Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis.

The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P(2) hydrolysis or increased PI(4,5)P(2) generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P(2) dictates the remodeling of actin necessary for completion of phagocytosis.

Membrane association facilitates the correct processing of pp220 during production of the major matrix proteins of African swine fever virus.

The African swine fever (ASF) virus polyprotein pp220 is processed at Gly-Gly-X sites by a virally encoded SUMO-like protease to produce matrix proteins p150, p37, p34, and p14. Four Gly-Gly-X sites are used to produce the matrix proteins, but the polyprotein contains an additional 15 sites potentially recognized by the protease. This study shows that cleavage occurs at many, if not all, Gly-Gly-X sites, and at steady state, p150 and p34 are minor products of processing. Significantly, only the final structural proteins, p150 and p34, were found in mature virions, suggesting that there is a mechanism for excluding incorrectly processed forms. ASF virus is assembled on the cytoplasmic face of the endoplasmic reticulum, and the distribution of pp220 products between membranes and cytosol was studied. Incorrectly processed forms of p34 were recovered from both the cytosol and membrane fractions. Interestingly, p34 was only detected in the membrane fraction, and of the many processed forms bound to membranes, only p34 was protected from trypsin, suggesting envelopment. The majority of the incorrectly processed forms of p150 were recovered from the cytosol. Again, the correct product of processing, p150, was selectively recruited to membranes. Sucrose density centrifugation showed that membrane-associated forms of p34 and p150 assembled into large structures suggestive of a viral matrix, while cytosolic and/or incorrectly processed forms of pp220 did not. Taken together, these results suggest that association with cellular membranes is important for regulating the correct processing of pp220 and the packaging of matrix proteins into virions.