Covering common ground

Abstract

Plant viruses are composed of diverse genomes (e.g., RNA or DNA) encoding proteins that vary widely in sequence. It is becoming clear, however, that some apparently unrelated viral proteins have similar functions. The P6 protein encoded by Cauliflower mosaic virus (CaMV) and the 126-kDa protein encoded by Tobacco mosaic virus (TMV) are examples of this convergence in protein function. Although having no apparent sequence similarity, both proteins are pathogenicity determinants during infection, are components of novel intracellular cytoplasmic inclusions and suppress RNA silencing. Here we review our recent results demonstrating an additional novel convergent activity between these proteins: both proteins traffic along the actin cytoskeleton (microfilaments). We also discuss results showing a unique property of the P6 protein: a non-mobile strong association with microtubules. Lastly, we discuss the potential mechanism by which the P6 and 126-kDa proteins traffic along microfilaments. We provide new results suggesting that actin filament polymerization-driven movement does not support 126-kDa protein transport, thus leading to a focus on myosins as the driving force for this movement.

Multifunctional Proteins and Movement

Many plant viruses encode one or more proteins capable of multiple functions during the infection process. This ability to multitask has helped to create the compact viral genomes often observed for higher plant viruses. The 126-kDa protein from Tobacco mosaic virus (TMV) and the P6 protein from Cauliflower mosaic virus (CaMV) are examples of multifunctional proteins encoded by two very diverse viruses (the former a single-stranded RNA virus and the latter a double-stranded DNA virus). The 126-kDa protein and the P6 protein have no apparent sequence identity, yet both proteins significantly influence disease intensities caused by their encoding virus.^1–4 Both these proteins are major constituents of the intracellular non-crystalline inclusion bodies containing viral and host factors associated with virus accumulation.^5,6 The influence on disease intensity shared by these two proteins may be mediated by their presence in these intracellular cytoplasmic inclusions since the size of these inclusions positively correlates with the disease intensity displayed by the host.^7–9 Interestingly, both proteins are capable of forming inclusion bodies in the absence of other viral components (P6-bodies or 126-bodies).^9,10 In addition, both proteins are silencing suppressors.^9,11 This convergence in function by two disparate viral proteins is an example of the concept of functional equivalence so clearly presented by Scholthof.¹²

In recent work we revealed further similarities between CaMV P6 and the TMV 126-kDa protein.¹³ In that study we utilized a fusion between P6 and the green fluorescent protein (P6-GFP) to show that, like 126-bodies, P6-bodies are highly motile structures that associate with and traffic along the actin cytoskeleton (microfilaments).^8,13 We found that the majority of P6-body movement was inhibited by disruption of microfilaments with the actin inhibitor, Latrunculin B (LatB), underscoring the importance of actin filaments for body movement. Treatment with LatB also inhibited CaMV infection, suggesting that the movement of P6-containing inclusion bodies along microfilaments is essential for sustained CaMV intercellular spread. Interestingly, movement of TMV 126-kDa protein-containing inclusions and sustained TMV cell-to-cell spread are also inhibited by LatB treatment.^8,14

The potentially similar roles of the P6 and 126-kDa proteins in intercellular virus movement is particularly intriguing given that neither protein is the classically defined movement protein for their respective viruses. However, the 126-kDa protein and/or its 183-kDa protein read-through product (containing the additional polymerase domain) have been shown to be essential for TMV movement irrespective of their function during replication.^15,16 It will be worthwhile to determine if a role for the P6 protein in CaMV movement can be verified through similar genetic studies. If findings from genetic studies support those from the pharmacological and cell biological studies indicating a role of the P6 protein in CaMV intercellular movement, it would be decisive evidence for an additional convergent function between the P6 and 126-kDa proteins. A bottleneck in the host cell-to-cell transport system used by viruses may have forced this functional convergence to occur.

A Unique Role for the P6 Protein: Microtubule Association

Interestingly, we found that unlike the 126-kDa protein, P6 co-localized abundantly with microtubules even though we did not observe movement of inclusions in association with the microtubule network. The idea that this interaction between P6 and microtubules was not involved with P6 body movement was supported by the fact that treatment with the microtubule disrupting drug, oryzalin, did not significantly inhibit P6 body motility. Although the importance of this interaction during virus infection is unclear, we observed stable microtubules in the presence of P6 following oryzalin treatment, suggesting a strong binding between these proteins. One possible function of this association is that the microtubule network may provide a scaffold for body assembly. It should be noted that the CaMV P2 protein, known to be involved with aphid transmission, also forms bodies that localize to microtubules. Recent work demonstrated that P2 bodies require intact microtubules for their efficient formation,¹⁷ lending support to the possibility that a similar mechanism may be utilized for the formation of inclusions containing P6. Once formed on the microtubules, inclusions could then move from the microtubule network to the microfilaments for long-distance transport. Interestingly, the use of microfilaments and not microtubules for long-distance intracellular movement of these plant virus inclusions would be the reverse of what is observed for many animal viruses.^18,19

Dissecting the Mechanism of Actin-Dependent Transport

Given the growing number of plant viral proteins found to associate with and traffic along microfilaments, a determination of the mechanism of actin-dependent transport is an important next step. One obvious possibility for such movement is that myosin motor proteins carry the viral proteins/bodies as cargo as they traverse the microfilament highways. For example, it was recently shown that the Hsp70 homolog from Beet yellows virus, Hsp70 h, requires a specific class of myosins (class VIII) for localization to plasmodesmata.²⁰ However, it is also conceivable that in some cases movement could be driven by actin polymerization. The actin cytoskeleton is a dynamic structure composed of actin monomers that can polymerize rapidly. In some cases this polymerization provides the driving force for the movement of micro-organisms. For example, the bacterial pathogens Listeria and Shigella as well as vaccinia virus have all been shown to utilize the force generated by actin polymerization for at least some aspects of their movement.^21–23 In addition, polymerization-depolymerization (treadmilling) has been suggested as a movement mechanism, although recent results indicate it is a minor activity in plants and fission yeast.^24,25

The driving force behind the actin-associated movement of P6 inclusions remains to be determined, but early evidence suggests that the actin-dependent movement of TMV 126-bodies is not polymerization-driven. To reach this conclusion we utilized the actin inhibitor LatB. At very low concentrations LatB inhibits actin polymerization without disrupting the overall structure of the actin cytoskeleton, thus allowing myosin-driven movement to continue.²⁶ However, at higher concentrations it can rapidly disrupt the structure of existing actin filaments. We tested the effect of decreasing doses of LatB (from 5 µM to 10 nM) on the movement of 126-bodies and only observed inhibition of 126-body movement at concentrations that disrupted microfilaments (data not shown). If actin polymerization were indeed responsible for 126-body movement we anticipated that we also would observe an inhibition of body movement in the presence of lower doses of LatB that did not affect overall actin structure. This preliminary evidence suggests that polymerization-driven movement is not a major force for 126-body movement and that further analysis of the role of myosins in virus transport will be important.

Addendum to: Harries PA, Palanichelvam K, Yu W, Schoelz JE, Nelson RS. The Cauliflower mosaic virus protein P6 forms motile inclusions that traffic along actin microfilaments and stabilize microtubules. Plant Physiol. 2009;149:1005–1016. doi: 10.1104/pp.108.131755.