PVA) is a single-stranded positive-sense RNA virus and a member of the family Potyviridae. The PVA coat protein (CP) has an intrinsic capacity to self-assemble into filamentous virus-like particles, but the mechanism responsible for the initiation of viral RNA encapsidation in vivo remains unclear. Apart from virion assembly, PVA CP is also involved in the inhibition of viral RNA translation. In this study, we show that CP inhibits PVA RNA translation in a dose-dependent manner, through a mechanism involving the CP-encoding region. Analysis of this region, however, failed to identify any RNA secondary structure(s) preferentially recognized by CP, suggesting that the inhibition depends on CP-CP rather than CP-RNA interactions. In agreement with this possibility, insertion of an in-frame stop codon upstream of the CP sequence led to a marked decrease in the inhibition of viral RNA translation. Based on these results, we propose a model in which the cotranslational interactions between excess CP accumulating in trans and CP translated from viral RNA in cis are required to initiate the translational repression. This model suggests a mechanism for how viral RNA can be sequestered from translation and specifically selected for encapsidation at the late stages of viral infection. IMPORTANCE: The main functions of the CP during potyvirus infection are to protect viral RNA from degradation and to transport it locally, systemically, and from host to host. Although virion assembly is a key step in the potyviral infectious cycle, little is known about how it is initiated and how viral RNA is selected for encapsidation. The results presented here suggest that CP-CP rather than CP-RNA interactions are predominantly involved in the sequestration of viral RNA away from translation. We propose that the cotranslational nature of these interactions may represent a mechanism for the selection of viral RNA for encapsidation. A better understanding of the mechanism of virion assembly may lead to development of crops resistant to potyviruses at the level of viral RNA encapsidation, thereby reducing the detrimental effects of potyvirus infections on food production.

Transcriptional response of virus-infected cassava and identification of putative sources of resistance for cassava brown streak disease.

Cassava (Manihot esculenta) is a major food staple in sub-Saharan Africa, which is severely affected by cassava brown streak disease (CBSD). The aim of this study was to identify resistance for CBSD as well as to understand the mechanism of putative resistance for providing effective control for the disease. Three cassava varieties; Kaleso, Kiroba and Albert were inoculated with cassava brown streak viruses by grafting and also using the natural insect vector the whitefly, Bemisia tabaci. Kaleso expressed mild or no disease symptoms and supported low concentrations of viruses, which is a characteristic of resistant plants. In comparison, Kiroba expressed severe leaf but milder root symptoms, while Albert was susceptible with severe symptoms both on leaves and roots. Real-time PCR was used to estimate virus concentrations in cassava varieties. Virus quantities were higher in Kiroba and Albert compared to Kaleso. The Illumina RNA-sequencing was used to further understand the genetic basis of resistance. More than 700 genes were uniquely overexpressed in Kaleso in response to virus infection compared to Albert. Surprisingly, none of them were similar to known resistant gene orthologs. Some of the overexpressed genes, however, belonged to the hormone signalling pathways and secondary metabolites, both of which are linked to plant resistance. These genes should be further characterised before confirming their role in resistance to CBSD.

The C-terminus of Wheat streak mosaic virus coat protein is involved in differential infection of wheat and maize through host-specific long-distance transport.

Viral determinants and mechanisms involved in extension of host range of monocot-infecting viruses are poorly understood. Viral coat proteins (CP) serve many functions in almost every aspect of the virus life cycle. The role of the C-terminal region of Wheat streak mosaic virus (WSMV) CP in virus biology was examined by mutating six negatively charged aspartic acid residues at positions 216, 289, 290, 326, 333, and 334. All of these amino acid residues are dispensable for virion assembly, and aspartic acid residues at positions 216, 333, and 334 are expendable for normal infection of wheat and maize. However, mutants D289N, D289A, D290A, DD289/290NA, and D326A exhibited slow cell-to-cell movement in wheat, which resulted in delayed onset of systemic infection, followed by a rapid recovery of genomic RNA accumulation and symptom development. Mutants D289N, D289A, and D326A inefficiently infected maize, eliciting milder symptoms, while D290A and DD289/290NA failed to infect systemically, suggesting that the C-terminus of CP is involved in differential infection of wheat and maize. Mutation of aspartic acid residues at amino acid positions 289, 290, and 326 severely debilitated virus ingress into the vascular system of maize but not wheat, suggesting that these amino acids facilitate expansion of WSMV host range through host-specific long-distance transport.

Nucleo-cytoplasmic shuttling of VPg encoded by Wheat yellow mosaic virus requires association with the coat protein.

VPg (virus protein, genome-linked) is a multifunctional protein that plays important roles in viral multiplication in the cytoplasm. However, a number of VPgs encoded by plant viruses target the nucleus and this appears to be biologically significant. These VPgs may therefore be translocated between nuclear and cytoplasmic compartments during virus infection, but such nucleo-cytoplasmic transport has not been demonstrated. We report that VPg encoded by Wheat yellow mosaic virus (WYMV, genus Bymovirus, family Potyviridae) accumulated in both the nucleus and cytoplasm of infected cells, but localized exclusively in the nucleus when expressed alone in plants. Computational analyses predicted the presence of a nuclear localization signal (NLS) and a nuclear export signal (NES) in WYMV VPg. Mutational analyses showed that both the N-terminal and the NLS domains of VPg contribute to the efficiency of nuclear targeting. In vitro and in planta assays indicated that VPg interacts with WYMV coat protein (CP) and proteinase 1 (P1) proteins. Observation of VPg fused to a fluorescent protein and subcellular fractionation experiments showed that VPg was translocated to the cytoplasm when co-expressed with CP, but not with P1. Mutations in the NES domain or treatment with leptomycin B prevented VPg translocation to the cytoplasm when co-expressed with CP. Our results suggest that association with CP facilitates the nuclear export of VPg during WYMV infection.

P1 peptidase--a mysterious protein of family Potyviridae.

The Potyviridae family, named after its type member, Potato virus Y (PVY), is the largest of the 65 plant virus groups and families currently recognized. The coding region for P1 peptidase is located at the very beginning of the viral genome of the family Potyviridae. Until recently P1 was thought of as serine peptidase with RNA-binding activity and with possible influence in cell-to-cell viral spreading. This N-terminal protein, among all of the potyviruses, is the most divergent protein: varying in length and in its amino acid sequence. Nevertheless, P1 peptidase in many ways is still a mysterious viral protein. In this review, we would like to offer a comprehensive overview, discussing the proteomic, biochemical and phylogenetic views of the P1 protein.