Identification of Inhibitory Mutants of Cauliflower mosaic virus Movement Protein Function after Expression in Insect Cells

Abstract

Cauliflower mosaic virus (CaMV) encodes a movement protein (MP) which forms tubules in vivo and mediates the translocation of virus particles through plasmodesmata. The relationship between CaMV MP structure and function, in isolation from the complete virus infection, was studied by using MP expression in insect cells. The study allowed the MP domains necessary for tubule formation to be identified and potential MP-MP interactions to be investigated by using double infections with recombinant baculoviruses. Two MP domains which interfered with the ability of the wild-type MP to form tubules were identified. These mutant domains appeared to act as competitive, rather than dominant negative, inhibitors.

Identification of distinct steps during tubule formation by the movement protein of Cowpea mosaic virus

“…Mutants that could not be complemented by MP WT might be specifically disturbed in the MP-MP interaction domain but we cannot rule out that introduction of these mutations disturbed the overall folding of the MP, although the mutations were very subtle. Although interaction between MP molecules has previously been implicated in tubule formation of Alfalfa mosaic virus (Sanchez-Navarro & Bol, 2001) and CaMV (Huang et al, 2001;Thomas & Maule, 1999) this is the first report to indicate that MP-MP interaction is involved in targeting to the plasma membrane.…”

“…The data obtained with MP D1-4 fGFP suggest that the N terminus of MP is essential for targeting MP to the plasma membrane. The N terminus of CaMV MP, however, is not important for targeting to peripheral punctate structures, although it is important for tubule formation (Huang et al, 2000;Thomas & Maule, 1999), indicating that the N termini of CPMV and CaMV play different roles in targeting of MP. Possibly a domain similar in function to the N terminus of CPMV MP is present in another part of CaMV MP.…”

Cauliflower mosaic virus: still in the news

“…P1 (40 kDa) is a cell‐to‐cell movement protein which forms tubules through the plasmodesmata, allowing CaMV particles to move from one cell to another (Perbal et al ., 1993). A central domain of P1 is needed for targeting the protein to the cell periphery (Huang et al ., 2001a), whereas most of the protein, except for the C‐terminal region is required for tubule formation (Thomas and Maule, 1999). The N‐ and C‐termini, which are the most variable sequences among P1 proteins from different Caulimoviruses , are exposed at the outer and inner (lumen) faces of the tubules, respectively (Thomas and Maule, 1995a, 1999).…”

“…A central domain of P1 is needed for targeting the protein to the cell periphery (Huang et al ., 2001a), whereas most of the protein, except for the C‐terminal region is required for tubule formation (Thomas and Maule, 1999). The N‐ and C‐termini, which are the most variable sequences among P1 proteins from different Caulimoviruses , are exposed at the outer and inner (lumen) faces of the tubules, respectively (Thomas and Maule, 1995a, 1999). P1 also contains an RNA‐binding domain (Citovsky et al ., 1991; Thomas and Maule, 1995b) that partially overlaps the sequence involved in its localization to the cell periphery.…”

Rapid functional analysis of protein–protein interactions by fluorescent C‐terminal labeling and single‐molecule imaging

“…The molecular weight of puromycin is so smaller than GFP that it significantly reduces the possibility of steric hindrance of protein–protein interactions. (4) Many commonly used variants of GFP are insoluble when expressed, thus proteins of interest are often rendered insoluble upon fusion to GFP [22,23]. The small size of puromycin is unlikely to affect the solubility properties of target proteins.…”