The Cauliflower Mosaic Virus Protein P6 Forms Motile Inclusions That Traffic along Actin Microfilaments and Stabilize Microtubules    

Abstract

The gene VI product (P6) of Cauliflower mosaic virus (CaMV) is a multifunctional protein known to be a major component of cytoplasmic inclusion bodies formed during CaMV infection. Although these inclusions are known to contain virions and are thought to be sites of translation from the CaMV 35S polycistronic RNA intermediate, the precise role of these bodies in the CaMV infection cycle remains unclear. Here, we examine the functionality and intracellular location of a fusion between P6 and GFP (P6-GFP). We initially show that the ability of P6-GFP to transactivate translation is comparable to unmodified P6. Consequently, our work has direct application for the large body of literature in which P6 has been expressed ectopically and its functions characterized. We subsequently found that P6-GFP forms highly motile cytoplasmic inclusion bodies and revealed through fluorescence colocalization studies that these P6-GFP bodies associate with the actin/endoplasmic reticulum network as well as microtubules. We demonstrate that while P6-GFP inclusions traffic along microfilaments, those associated with microtubules appear stationary. Additionally, inhibitor studies reveal that the intracellular movement of P6-GFP inclusions is sensitive to the actin inhibitor, latrunculin B, which also inhibits the formation of local lesions by CaMV in Nicotiana edwardsonii leaves. The motility of P6 along microfilaments represents an entirely new property for this protein, and these results imply a role for P6 in intracellular and cell-to-cell movement of CaMV.

Association of the P6 Protein of Cauliflower mosaic virus with Plasmodesmata and Plasmodesmal Proteins

“…3A), whereas P6-GFP formed well-defined P6-GFP I-LBs of varying sizes (Fig. 3B) as previously described (Harries et al, 2009a;Angel et al, 2013). Upon coagroinfiltration of AtSRC2.2-RFP with P6-GFP into leaf panels of N. benthamiana, the AtSRC2.2-RFP protein was frequently colocalized with P6-GFP in both small and large aggregates (Fig.…”

“…Ectopic expression of a P6-GFP fusion protein in N. benthamiana had shown that P6-GFP I-LBs were capable of associating with and moving on microfilaments (Harries et al, 2009a). Furthermore, treatment of plant tissues with latrunculin B blocked the development of CaMV local lesions in N. edwardsonii.…”

“…To visualize P6 I-LBs, we utilized P6-GFP, a construct in which the C terminus of the full-length P6 coding sequence of CaMV strain W260 was fused with GFP ( Fig. 1; Harries et al, 2009a;Angel et al, 2013). Each construct was individually agroinfiltrated into N. benthamiana leaves to determine expression patterns.…”

“…Furthermore, treatment of plant tissues with latrunculin B blocked the development of CaMV local lesions in N. edwardsonii. Because latrunculin B disrupts the structure of microfilaments, Harries et al (2009a) suggested that intracellular movement of P6 IBs on actin microfilaments might be essential for the CaMV infection process. A later study showed that the P6 protein interacts with the host protein CHUP1, a protein necessary for movement of chloroplasts on microfilaments in response to changes in light intensity (Angel et al, 2013).…”

“…Ectopic expression of P6 in Nicotiana benthamiana leaves resulted in the formation of inclusion-like bodies (I-LBs) that were capable of intracellular movement along actin microfilaments. Furthermore, treatment of Nicotiana edwardsonii leaves with latrunculin B abolished the formation of CaMV local lesions, suggesting that intact microfilaments are required for CaMV infection (Harries et al, 2009a). A subsequent paper showed that P6 physically interacts with Chloroplast Unusual Positioning1 (CHUP1), a plant protein localized to the chloroplast outer membrane that contributes to movement of chloroplasts on microfilaments in response to changes in light intensity (Oikawa et al, 2003(Oikawa et al, , 2008Angel et al, 2013).…”