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Cauliflower mosaic virus (CaMV) is a member part of the Caulimoviridae, the virions of which, contain a circular, double-stranded DNA (dsDNA) genome. The 8kb CaMV genome encodes 7 different proteins (P1-P7). The non-enveloped, 50nm in diameter icosahedral (T=7) virions are mainly composed of the capsid protein (P4). The virion-associated protein, P3 acts as a linker joining virus particles to either the aphid transmission factor, P2 or the movement protein, P1. CaMV particles can be transmitted either by mechanical inoculation or by an insect vector (aphid). CaMV mainly infects members of the Brassicaceae (including: radish, canola, mustard, cauliflower, cabbage and broccoli). Two strains of CaMV (W260, D4) also have been found to infect species within the Solanaceae species such as Datura stramonium (devils’s trumpet) and Nicotiana tabacum (tobacco). In agriculture, CaMV is a widely-distributed virus that is primarily found in temperate regions. CaMV-infected plants show yield reductions of 20-50% and can be detected in about >70% of crops. Unfortunately, tools for managing CaMV infections (isolation and aphid management) are of limited efficacy. However, to develop better ways to manage CaMV infection, we must first explore how the virus propagates within its host. A characteristic cytopathic effect of a CaMV infection is the formation of viroplasms or more commonly known as inclusion bodies (IBs). IBs are cytoplasmic structures, formed by CaMV where virus protein synthesis, genome replication, and virion assembly occur within an cell.CaMV P6 protein is the major constituent of IBs. Expression of P6 tagged on its C-terminus with GFP (P6-GFP) produces fluorescent green foci, visible by confocal laser-scanning microscopy, that were later demonstrated to be IBs. P6 contains 4 self-association domains (D1-D4). Here we show that deletion of any of these domains alters the formation of IBs. Surprisingly, deletion of D1, D2, or D3 individually did not abolish the ability of P6 to form foci, but the characteristics of the foci was different from those produced by the wild-type protein. Deletion of D1 produced small intra-nuclear foci, while the D2 deletion produced mainly a single very large focus per cell. The small intra-nuclear foci formed by P6 lacking D1 localized adjacent to nuclear aggregates containing coilin, a marker for Cajal bodies. The single large IB formed by P6 lacking D2 resembles late stage viral infection in chronically-infected leaves. The D3 deletion produced several large foci per cell. Only the D4 deletion showed no focus formation whatsoever and a cytoplasmic distribution near the plasma membrane. These results provide useful knowledge regarding the role of the self-association domains in IB formation.To delve more deeply into formation, we examined characteristics and dynamics of IBs within a cell. Size distribution analysis of P6-GFP expression in cells, showed that small IBs (0.07-1.0µm2) predominated. The number of IBs greater than 5 µm2 dropped off considerably, but still exhibited a long-tailed distribution, with one or two IBs >50 µm2 in area. Such a distribution is characteristic of systems where the components interact. Indicative of this interaction, small (2) IBs were observed dynamically fusing and dissociating into smaller IBs. Furthermore, these dynamic IBs appeared pleomorphic as they changed shape as they went through the process of fusion/dissociation. Nonetheless, fusion/dissociation seem to be regulated considering that some IBs were found in close proximity but did not achieve fusion. IBs larger than 5 µm2 did not appear to undergo much fusion/dissociation. However, they can still fuse when they get larger, these larger IBs are just not as dynamic as smaller IBs. The properties that IBs displayed within a cell (spheroidal shape, shape changes, fusion/dissociation), led us to hypothesize that P6 IBs have liquid-like properties and may suggest that IBs form via. When P6 IBs where exposed to 1,6 hexanediol, small IBs were disrupted. Large IBs (>5 µm2) were not disrupted but decreased in size. This data suggests that these large IBs exhibit a liquid-like outer layer, with a gel-like property that is resistant to the solvent. Such a structure has been proposed for P-bodies.In order for small IBs to fuse and dissociate, they need to move. As previously reported by others, we found that small IBs (2) were associated with actin filaments and were highly mobile, while the more stationary, larger IBs (>5 µm2) appeared to be associated with microtubules. Interestingly, when co-expressing an actin-binding polypeptide with P6, we found that the IB size distribution and velocity was significantly altered. Additionally, the effects were quite drastic on the P6ΔD2 mutant in that instead of producing a single large IB, multiple small IBs were observed. Particles can move in association with actin in two ways, either via myosin motor proteins or by actin filament polymerization. After treatment with myosin inhibiting drugs, we found that as expected, mitochondrial movement was affected but IB movement was not significantly altered. However, when exposed to an actin polymerization inhibiting drugs, we observed that IB movement and size distribution were altered. Our data suggest that fusion of smaller IBs to form larger ones is dependent on the movement along actin filaments.As reported previously, we found that large IBs (>5 µm2) associate with microtubules, are poorly mobile and are primarily located at the periphery of a nucleus. Therefore, we hypothesized that the formation of large IBs is aided by the aggresome pathway. As expected we found co-localization of some intermediate (1-5 µm2) and most large IBs (5>µm2) with proteins expected to be located with the aggresome pathway including HSC70 and GFP-250. Large IBs were also surrounded by mitochondria, another common aggresomal characteristic. The single large IB formed by P6 lacking D2 exhibited the same characteristics as the large IBs formed by wild-type P6 namely, its formation was influenced by factors affecting the actin cytoskeleton and it co-localized with aggresomal markers.Taken together, these data imply that IB body formation is a dynamic and intricate process that is divided by phases. Formation of these large IBs rely on the ability of small IBs to move on actin filaments and fuse. Because of the co-localization of the aggresomal marker and variable movement shown by intermediate IBs, this indicates that at some point the IB gets to a certain size where it is recognized and translocated to microtubules. This process is possibly achieved through the aggresome pathway, which is supported by the aggresome marker co-localization data from the large IBs. Serendipitously, we discovered that temperature affected the development of systemic symptoms by CaMV. Plants of two different species (Brassica rapa, turnips and Orychophragmus violaceus, Chinese violet) showed a delay in systemic symptom formation when grown at 18°C in comparison to 22°C. Interestingly, levels of CaMV DNA and RNA in systemically-infected leaves, as determined by qPCR, RT-qPCR, were higher in plants propagated at 18°C in comparison to 22°C. However, coat protein levels were the same at both temperatures. Physiological analysis of plants at both temperatures showed that chlorophyll measurements were not significantly altered. However, the cooler temperature appeared to disrupt the actin cytoskeleton. The size distribution of IBs under the cooler temperature conditions was altered. Taken together, our current hypothesis is that the cooler temperatures alter the actin cytoskeleton, which subsequently alters IB movement, and the fusion-dissociation equilibrium, altering the size distribution. Because IB structure is altered under the cooler temperatures, events occurring in IBs such as virion, formation are reduced and less virions are formed. The higher concentrations of viral DNA and RNA could be due to the reduced RNAi pathway caused by a reduction in temperature as observed in other systems.Additionally, P6 is known to be a multifunctional protein with the capability to bind to an array of host proteins. Furthermore, a study done by Dr. Lyubov Ryabiva and Nina Lukhovitskaya showed that P6 can act as a suppressor of nonsense-mediated-decay (NMD) by interaction with a scaffold protein of the decapping complex VARICOSE (VCS). Interestingly, we have found that P6 IBs co-localize with the ATP-dependent RNA helicase upstream frameshift1 (UPF1) protein. UPF1 is a mobile protein that is involved in NMD and are found in stress granules as well as P-bodies. Our data showed that IBs, specifically the small IBs (2), move alongside foci formed by UPF1. Because P6 interacts with VCS to suppress NMD, we hypothesize that P6 might recruit UPF1 for aid in movement or IB formation. Furthermore, possible association with UPF1 may permit P6 to shuttle between P-bodies and stress granules.To summarize, P6 IBs are highly dynamic structures that display a long-tailed distribution according to their size. The mechanism by which IBs form is dependent on movement by actin filaments. Once IBs attain a certain size, they are recognized by the aggresome pathway and translocated to microtubules. Finally, large IBs seem to be formed via the aggresome pathway as they reach their destination, the periphery of the nucleus.