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, 83 (11), 5419-29

The C Terminus of the Polerovirus p5 Readthrough Domain Limits Virus Infection to the Phloem

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The C Terminus of the Polerovirus p5 Readthrough Domain Limits Virus Infection to the Phloem

Kari A Peter et al. J Virol.

Abstract

Poleroviruses are restricted to vascular phloem tissues from which they are transmitted by their aphid vectors and are not transmissible mechanically. Phloem limitation has been attributed to the absence of virus proteins either facilitating movement or counteracting plant defense. The polerovirus capsid is composed of two forms of coat protein, the major P3 protein and the minor P3/P5 protein, a translational readthrough of P3. P3/P5 is required for insect transmission and acts in trans to facilitate long-distance virus movement in phloem tissue. Specific potato leafroll virus mutants lacking part or all of the P5 domain moved into and infected nonvascular mesophyll tissue when the source-sink relationship of the plant (Solanum sarrachoides) was altered by pruning, with the progeny virus now being transmissible mechanically. However, in a period of months, a phloem-specific distribution of the virus was reestablished in the absence of aphid transmission. Virus from the new phloem-limited infection showed compensatory mutations that would be expected to restore the production of full-length P3/P5 as well as the loss of mechanical transmissibility. The data support our hypothesis that phloem limitation in poleroviruses presumably does not result from a deficiency in the repertoire of virus genes but rather results from P3/P5 accumulation under selection in the infected plant, with the colateral effect of facilitating transmission by phloem-feeding aphid vectors.

Figures

FIG. 1.
FIG. 1.
Genome organization of PLRV and illustration of P5 mutant viruses. (A) The 6-kb monopartite, positive-sense, single-stranded RNA genome consisting of eight ORFs. ORFs P3 to P5, which are involved in structure and movement, are conserved among luteoviruses and are expressed from subgenomic RNA-1. Virions are composed mainly of P3 coat proteins and a small number of P3/P5 readthrough proteins, which are anchored into the virion by the P3 moiety. The P5 domain is exposed on the surface and has a conserved N-terminal region and a variable C-terminal region. (B) Illustration of the P3/P5 translation of the mutant viruses described in this study. Deletion of amino acids is specified as a line, and a lack of protein expression is shown as a dotted line. WT PLRV with expression of the entire P3/P5 is depicted. For the SST and QSS mutant viruses, the 9 nt comprising the SST sequence (nt 4531 to 4539) and the 9-nt QSS sequence (nt 4438 to 4446) were deleted, respectively, while full-length P3/P5 translation was maintained. The resulting P3/P5 protein was incorporated into the virion for the SST mutant virus but not for the QSS mutant virus. The ΔP5 mutant virus contains two stop codons at the end of the P3 sequence followed by a 100-nt deletion in P5 (double Xs); no P3/P5 translation occurs. The SYG mutation is located 18 amino acids from the C terminal region. In addition to the 9-nt deletion to create the SYG deletion (nt 4828 to 4836), a single nucleotide was deleted 14 nt downstream (nt 4850) (triangle). This created a frameshift, which altered the subsequent amino acids (diagonal lines) and eventually produced a stop codon (X) 17 nt downstream from the single-nucleotide deletion. As a result, a truncated P3/P5 was translated. ΔP5 and SYG mutant viruses do not have P3/P5 incorporated into their virions.
FIG. 2.
FIG. 2.
Representative S. sarrachoides plant 3 weeks p.i. Samples were taken for tissue immunoblots. (A) Stem tissue sample below inoculation sites; (B to I) stem samples above the inoculation sites, with stem samples taken at nodes to apex of branches; (J) mature leaf and petiole samples; (K) developing leaf and petiole samples; (L) seed pod peduncles. The inset picture shows a plant (8- to10-leaf stage) at the time of inoculation. Stars indicate the general area where 3- to 4-week-old plants were inoculated. Plants were pruned at 3 to 4 weeks p.i. by removing 7.5 to 10 cm of stem from each branch.
FIG. 3.
FIG. 3.
S. sarrachoides stem and leaf tissue immunoblots. Plants were agroinoculated with WT virus or the ΔP5 mutant 3 weeks prior to destructive sampling from many sites on the plant. Samples from similar regions on healthy plants were also collected. Virus antigen is detected as a purplish-blue immunostained region, some of which are indicated by arrowheads. The internal (Int) and external (Ext) phloem bundles are labeled. Immunoblots on tissue collected from plants infected with the SST, QSS, and SYG mutants were also completed (data not shown). The level and distribution of virus for SST and QSS were similar to those of the WT, and SYG was similar to ΔP5.
FIG. 4.
FIG. 4.
Symptoms observed on S. sarrachoides leaves infected systemically with WT (A) and SYG (E) viruses at 4 and 10 weeks p.i., respectively. These leaves were used to generate whole-leaf tissue immunoblots of the abaxial surfaces. (B) Enlarged view of the immunoblot of WT-infected leaf tissue showing virus associated with only the veins (arrows pointing to small purple regions). The area shown is approximately a ×2.5 magnification of what is viewed in F. (C) Transmission electron microscopy analysis of similar WT-infected tissues (magnification, ×3,000) illustrating virions present in phloem-related cells, companion cells (CC), and sieve elements (SE). (D) Higher magnification (magnification, × 30,000) of the boxed region in C displaying virions (arrows) within the cell cytoplasm. (F) Enlarged view of the whole-leaf immunoblot of SYG-infected tissue revealing extensive spread of virus outside the phloem tissues and into mesophyll tissues (purple regions). Similar results were observed for ΔP5-infected leaves (not shown). (G) Transmission electron microscopy analysis of SYG-infected tissue (magnification, ×2,000) confirming immunoblot data for the presence of virions in mesophyll cells (Me). (H) Higher magnification (magnification, ×30,000) of the boxed region in G where crystalline arrays of virions (arrows) were commonly observed to be associated with the chloroplast outer membranes.
FIG. 5.
FIG. 5.
Immunoblots of various tissue sections from pruned or nonpruned S. sarrachoides plants infected previously with WT or the ΔP5 mutant at 9 weeks p.i. Areas positive for virus are indicated by purplish-blue staining. Virus levels and distribution were similar for pruned and nonpruned (not shown) plants infected with WT and QSS virus (pruned WT samples are shown). Virus levels and distribution for the SYG mutant (not shown) were similar to those of images shown for ΔP5. Similar to the images of healthy tissue shown in Fig. 3, no labeling was observed in healthy control tissue used in this experiment (not shown).
FIG. 6.
FIG. 6.
Measurement of virus and P3 protein accumulation in WT-, ΔP5-, and SYG-infected leaves using Western blot analysis and DAS-ELISA. Three leaves each from pruned WT-, ΔP5-, and SYG-infected plants were divided into two halves. (A) The P3 protein was detected in total protein extracts in one half of the leaf by Western blot analysis. (B) Virus accumulation was detected in the other half of the leaf using DAS-ELISA. The mean absorbance values (A405) ± standard errors are shown. One-way analysis of variance indicated a significant effect of virus type on antigen accumulation (F = 69.53; df = 2; P < 0.001), and treatment means were all significantly different from each other according to the Newman-Keuls test (P = 0.05).
FIG. 7.
FIG. 7.
Mechanically inoculated S. sarrachoides leaves and their respective immunoblots approximately 5 days p.i. using WT-, SYG revertant (exhibiting WT-like symptoms)-, or SYG-infected S. sarrachoides leaves as the source of plant sap for virus inoculation. The immunoblots for the WT and the SYG revertant are approximately a ×2.5 magnification of the SYG immunoblot.
FIG. 8.
FIG. 8.
Analysis of SYG revertant-infected leaf tissue. (A) SYG-infected S. sarrachoides leaf exhibiting WT-like symptoms. (B) Enlarged view of a SYG-infected whole-leaf tissue immunoblot from the same leaf showing virus confined to phloem tissue. (C) SYG revertant sequences (sequences SYG-1 to -4) obtained from progeny virus in four different infected leaves. WT and SYG sequences are shown for comparison. In the SYG sequence, the codon containing a deleted nucleotide (14 nt downstream from the SYG deletion) that resulted in a frameshift mutation is underlined. Amino acids and nucleotides that were altered in the pseudorevertants are shown in red type. Sequences SYG-1 to -4 originate from leaves manifesting WT-like symptoms.
FIG. 9.
FIG. 9.
RT-PCR to detect the presence of virus in aphids after 24- and 72-h periods of feeding on virus-infected tissue to determine if aphids ingested virus from infected leaves. Primers amplify a 624-bp fragment of the P3 ORF. Primers that amplify a 301-bp fragment of the 18S rRNA were used as an RNA control. Aphids were fed on tissue systemically infected with WT, QSS, ΔP5, or SYG virus. WT and QSS viruses were identical, with the WT being the representative sample shown. Two groups of three aphids were collected at each time point for each sample.

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