Systemic infection by plant viruses results from the complex molecular interplay between the host plant and the invading virus [
1]. To establish systemic infection, plant viruses must have the ability to generate progeny viruses in the primarily infected cell, move from therein to neighboring cells and further transport long-distance within the plant. Viral cell-to-cell or local movement mainly occurs in mesophyll and epidermal cells through plasmodesmata (PD), a specialized intercellular organelle that crosses the cell wall to establish cytoplasmic and endomembrane continuity between adjacent cells [
2], whereas the phloem-dependent long-distance transport allows the virus to reach remote tissues through the vascular system [
3]. Currently, it is generally accepted that viral intercellular movement is mediated by virus-encoded movement protein (MP) [
4], although the number of MPs, their interactions with host cellular structure and mode of action are different from virus to virus.
Potyviruses represent the largest group of known plant viruses and include many agriculturally important viruses such as
Turnip mosaic virus (TuMV),
Plum pox virus,
Tobacco etch virus (TEV), and
Soybean mosaic virus [
5]. The potyviral genome is a single positive-strand RNA and encodes a long polyprotein that is processed by three proteinases (P1, HC-Pro and NIaPro) to release 10 mature proteins [
5]. A frameshift resulting from replication slippage in the P3 cistron leads to the production of an additional protein P3N-PIPO [
6‐
8]. Although five viral proteins, i.e., the cylindrical inclusion protein (CI), the coat protein (CP), the helper component proteinase (HC-Pro), the viral genome-linked protein (VPg) and P3N-PIPO have been implicated in viral intercellular movement, CI seems to play a direct role in viral cell-to-cell movement [
3,
9].
Previous studies have shown that potyviral CI is an RNA helicase [
10]. As highly conserved enzymes, RNA helicases can utilize ATP to catalyze the separation of RNA duplexes and the structural rearrangement of RNA and RNA/protein complexes (ribonucleoprotein (RNP) complexes) in all aspects of RNA metabolism, from transcription, mRNA splicing and translation, RNA modification and transport, ribosome biogenesis, RNP complex assembly to mRNA degradation [
11,
12]. RNA helicases are present in all eukaryotic cells as well as many bacteria and some viruses [
13]. Based on sequence and structural features, RNA helicases are classified into five main groups, namely, superfamily (SF) 1 to SF5 [
14]. The potyviral CI RNA helicase belongs to SF2 and contains seven highly conserved motifs, I, Ia, II, III, IV, V, and VI. Motifs Ia, III, and IV are the least conserved, whereas motif IV as well as motifs I, II, and V are the best conserved [
15]. The helicase domain of the CI protein is located at the N-terminal and central region. The C-terminal region shows no homology with any known proteins. Genetic analyses conducted with a TEV full-length infectious clone tagged with the marker gene encoding b-glucuronidase (GUS) provided genetic evidence that the CI protein plays an essential role in viral genome replication and cell-to-cell movement [
3]. Consistent with the genetic data, CI has been found to be present in the viral replication complex, presumably assisting viral genome amplification through its RNA binding and duplex unwinding activities [
16,
17]. CI also interacts with CP in the cytoplasm [
9,
18] and forms the conical structures at PD [
9,
19,
20]. Recently we have shown that P3N-PIPO is a PD-located protein that modulates the targeting of CI to the PD to facilitate potyviral cell-to-cell movement [
9]. The structure and function of potyviral CI have been reviewed recently [
21]. Despite this progress, the mode of action by CI and its underlying mechanism in the potyviral infection process are still far from being understood.
In this study, we generated a serial of double-substitutions at the clustered charged residues of the CI protein of TuMV and analyzed the effect of these mutations on viral genome amplification, cell-to-cell movement and systemic infection. Our data support that the potyviral CI protein has essential, yet distinct roles in viral replication and intercellular movement.