High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility

Polyomavirus middle T-antigen is a transmembrane protein that binds signaling proteins in discrete subcellular membrane sites

Murine polyomavirus middle T-antigen (MT) induces tumors by mimicking an activated growth factor receptor. An essential component of this action is a 22-amino-acid hydrophobic region close to the C terminus which locates MT to cell membranes. Here, we demonstrate that this sequence is a transmembrane domain (TMD) by showing that a hemagglutinin (HA) tag added to the MT C terminus is exposed on the outside of the cells, with the N terminus inside. To determine whether this MT TMD is inserted into the endoplasmic reticulum (ER) membrane, we added the ER retention signal KDEL to the MT C terminus (MTKDEL). This mutant protein locates only in the ER, demonstrating that MT does insert into membranes solely at this location. In addition, this ER-located MT failed to transform. Examination of the binding proteins associated with the MTKDEL protein demonstrated that it associates with PP2A and c-Src but fails to interact with ShcA, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLC-γ1), despite being tyrosine phosphorylated. Additional mutant and antibody studies show that MT binding to PP2A is probably required for MT to efficiently exit the ER and migrate to the plasma membrane though the TMD also plays a role in this relocation. Overall, these data, together with previous publications, illustrate that MT associates with signaling proteins at different sites in its maturation pathway. MT binds to PP2A in the cytoplasm, to c-Src at the endoplasmic reticulum, and to ShcA, PI3K, and PLC-γ1 at subsequent locations en route to the plasma membrane

Polyomavirus Middle T-Antigen Is a Transmembrane Protein That Binds Signaling Proteins in Discrete Subcellular Membrane Sites▿

Murine polyomavirus middle T-antigen (MT) induces tumors by mimicking an activated growth factor receptor. An essential component of this action is a 22-amino-acid hydrophobic region close to the C terminus which locates MT to cell membranes. Here, we demonstrate that this sequence is a transmembrane domain (TMD) by showing that a hemagglutinin (HA) tag added to the MT C terminus is exposed on the outside of the cells, with the N terminus inside. To determine whether this MT TMD is inserted into the endoplasmic reticulum (ER) membrane, we added the ER retention signal KDEL to the MT C terminus (MTKDEL). This mutant protein locates only in the ER, demonstrating that MT does insert into membranes solely at this location. In addition, this ER-located MT failed to transform. Examination of the binding proteins associated with the MTKDEL protein demonstrated that it associates with PP2A and c-Src but fails to interact with ShcA, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLC-γ1), despite being tyrosine phosphorylated. Additional mutant and antibody studies show that MT binding to PP2A is probably required for MT to efficiently exit the ER and migrate to the plasma membrane though the TMD also plays a role in this relocation. Overall, these data, together with previous publications, illustrate that MT associates with signaling proteins at different sites in its maturation pathway. MT binds to PP2A in the cytoplasm, to c-Src at the endoplasmic reticulum, and to ShcA, PI3K, and PLC-γ1 at subsequent locations en route to the plasma membrane