Analysis of Mason-Pfizer monkey virus Gag domains required for capsid assembly in bacteria: role of the N-terminal proline residue of CA in directing particle shape.

Mason-Pfizer monkey virus (M-PMV) preassembles immature capsids in the cytoplasm prior to transporting them to the plasma membrane. Expression of the M-PMV Gag precursor in bacteria results in the assembly of capsids indistinguishable from those assembled in mammalian cells. We have used this system to investigate the structural requirements for the assembly of Gag precursors into procapsids. A series of C- and N-terminal deletion mutants progressively lacking each of the mature Gag domains (matrix protein [MA]-pp24/16-p12-capsid protein [CA]-nucleocapsid protein [NC]-p4) were constructed and expressed in bacteria. The results demonstrate that both the CA and the NC domains are necessary for the assembly of macromolecular arrays (sheets) but that amino acid residues at the N terminus of CA define the assembly of spherical capsids. The role of these N-terminal domains is not based on a specific amino acid sequence, since both MA-CA-NC and p12-CA-NC polyproteins efficiently assemble into capsids. Residues N terminal of CA appear to prevent a conformational change in which the N-terminal proline plays a key role, since the expression of a CA-NC protein lacking this proline results in the assembly of spherical capsids in place of the sheets assembled by the CA-NC protein.

The three-dimensional solution structure of the matrix protein from the type D retrovirus, the Mason-Pfizer monkey virus, and implications for the morphology of retroviral assembly.

The Mason-Pfizer monkey virus (M-PMV) is the prototype of the type D retroviruses. In type B and D retroviruses, the Gag protein pre-assembles before association with the membrane, whereas in type C retroviruses (lentiviruses, BLV/HTLV group) Gag is targeted efficiently to the plasma membrane, where the particle formation occurs. The N-terminal domain of Gag, the matrix protein (MA), plays a critical role in determining this morphogenic difference. We have determined the three-dimensional solution structure of the M-PMV MA by heteronuclear nuclear magnetic resonance. The protein contains four alpha-helices that are structurally similar to the known type C MA structures. This similarity implies possible common assembly units and membrane-binding mechanisms for type C and B/D retroviruses. In addition to this, the interpretation of mutagenesis data has enabled us to identify, for the first time, the structural basis of a putative intracellular targeting motif.

Efficient in vivo and in vitro assembly of retroviral capsids from Gag precursor proteins expressed in bacteria.

The capsid precursor protein (Gag) of Mason-Pfizer monkey virus, the prototype type D retrovirus, has been expressed to high levels in bacteria under the control of the phage T7 promoter. Electron microscopic studies of induced cells revealed the assembly of capsid-like structures within inclusion bodies that formed at the poles of the cells 6 h after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). The inclusion bodies and enclosed capsid-like structures were solubilized completely in 8 M urea, but following renaturation, we observed assembly in vitro of capsid-like structures that demonstrated apparent icosahedral symmetry. These results demonstrate for the first time that retroviral capsid precursors have the propensity to self-assemble in vitro and point to new approaches for the analysis of retroviral assembly and structure.