Minna-Liisa Rajamäki

The 6K2 Protein and the VPg of Potato Virus A Are Determinants of Systemic Infection in Nicandra physaloides

Infection with the isolate PVA-M of potato virus A (PVA; genus Potyvirus) is restricted to the inoculated leaves of Nicandra physaloides (Solanaceae), whereas the isolate PVA-B11 infects plants systemically by 10 days post inoculation. Resistance to systemic infection was shown to develop during plant growth. A recombinant virus (B11-M) in which a 1,208-nucleotide sequence of the full-length cDNA clone of PVA-B11 was replaced with the corresponding sequence from PVA-M displayed a phenotype similar to that of PVA-M. The replaced sequence contained four amino acid differences between the two isolates: one in the 6K2 protein and three in the viral genome-linked protein (VPg). Site-directed mutagenesis of the cDNA clones and inoculation of the mutants to N. physaloides indicated that the amino acid substitutions of Met5Val in the 6K2 protein or Leu185Ser in the VPg permitted vascular movement and systemic infection. However, resistance was only partially overcome by these changes, since systemic infection proceeded at a slower rate than with PVA-B11. The amino acid substitution Val116Met in the VPg alone was sufficient to overcome resistance and recover the phenotype of the isolate PVA-B11. These data indicated that both the 6K2 protein and the VPg were avirulence determinants of PVa-M in N. physaloides and suggested a possibly coordinated function of them in the vascular movement of PVA.

Towards a protein interaction map of potyviruses: protein interaction matrixes of two potyviruses based on the yeast two-hybrid system.

A map for the interactions of the major proteins from Potato virus A (PVA) and Pea seed-borne mosaic virus (PSbMV) (members of the genus POTYVIRUS:, family POTYVIRIDAE:) was generated using the yeast two-hybrid system (YTHS). Interactions were readily detected with five PVA protein combinations (HC-HC, HC-CI, VPg-VPg, NIa-NIb and CP-CP) and weak but reproducible interactions were detected for seven additional combinations (P1-CI, P3-NIb, NIaPro-NIb, VPg-NIa, VPg-NIaPro, NIaPro-NIa and NIa-NIa). In PSbMV, readily detectable interactions were found in five protein combinations (HC-HC, VPg-VPg, VPg-NIa, NIa-NIa and NIa-NIb) and weaker but reproducible interactions were detected for three additional combinations (P3-NIa, NIa-NIaPro and CP-CP). The self-interactions of HC, VPg, NIa and CP and the interactions of VPg-NIa, NIa-NIaPro and NIa-NIb were, therefore, common for the two potyviruses. The multiple protein interactions revealed in this study shed light on the co-ordinated functions of potyviral proteins involved in virus movement and replication.

Helper Component Proteinase of the Genus Potyvirus Is an Interaction Partner of Translation Initiation Factors eIF(iso)4E and eIF4E and Contains a 4E Binding Motif

ABSTRACT The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.

Detection of the Potyviral Genome-Linked Protein VPg in Virions and Its Phosphorylation by Host Kinases

ABSTRACT The multifunctional genome-linked protein (VPg) of Potato virus A (PVA; genus Potyvirus) was found to be phosphorylated as a part of the virus particle by a cellular kinase activity from tobacco. Immunoprecipitation, immunolabeling, and immunoelectron microscopy experiments showed that VPg is exposed at one end of the virion and it is accessible to protein-protein interactions. Substitution Ser185Leu at the C-proximal part of VPg reduces accumulation of PVA in inoculated leaves of the wild potato species Solanum commersonii and delays systemic infection, which is not observed in tobacco plants. Our data show that kinases of S. commersonii differentially recognize the VPg containing Ser or Leu at position 185, whereas both forms of VPg are similarly recognized by tobacco kinases. Taken together, our data imply that the virion-bound VPg may interact with host proteins and that phosphorylation of VPg may play a role in the VPg-mediated functions during the infection cycle of potyviruses.

Proteolytic processing of potyviral proteins and polyprotein processing intermediates in insect and plant cells

Processing of the polyprotein encoded by Potato virus A (PVA; genus Potyvirus) was studied using expression of the complete PVA polyprotein or its mutants from recombinant baculoviruses in insect cells. The time-course of polyprotein processing by the main viral proteinase (NIaPro) was examined with the pulse-chase method. The sites at the P3/6K1, CI-6K2 and VPg/NIaPro junctions were processed slowly, in contrast to other proteolytic cleavage sites which were processed at a high rate. The CI-6K2 polyprotein was observed in the baculovirus system and in infected plant cells. In both cell types the majority of CI-6K2 was found in the membrane fraction, in contrast to fully processed CI. Deletion of the genomic region encoding the 6K1 protein prevented proper proteolytic separation of P3 from CI, but did not affect processing of VPg, NIaPro, NIb or CP from the polyprotein. The 6K2-encoding sequence could be removed without any detectable effect on polyprotein processing. However, deletion of either the 6K1 or 6K2 protein-encoding regions rendered PVA non-infectious. Mutations at the 6K2/VPg cleavage site reduced virus infectivity in plants, but had a less pronounced, albeit detectable, effect on proteolytic processing in the baculovirus system. The results of this study indicate that NIaPro catalyses proteolytic cleavages preferentially in cis, and that the 6K1/CI and NIb/CP sites can also be processed in trans. Both 6K peptides are indispensable for virus replication, and proteolytic separation of the 6K2 protein from the adjacent proteins by NIaPro is important for the rate of virus replication and movement.

Involvement of the plant nucleolus in virus and viroid infections: parallels with animal pathosystems.

Abstract The nucleolus is a dynamic subnuclear body with roles in ribosome subunit biogenesis, mediation of cell-stress responses, and regulation of cell growth. An increasing number of reports reveal that similar to the proteins of animal viruses, many plant virus proteins localize in the nucleolus to divert host nucleolar proteins from their natural functions in order to exert novel role(s) in the virus infection cycle. This chapter will highlight studies showing how plant viruses recruit nucleolar functions to facilitate virus translation and replication, virus movement and assembly of virus-specific ribonucleoprotein (RNP) particles, and to counteract plant host defense responses. Plant viruses also provide a valuable tool to gain new insights into novel nucleolar functions and processes. Investigating the interactions between plant viruses and the nucleolus will facilitate the design of novel strategies to control plant virus infections. The nucleolus is a dynamic subnuclear body with roles in ribosome subunit biogenesis, mediation of cell-stress responses, and regulation of cell growth. An increasing number of reports reveal that similar to the proteins of animal viruses, many plant virus proteins localize in the nucleolus to divert host nucleolar proteins from their natural functions in order to exert novel role(s) in the virus infection cycle. This chapter will highlight studies showing how plant viruses recruit nucleolar functions to facilitate virus translation and replication, virus movement and assembly of virus-specific ribonucleoprotein (RNP) particles, and to counteract plant host defense responses. Plant viruses also provide a valuable tool to gain new insights into novel nucleolar functions and processes. Investigating the interactions between plant viruses and the nucleolus will facilitate the design of novel strategies to control plant virus infections.

Diagnosis and discovery of fungal viruses using deep sequencing of small RNAs.

Analysis of virus-derived small RNAs with high-throughput sequencing has been successful for detecting novel viruses in plants and invertebrates. However, the applicability of this method has not been demonstrated in fungi, although fungi were among the first organisms reported to utilize RNA silencing. Here, we used virus-infected isolates of the fungal species complex Heterobasidion annosum sensu lato as a model system to test whether mycovirus genome segments can be detected with small RNA deep sequencing. Species of the genus Heterobasidion are some of the most devastating forest pathogens in boreal forests. These fungi cause wood decay and are commonly infected with species of the family Partitiviridae and the unassigned virus species Heterobasidion RNA virus 6. Small RNA deep sequencing allowed the simultaneous detection of all eight double-stranded RNA virus strains known to be present in the tested samples and one putative mitovirus species (family Narnaviridae) with a single-stranded RNA genome, designated here as Heterobasidion mitovirus 1. Prior to this study, no members of the family Narnaviridae had been described as infecting species of Heterobasidion. Quantification of viral double- and single-stranded RNA with quantitative PCR indicated that co-infecting viral species and viruses with segmented genomes can be detected with small RNA deep sequencing despite vast differences in the amount of RNA. This is the first study demonstrating the usefulness of this method for detecting fungal viruses. Moreover, the results suggest that viral genomes are processed into small RNAs by different species of Heterobasidion.

Interaction of the Microtubule-Associated Host Protein HIP2 with Viral Helper Component Proteinase Is Important in Infection with Potato virus A

Microtubules (MT) outline and maintain the overall shape of cells and can reorganize cellular membranes to serve as sites of RNA virus replication. Here, we provide data on involvement of an MT-associated protein in infection of plants with a potyvirus, Potato virus A (PVA), representing the largest family of plant-infecting RNA viruses. Our results showed that helper-component proteinase (HCpro)-interacting protein 2 (HIP2) of potato (Solanum tuberosum) is an MT-associated protein similar to Arabidopsis SPR2. Virus-induced silencing of HIP2 in Nicotiana benthamiana resulted in a spiral-like growth phenotype, similar to the Arabidopsis spr2 mutant, and the spr2 phenotype in Arabidopsis was complemented with potato HIP2. HCpro of PVA interacted with HIP2 of potato and tobacco (Nicotiana tabacum). The interaction was detected by bimolecular fluorescence complementation in PVA-infected leaves on MT and MT intersections at the cell cortex. HIP2-HCpro interaction was determined by the C-proximal α-helix-rich domain of HIP2, whereas the N-proximal putative TOG domain and the central coiled-coil domain of HIP2 controlled HIP2 dimerization and binding to MT. Accumulation of PVA was significantly reduced in the HIP2-silenced leaves of N. benthamiana, which indicates that HIP2-HCpro interactions are important for virus infection.

Silencing Suppressor Protein VPg of a Potyvirus Interacts With the Plant Silencing-Related Protein SGS3

Viral genome-linked protein (VPg) of potyviruses is involved in multiple steps of the potyvirus infection cycle, including viral multiplication and movement in plants. Recently, we showed that VPg of Potato virus A (PVA; genus Potyvirus) suppresses sense-mediated RNA silencing, which is linked to one or both nuclear or nucleolar localization. Here, we studied interactions between VPg and components of the plant RNA silencing pathway. Results showed that VPg interacts with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana, as shown by yeast two-hybrid analysis and bimolecular fluorescence complementation assays. VPg-SGS3 interactions co-localized with small cytoplasmic bodies that contained plant RNA-dependent RNA polymerase 6 (RDR6) (likely SGS3/RDR6 bodies). The N-terminal zinc finger (ZF) domain of SGS3 was the main determinant of the VPg interaction. Our data also suggest that the ZF domain controls SGS3 localization. SGS3 homodimerization was controlled by multiple protein regions. The VPg-SGS3 interaction appeared beneficial for PVA, as viral RNA levels correlated positively with sgs3 mRNA levels in the SGS3-silenced and SGS3-overexpressing leaves of Nicotiana benthamiana. The data support the idea that VPg acts as a suppressor of RNA silencing and suggest that an interaction with SGS3 may be important, especially in suppression of sense-mediated RNA silencing.

Mutation of a Short Variable Region in HCpro Protein of Potato virus A Affects Interactions with a Microtubule-Associated Protein and Induces Necrotic Responses in Tobacco

Helper component proteinase (HCpro) is a multifunctional protein of potyviruses (genus Potyvirus). HCpro of Potato virus A (PVA) interacts with the microtubule-associated protein HIP2 in host cells, and depletion of HIP2 reduces virus accumulation. This study shows that HCpro of Potato virus Y and Tobacco etch virus also interact with HIP2. The C-proximal portion of PVA HCpro determines the interaction with HIP2 and was found to contain a stretch of six residues comprising a highly variable region (HVR) in potyviruses. Mutations in HVR reduced PVA accumulation in tobacco plants and induced necrotic symptoms novel to PVA. Microarray and quantitative reverse transcription polymerase chain reaction analyses revealed induction of many defense-related genes including ethylene- and jasmonic acid-inducible pathways in systemically infected leaves at necrosis onset. Salicylic acid-mediated signaling was dispensable for the response. Genes related to microtubule functions were down-regulated. Structural modeling of HCpro suggested that all mutations in HVR caused conformational changes in adjacent regions containing functionally important motifs conserved in potyviruses. Those mutations, which also caused conformational changes in HVR, led to the greatest reduction of fitness. Our results implicate HVR in the regulation of HCpro conformation and virus-host interactions and suggest that mutation of HVR induces host defense.