Ron Reade

A 38-Amino-Acid Sequence Encompassing the Arm Domain of the Cucumber Necrosis Virus Coat Protein Functions as a Chloroplast Transit Peptide in Infected Plants

ABSTRACT Experiments to determine the subcellular location of the coat protein (CP) of the tombusvirus Cucumber necrosis virus (CNV) have been conducted. By confocal microscopy, it was found that an agroinfiltrated CNV CP-green fluorescent protein (GFP) fusion targets chloroplasts in Nicotiana benthamiana leaves and that a 38-amino-acid (aa) region that includes the complete CP arm region plus the first 4 amino acids of the shell domain are sufficient for targeting. Western blot analyses of purified and fractionated chloroplasts showed that the 38-aa region directs import to the chloroplast stroma, suggesting that the CNV arm can function as a chloroplast transit peptide (TP) in plants. Several features of the 38-aa region are similar to features typical of chloroplast TPs, including (i) the presence of an alanine-rich uncharged region near the N terminus, followed by a short region rich in basic amino acids; (ii) a conserved chloroplast TP phosphorylation motif; (iii) the requirement that the CNV 38-aa sequence be present at the amino terminus of the imported protein; and (iv) specific proteolytic cleavage upon import into the chloroplast stroma. In addition, a region just downstream of the 38-aa sequence contains a 14-3-3 binding motif, suggesting that chloroplast targeting requires 14-3-3 binding, as has been suggested for cellular proteins that are targeted to chloroplasts. Chloroplasts of CNV-infected plants were found to contain CNV CP, but only the shell and protruding domain regions were present, indicating that CNV CP enters chloroplasts during infection and that proteolytic cleavage occurs as predicted from agroinfiltration studies. We also found that particles of a CNV CP mutant deficient in externalization of the arm region have a reduced ability to establish infection. The potential biological significance of these findings is discussed.

The p33 auxiliary replicase protein of Cucumber necrosis virus targets peroxisomes and infection induces de novo peroxisome formation from the endoplasmic reticulum.

Tombusviruses replicate on pre-existing organelles such as peroxisomes or mitochondria, the membranes of which become extensively reorganized into multivesicular bodies (MVBs) during the infection process. Cucumber necrosis virus (CNV) has previously been shown to replicate in association with peroxisomes in yeast. We show that CNV induces MVBs from peroxisomes in infected plants and that GFP-tagged p33 auxiliary replicase protein colocalizes with YFP(SKL), a peroxisomal marker. Most remarkably, the ER of CNV infected Nicotiana benthamiana 16C plants undergoes a dramatic reorganization producing numerous new peroxisome-like structures that associate with CNV p33, thus likely serving as a new site for viral RNA replication. We also show that plants agroinfiltrated with p33 develop CNV-like necrotic symptoms which are associated with increased levels of peroxide. Since peroxisomes are a site for peroxide catabolism, and peroxide is known to induce plant defense responses, we suggest that dysfunctional peroxisomes contribute to CNV induced necrosis.

Induction of Particle Polymorphism by Cucumber Necrosis Virus Coat Protein Mutants In Vivo

ABSTRACT The Cucumber necrosis virus (CNV) particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. Plants infected with wild-type CNV accumulate a high number of T=3 particles, but other particle forms have not been observed. Particle polymorphism in several T=3 icosahedral viruses has been observed in vitro following the removal of an extended N-terminal region of the CP subunit. In the case of CNV, we have recently described the structure of T=1 particles that accumulate in planta during infection by a CNV mutant (R1+2) in which a large portion of the N-terminal RNA binding domain (R-domain) has been deleted. In this report we further describe properties of this mutant and other CP mutants that produce polymorphic particles. The T=1 particles produced by R1+2 mutants were found to encapsidate a 1.9-kb RNA species as well as smaller RNA species that are similar to previously described CNV defective interfering RNAs. Other R-domain mutants were found to encapsidate a range of specifically sized less-than-full-length CNV RNAs. Mutation of a conserved proline residue in the arm domain near its junction with the shell domain also influenced T=1 particle formation. The proportion of polymorphic particles increased when the mutation was incorporated into R-domain deletion mutants. Our results suggest that both the R-domain and the arm play important roles in the formation of T=3 particles. In addition, the encapsidation of specific CNV RNA species by individual mutants indicates that the R-domain plays a role in the nature of CNV RNA encapsidated in particles.

A highly basic KGKKGK sequence in the RNA-binding domain of the Cucumber necrosis virus coat protein is associated with encapsidation of full-length CNV RNA during infection

The Cucumber necrosis virus particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. The N-terminal 58 aa residue segment of the CP R domain is believed to bind viral RNA within virions and during assembly. We report results of in vivo experiments that examine the role of the R domain in assembly. Deletion analyses identified 3 conserved 5-10 aa regions as playing critical roles. A highly basic KGKKGK sequence was found to be both necessary and sufficient for encapsidation of the full-length genome and polymorphic virions were produced in mutants lacking the KGKKGK sequence. The amount of full-length RNA present in virions was substantially reduced in R domain mutants where 2 of the 4 lysine residues were substituted with alanine, whereas substitution of 4 lysines by arginine had only a modest effect. The potential role of the R domain in formation of a scaffold for particle assembly is discussed.

Molecular analysis of the cucumber leaf spot virus genome

Full-length clones of the genome of the Aureusvirus, Cucumber leaf spot virus (CLSV), have been constructed and infectious T7 polymerase derived synthetic transcripts have been produced. Mutational analysis of the genome indicates a role for p84 in viral RNA replication, the CP in systemic movement, p27 in viral cell-to-cell movement and p17 in symptom induction. A CLSV mutant lacking ORFs for the CP, p27 and p17 (CLSV YX) was capable of replication and systemic movement in transgenic Nicotiana benthamiana plants expressing the Red clover necrotic mosaic virus (RCNMV) movement protein (MP) suggesting that p25 and p84 are sufficient for viral RNA replication and that the RCNMV MP can permit CLSV cell-to-cell as well as systemic movement. Moreover, CLSV YX induced severe necrosis in both inoculated and uninoculated leaves of transgenic plants suggesting that CLSV p25 and/or p84 are important determinants of the necrotic phenotype. Another mutant similar to CLSV YX but expressing only limited amino-terminal portions of CP, p27 and p17 failed to produce necrosis or to move systemically in RCNMV MP transgenic N. benthamiana plants. These results suggest that these short translated regions or cis-acting sequences present in the CLSV CP, p27 and/or p17 ORFs suppress the necrosis induced by p25/p84 and also suppress systemic movement mediated by the RCNMV MP.

Cucumber necrosis virus p20 is a viral suppressor of RNA silencing

The p20 protein encoded by the tombusvirus, Cucumber necrosis virus has previously been shown to be involved in host pathogenicity and shares sequence similarity with the Tomato bushy stunt virus p19 suppressor of silencing. Using a virus-induced gene silencing (VIGS) assay, we show that p20 is a viral suppressor of RNA silencing (VSR) in infected plants. In addition, a CNV p20-knockout mutant showed a decline in viral RNA accumulation in infected plants, consistent with the role of p20 in suppression of RNA silencing. However, unexpectedly, all GFP transgenic plants co-infiltrated with p20 and GFP displayed RNA silencing using an Agrobacterium-mediated silencing assay. Detailed RNA analysis of GFP mRNA levels in p20 agro-infiltrated plants revealed that p20 did initially display suppressor activity but this was rapidly overcome by RNA silencing. p20 expression levels in agro-infiltrated plants were shown to be approximately 50-fold lower than that of the TBSV p19 silencing suppressor, consistent with the notion that p20 dosage levels are not sufficient to suppress RNA silencing in the Agrobacterium-mediated system. Our results suggest that a viral-based VIGS assay may be required for identifying VSRs encoded by some plant viruses. Based on bioinformatics studies the mechanism of suppression of silencing by p20 is predicted to be similar to that of the TBSV p19 suppressor.

Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection

ABSTRACT Next-generation sequence analysis of virus-like particles (VLPs) produced during agroinfiltration of cucumber necrosis virus (CNV) coat protein (CP) and of authentic CNV virions was conducted to assess if host RNAs can be encapsidated by CNV CP. VLPs containing host RNAs were found to be produced during agroinfiltration, accumulating to approximately 1/60 the level that CNV virions accumulated during infection. VLPs contained a variety of host RNA species, including the major rRNAs as well as cytoplasmic, chloroplast, and mitochondrial mRNAs. The most predominant host RNA species encapsidated in VLPs were chloroplast encoded, consistent with the efficient targeting of CNV CP to chloroplasts during agroinfiltration. Interestingly, droplet digital PCR analysis showed that the CNV CP mRNA expressed during agroinfiltration was the most efficiently encapsidated mRNA, suggesting that the CNV CP open reading frame may contain a high-affinity site or sites for CP binding and thus contribute to the specificity of CNV RNA encapsidation. Approximately 0.09% to 0.7% of the RNA derived from authentic CNV virions contained host RNA, with chloroplast RNA again being the most prominent species. This is consistent with our previous finding that a small proportion of CNV CP enters chloroplasts during the infection process and highlights the possibility that chloroplast targeting is a significant aspect of CNV infection. Remarkably, 6 to 8 of the top 10 most efficiently encapsidated nucleus-encoded RNAs in CNV virions correspond to retrotransposon or retrotransposon-like RNA sequences. Thus, CNV could potentially serve as a vehicle for horizontal transmission of retrotransposons to new hosts and thereby significantly influence genome evolution. IMPORTANCE Viruses predominantly encapsidate their own virus-related RNA species due to the possession of specific sequences and/or structures on viral RNA which serve as high-affinity binding sites for the coat protein. In this study, we show, using next-generation sequence analysis, that CNV also encapsidates host RNA species, which account for ∼0.1% of the RNA packaged in CNV particles. The encapsidated host RNAs predominantly include chloroplast RNAs, reinforcing previous observations that CNV CP enters chloroplasts during infection. Remarkably, the most abundantly encapsidated cytoplasmic mRNAs consisted of retrotransposon-like RNA sequences, similar to findings recently reported for flock house virus (A. Routh, T. Domitrovic, and J. E. Johnson, Proc Natl Acad Sci U S A 109:1907–1912, 2012). Encapsidation of retrotransposon sequences may contribute to their horizontal transmission should CNV virions carrying retrotransposons infect a new host. Such an event could lead to large-scale genomic changes in a naive plant host, thus facilitating host evolutionary novelty.

The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains

Abstract Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication.