The TLS element is essential for viral RNAs to enhance satRNA replication in <i>trans</i>-replication assays.

(A) Schematic diagrams of L3, ncL3 and their derivatives. The 3′ UTR of CMV RNAs is divided into three regions: a variable region (VR) at the 5′ end, a conserved TLS at the 3′ end, and a highly conserved region (CR) separating them. Deleted sequences in the constructed mutants were indicated by dashed lines. The TLS in ncL3 was substituted with the TLS of BMV, PSV, TAV, TMV, or TYMV, to generate six chimeric ncL3 mutants. (B-D, F) Northern blotting analyses of the accumulation of sat-T1, L3 and its mutants in the 5th true leaves of Nicotiana benthamiana transiently expressing LS replication proteins (L1a+L2a) and the RNA silencing suppressor P19. The mutants of L3 or ncL3 tested in these experiments are shown above. (E) Northern blotting analyses of TLS, RNA4 (L4) and its noncoding version (ncL4) of L3, as well as both polarities of sat-T1 in the trans-replication assays. In this replication assay, the TLS, L4 and ncL4 were provided separately in trans via agroinfiltration. It is worth mentioning that the probe used to detect RNA3 and its subgenomic RNA4 in (C) & (D) is the digoxin-labeled oligonucleotide complementary to the sequence spanning from nt 1200 to 1333 of L3. The digoxin-labeled oligonucleotide probe used to detect these RNAs in (E) is complementary to the sequence positioning at nt 2128–2167 of L3. Mock plants were treated by infiltration solution alone. The relative accumulation levels of RNA3, and positive-sense or negative-sense RNA of sat-T1 in (B-D, F) are shown below. Ethidium bromide-stained ribosomal RNAs served as the loading control.</p

Primers used for constructing plasmids and strand-specific reverse transcription-polymerase chain reactions.

Primers used for constructing plasmids and strand-specific reverse transcription-polymerase chain reactions.</p

RNA structures of the TLS elements from six RNA viruses.

(A) TLSBMV: the TLS from brome mosaic virus (BMV) RNA3, (B) TLSPSV: the TLS from peanut stunt virus (PSV) RNA3. (C) TLSTAV: the TLS from tomato aspermy virus (TAV) RNA3. (D) TLSTMV: the TLS from tobacco mosaic virus (TMV). (E) TLSTYMV: the TLS from turnip yellow mosaic virus (TYMV). (F) TLSCMV: the TLS from RNA3 of cucumber mosaic virus LS strain. The RNA structures of TLSBMV, TLSTYMV and TLSTMV were reported previously [63–64]. The RNA structures of TLSPSV, TLSTAV and TLSCMV were predicted based on that of TLSBMV. All these structures were redrawn using RNA2Drawer (available at https://rna2drawer.app/). The dash lines with arrows indicate pseudoknots formed by the base-paring of the nucleotide sequences colored red. (TIF)</p

Both replicase components of LS-CMV are responsible for the defect in supporting satRNA replication.

(A-C) Northern blotting analyses of the accumulation of L3 (A), sat-T1 (B), or both together (C). L3, sat-T1, or both were co-expressed with the replication proteins of Fny or LS, or their heterologous recombinants (F1a + L2a, L1a + F2a) in the 5th true leaves of Nicotiana benthamiana plants. Co-expression of sat-T1 with F1a or F2a served as negative controls in (B). At 3 days post-infiltration, total RNAs were extracted from the infiltrated leaves and subjected to northern blot hybridization. Mock plants were treated by infiltration solution alone. The relative accumulation levels of L3, and positive-sense or negative sense RNAs of sat-T1 were presented below. Ethidium bromide-stained ribosomal RNAs were used to assess the relative loading amounts of the RNA samples.</p

A proposed model of satRNA replication stimulated by viral RNAs in plants.

During the initial replication stage, CMV replication proteins localize to tonoplast and remodel it to create viral replication organelles (VROs). This process involves the recruitment of satRNAs, viral RNAs, or both to assemble viral replication complexes (VRCs). Notably, VROs could be free of both CMV and satellite RNAs, as viral replication proteins themselves can form VRO-like spherules, as reported previously [38]. VRCs assembled with satRNAs exhibit lower replication activity (indicated by VROs enclosed in green circles), whereas those formed with viral RNAs exhibit high activity (indicated by VROs enclosed in red circles). These highly active VRCs replicate not only viral RNAs, but also satRNAs when satRNAs are recruited alongside viral RNAs into the same VROs. However, considering that LS replication proteins have limited capability for satRNA recruitment, some VROs may lack satRNAs (indicated by VROs enclosed in gray circles) in the absence of viral RNAs. Consequently, VRCs formed in the presence of satRNAs alone would produce less satRNAs compared to those formed in the presence of both viral and satellite RNAs. Following the initial replication, more satRNAs, along with viral RNAs produced during the initial replication, participate in the creation of new VROs. This is expected to contribute to the enhanced proliferation of satRNAs with the assistance of viral RNAs.</p

The recruitment element Box-B is indispensable for viral RNAs to enhance satRNA replication.

(A) Schematic diagrams of L3 and its derivatives. The sequence from positions 47–96 nt in the 5′ UTR was removed from L3, to create L3-Δ5UII, as shown in the rectangle with green dashed lines. The Box-B motif in the IGR was removed from L3 to generate the mutant L3-ΔBoxB, as shown in the rectangle with blue dashed lines. The stem-loop C in the TLS was mutated with the nucleotides (AGUCAC) to create the mutant L3-mSLC, as shown in the rectangle with brown dashed lines. (B) Northern blotting analyses of the accumulation of RNA3 and sat-T1 in the presence of LS replication proteins (L1a+L2a). In the trans-replication assays, sat-T1 was co-expressed with L3, the aforementioned mutants, or the vector pCB301 in the 5th true leaves of Nicotiana benthamiana plants. At 3 days post-infiltration, total RNAs were extracted from the infiltrated leaves and subjected to northern blot hybridization. Mock plants were treated by infiltration solution alone. Ethidium bromide-stained ribosomal RNAs were used to ensure the equal loading for all RNA samples. The relative accumulation levels of sat-T1 or RNA3 are depicted in the graph on the right. Mean values with standard errors were calculated from three independent biological experiments.</p

The replication proteins of Fny-CMV and LS-CMV exhibit significant differences in their ability to recruit positive-sense RNA of sat-T1.

(A) The hairpin structures containing the Box-B sequence in blue and the mutated Box-B (mBox-B) in red. mF3 denotes the F3 mutant, in which the Box-B was substituted with mBox-B. mF3-T1(+) and mF3-T1(-) are mF3 derivatives with positive-sense or negative-sense RNA of sat-T1 inserted between the CP and 3′ UTR in mF3, respectively. A fragment of the GUS gene (337 nt), equivalent in size of sat-T1, was introduced into F3 or mF3, resulting to the creation of F3-gus or mF3-gus, respectively. (B) The accumulation levels of F3 and mF3 in the trans-replication assay. Either F3 or mF3 was co-expressed with LS replication proteins and P19 in the 5th true leaves of N. benthamiana plants. Mock plants were treated with infiltration solution. At 3 days post-infiltration, total RNAs were extracted separately from three infiltrated leaves for each treatment and subjected to northern blotting analyses. The relative accumulation levels of F3 and mF3 are shown below as the mean values with standard errors from three independent biological samples. (C) Determination of the replication activities of mF3-T1(+), mF3-T1(-), and the controls F3-gus and mF3-gus. These four F3 derivatives was co-expressed with the P19 suppressor and the replication proteins of LS-CMV (upper panel) or Fny-CMV (the lower panel) in the 5th true leaves of Nicotiana benthamiana plants. Mock plants were treated with infiltration solution. At 3 days post-infiltration, total RNAs were extracted separately from three infiltrated leaves for each treatment, and analyzed by northern blot hybridization. The relative accumulation levels with standard errors shown below were calculated from three independent biological samples. “UD” denotes the undetectable level. Ethidium bromide-stained ribosomal RNAs were used as a loading control for normalization of the relative accumulation levels.</p

Subcellular distribution of F1a-mCherry when co-expressed with 6×MS2-satT1 in the leaf tissues of <i>Nicotiana benthamiana</i>.

The lower epidermis of the leaves was infiltrated with Agrobacterium cells harboring the binary plasmids to express F1a-mCherry, 6×MS2-satT1, and p19. At 2 days post-agroinfiltration, the infiltrated leaves were subjected to Laser confocal microscopy for visualizing red fluorescence omitted from F1a-mCherry. (TIF)</p

Substitution of the Box-B motif in RNA3 with sat-T1 inactivated the replication of the RNA3 variants by the replicase of LS-CMV.

(A) Schematic diagrams of RNA3 and its derivative (R3-ΔBox-T1), in which the Box-B sequence was substituted with the (+) strand of sat-T1. (B) Northern blotting analyses of the accumulation of RNA3 and its variants. RNA3 from Fny-CMV (F3) or LS-CMV (L3), as well as their mutants or vector pCB301 was separately co-expressed with the replicase of LS-CMV, together with the RNA silencing suppressor P19. At 3 days post-agroinfiltration, total RNAs were extracted from the infiltrated leaves and subjected to northern blot hybridization. Mock plants were treated by infiltration solution alone. Ethidium bromide-stained ribosomal RNAs were used to assess the loading amounts of all RNA samples. (TIF)</p

Sequence alignment of the 3′ UTRs from the genomic RNAs of Fny-CMV and LS-CMV.

The sequence alignment was carried out using the MegAlign program in DNAstar. The 3′ UTR sequence is divided into three regions: a variable region (VR) at the 5′ end, a conserved tRNA-like structure (TLS) at the 3′ end, and a highly conserved stretch region (CR) separating them. (TIF)</p