Deciphering the mechanism of defective interfering RNA (DI RNA) biogenesis reveals that a viral protein and the DI RNA act antagonistically in virus infection

Abstract

Potato mop-top virus (PMTV) produces a defective RNA (D RNA) encompassing the 5'-terminal 479 nucleotides (nt) and 3'-terminal 372 nt of RNA-TGB (where TGB is triple gene block). The mechanism that controls D RNA biogenesis and the role of D RNA in virus accumulation was investigated by introducing deletions, insertions, and point mutations into the sequences of the open reading frames (ORFs) of TGB1 and the 8-kilodalton (8K) protein that were identified as required for efficient production of the D RNA. Transient expression of RNA-TGB in the absence of RNA-Rep (which encodes the replicase) did not result in accumulation of D RNA, indicating that its production is dependent on PMTV replication. The D RNA could be eliminated by disrupting a predicted minus-strand stem-loop structure comprising complementary sequences of the 5' TGB1 ORF and the 3' 8K ORF, suggesting intramolecular template switching during positive-strand synthesis as a mechanism for the D RNA biogenesis. Virus accumulation was reduced when the 8K ORF was disrupted but D RNA was produced. Conversely, the virus accumulated at higher titers when the 8K ORF was intact and D RNA production was blocked. These data demonstrate that the D RNA interferes with virus infection and therefore should be referred to as a defective interfering RNA (DI RNA). The 8K protein was shown to be a weak silencing suppressor. This study provides an example of the interplay between a pathogen and its molecular parasite where virus accumulation was differentially regulated by the 8K protein and DI RNA, indicating that they play antagonistic roles and suggesting a mechanism by which the virus can attenuate replication, decreasing viral load and thereby enhancing its efficiency as a parasite.

Fig 1

Schematic representation of the RNA-TGB mutants engineered in this study or obtained by Savenkov et al. (12) and structure of D RNA. (A) Schematic of the D RNA formation through a single internal deletion in the parental RNA. Structure of the D RNA relative to the full-length RNA-TGB. The D RNA is composed of the fused ends of the viral RNA-TGB as described in the text. The length of the DI RNA (851 nt) is indicated. (B) Summary of the three sets of RNA-TGB mutants used in this study. (C) Graphical summary of RNA-TGB mutants. Small gray rectangles denote deletions engineered into infectious cDNA of RNA-TGB. Positions of the probes used for Northern blotting are indicated on the diagram. (D) Characteristic features of recombinants 8K-D, 8K-S, 8K-FLAG, No-N-Stop, and No-NΔC mutants compared to wt sequence. The sequences at the 3′ end of the 8K ORF (bottom sequence) and wt (top sequence) are shown. Translation of the ORFs is shown below the top and bottom sequences. Translation of the overlapping part of the TGB3 ORF is shown above the wt sequence. The mutated or inserted nucleotide residues in relation to the RNA-TGB wt sequence are shown in small script and in bold. The dashed line indicates the deletion of the sequence encompassing the 3′-proximal part of the 8K ORF, which does not overlap the TGB3 ORF. The sequence that is not shown is indicated with greater-than (>) symbols. Numbering refers to nucleotide positions in full-length RNA-TGB. Stop codons are indicated with asterisks. The restrictions sites used to facilitate cloning are underlined and refer to the cDNA (not RNA).

Fig 2

Identification and location of RNA-TGB sequences within D RNA. Northern blot analyses depicting the accumulation of D RNA for N-stop, No-N-stop, and wt and the absence of D RNA accumulation for NΔC and No-NΔC. Note that 8K-D, 8K-S, and 8K-FLAG do not produce detectable D RNA. Five ³²P-labeled RNA probes were used (shown schematically in Fig. 1). The identity of each probe and position of molecular size markers are given on the left. Note that the 8K probe does not detect No-NΔC because it completely lacks the 8K ORF.

Fig 3

Stem-loops in the D RNA and in the plus strand of full-length RNA-TGB as predicted by RNAfold. (A) Predicted secondary structure of the D RNA showing elements referred to in the text. The AUG and UGA codons are indicated by black bars. Numbering refers to nucleotide positions in the plus strand of full-length RNA-TGB. JP, junction point. (B) Predicted secondary structure of the plus strand of full-length RNA-TGB.

Fig 4

Effects of CSLIII mutations on accumulation of D RNA and the virus progeny. (A) Predicted stem-loop structure in the minus strand of RNA-TGB. The predicted secondary structure of the minus strand of full-length RNA-TGB is shown on the right. Numbering refers to nucleotide positions in the plus strand of full-length RNA-TGB. Mutations used to test CSLIII requirements for D RNA production are shown on the left for TGB1-mod (nine point mutations), and a deletion and point mutations in NΔC and No-NΔC are highlighted in gray. The free energies refer to the stem-loops in the wt CSLIII and in the TGB1-mod. (B) Northern analysis to measure the accumulation of D RNA in total RNA preparations recovered from the leaves of N. benthamiana inoculated with wt and TGB1-mod. The ³²P-labeled RNA probe specific for the 5′ UTR was used, and D RNA is indicated by an arrow. Lane m, mock. (C) Detection of PMTV by ELISA as indicated by absorbance values at 405 nm. Plant extracts were prepared from upper leaves at 14 dpi. The average absorbance of a healthy plant extract was equal to 0.18. ELISAs were conducted twice (n = 12).

Fig 5

Stem-loop II (SLII)-deficient mutants do not produce D RNA. (A) The RNAfold-predicted structures in the positive strand of D RNA and their calculated free energies in kcal/mole are shown. Deleted bases in ΔSLII^TGB1arm and ΔSLII^8Karm are represented by a delta (Δ). (B) Northern blot analysis to detect the DI in total RNA preparations recovered from the leaves of N. benthamiana inoculated with wt, ΔSLII^TGB1arm, and ΔSLII^8Karm inocula. The ³²P-labeled RNA probe specific for the 3′ UTR was used. (C) Accumulation of PMTV was measured by ELISA as indicated by absorbance values at 405 nm. Plant extracts were prepared from upper leaves at 14 dpi. The average absorbance of a healthy plant extract was equal to 0.15. ELISAs were conducted twice (n = 8).

Fig 6

Comparison of the absorbance values of the PMTV mutant deficient in 8K and DI RNA production. (A) Levels of PMTV virion accumulation detected by double-antibody sandwich ELISA as indicated by absorbance values at 405 nm. Plant extracts were prepared from upper leaves at 14 dpi. The average absorbance of a healthy plant extract was equal to 0.19. Asterisks indicate that the absorbance values were significantly different (P < 0.05, Student t test) from the wt. ELISAs were conducted three times (n = 18). (B) Effect of the lack/presence of 8K and DI on virus accumulation. Accumulation of wt progeny was considered to be 100%. The data were collected from three independent experiments (n = 18). Asterisks indicate that the absorbance values were significantly different (P < 0.05, Student t test) from the wt. The RNA-TGB mutants are also presented in the table (C). An upward arrow indicates statistically significant increase in virus accumulation compared to the wt. A downward arrow indicates statistically significant reduction in virus accumulation compared to the wt. Double-headed arrow, no change; ns, not significant (P > 0.05, Student t test).

Fig 7

Effects of CSLIII and SLII mutations on accumulation of PMTV DI RNA and the virions. (A and B) Mutations used to disrupt the stem-loops are shown on the left side of the secondary structures and refer to the No-CSLIII and No-SLII mutants, respectively. Mutations introduced into the No-CSLIII and No-SLII mutants to restore the stem-loops are shown on the right side of the secondary structures and refer to the CSLIII-restore and No-SLII-restore mutants, respectively. Numbering refers to nucleotide positions in the plus strand of full-length RNA-TGB. (C) RT-PCR of cDNA of N. benthamiana upper leaves to detect the accumulation of DI RNA and the virus genomic RNAs. The identity of each mutant is indicated above the gels. Twenty-four cycles were used for amplification. The constitutively expressed gene for N. benthamiana elongation factor 1α (Nb EF1α) served as a normalization control. The experiment was repeated twice with similar results. (D) Levels of PMTV virion accumulation detected by double-antibody sandwich ELISA as indicated by absorbance values at 405 nm. Plant extracts were prepared from upper leaves at 14 dpi. The average absorbance of a healthy plant extract was equal to 0.16. Asterisks indicate that the absorbance values were significantly different (P < 0.05, Student t test) from No-N-stop, which was used as a backbone to obtain all other constructs. ELISAs were conducted twice (n = 12).

Fig 8

Dependence of DI RNA production on replication and inefficient DI RNA movement into the upper leaves. (A) Electrophoresis of RT-PCR-amplified DNA fragments corresponding to RNA-TGB, RNA-Rep, and DI. N. benthamiana leaves were infiltrated autonomously with the RNA-TGB genomic component (lanes 1 and 2), or agro-transformants of RNA-TGB and RNA-Rep were coexpressed (lanes 3 and 4). Control infiltration was performed with an Agrobacterium strain transformed with an empty plasmid (EP). (B) Northern blot analysis of RNA extracted from inoculated and upper leaves of the N. benthamiana plants inoculated with wt (RNA-Rep plus RNA-CP plus RNA-TGB; lanes 2 and 3), No-NΔC (RNA-Rep plus RNA-CP plus No-NΔC; lanes 4 and 5), or No-NΔC inoculum supplemented with T7 RNA polymerase-generated transcripts of DI (lanes 6 and 7). The experiment was repeated three times with similar results. DI RNA is indicated by an arrow. (C) RT-PCR on cDNA of N. benthamiana upper leaves to detect accumulation of the DI RNA and the virus genomic RNAs at 21 dpi. The identity of each type of inoculum is indicated above the gels. Twenty-four cycles were used for amplification. The constitutively expressed gene for N. benthamiana elongation factor 1α (Nb EF1α) served as a normalization control. The data are from two independent experiments.

Fig 9

Knockout of the putative coding ORF within DI RNA does not affect its capability to interfere with virus accumulation. (A) Schematic representation of the DI mutant engineered to prevent translation from the DI-encoding ORF. The mutated nucleotide residues in relation to the DI RNA sequence are shown in small script and in bold. (B) Levels of PMTV CP antigen detected by double-antibody sandwich ELISA as indicated by absorbance values at 405 nm. Plant extracts were prepared from inoculated leaves at 14 dpi. The average absorbance of a healthy plant extract was equal to 0.18. Asterisks indicate that the absorbance values were significantly different (P < 0.0005, Student t test) from the control inoculation with TGB1-mod. The data were collected from two independent experiments (n = 8).

Fig 10

Analysis of local RNA silencing suppression activity in N. benthamiana plants. (A) Representative images of TCV-sGFP complementation assay with constructs indicated on the panels. Preinfiltrated Agrobacterium cultures carrying either an empty plasmid (pGWB) or TGB1 construct does not complement the movement of TCV-sGFP, while preinfiltrated 8K and PVA HC-Pro constructs complement the movement of TCV-sGFP at 3 dpi. Scale bar, 100 μm. (B) Representative image showing the lack of movement. Preinfiltrated Agrobacterium cultures carrying the 8K construct do not complement the movement of TCVΔ92-sGFP at 3 dpi. Scale bar, 100 μm. (C) Quantification of the movement of TCV-sGFP in the complementation assay. The diameter of 40 foci of infection were measured at 3 dpi for each construct indicated below the graph. An asterisk (*) indicates a statistically significant (P < 0.0005) difference in focus diameter compared to the empty plasmid control (pGWB). ns, not significant (P > 0.05). (C) GFP fluorometric analysis of the protein extracts from N. benthamiana leaves of the PZP-TCV-sGFP coinfiltration assay. Leaves were coinfiltrated with PZP-TCV-sGFP and various constructs, indicated below the graph. The baseline represents the level of fluorescence of the protein extract from the leaf expressing an empty T-DNA. Both PMTV 8K and PVA HC-Pro showed statistically significant increases in GFP fluorescence (P < 0.0005). ns, not significant (P > 0.05). Error bars denote standard deviation of the measurements.