Rescue of internal scaffold-deleted Mason-Pfizer monkey virus particle production by plasma membrane targeting.

The Mason-Pfizer monkey virus (M-PMV) Gag protein follows a morphogenesis pathway in which immature capsids are preassembled within the cytoplasm before interaction with and budding through the plasma membrane. Intracytoplasmic assembly is facilitated by sequences within the p12 domain of Gag that we have termed the Internal Scaffold Domain (ISD). If M-PMV utilizes an ISD then what provides the equivalent function for most other retroviruses that assemble at the plasma membrane? To investigate the possibility that the membrane itself fulfills this role, we have combined functional deletion of the ISD with a mutation that disrupts intracellular targeting or with a plasma membrane targeting signal. By either modification, targeting of ISD-deleted Gag to the plasma membrane restores particle production. These results provide support for a model in which the plasma membrane and the D-type ISD provide an interchangeable scaffold-like function in retrovirus assembly.

The Mason-Pfizer monkey virus internal scaffold domain enables in vitro assembly of human immunodeficiency virus type 1 Gag.

The Mason-Pfizer monkey virus (M-PMV) Gag protein possesses the ability to assemble into an immature capsid when synthesized in a reticulocyte lysate translation system. In contrast, the human immunodeficiency virus (HIV) Gag protein is incapable of assembly in parallel assays. To enable the assembly of HIV Gag, we have combined or inserted regions of M-PMV Gag into HIV Gag. By both biochemical and morphological criteria, several of these chimeric Gag molecules are capable of assembly into immature capsid-like structures in this in vitro system. Chimeric species containing large regions of M-PMV Gag fused to HIV Gag sequences failed to assemble, while species consisting of only the M-PMV p12 region, and its internal scaffold domain (ISD), fused to HIV Gag were capable of assembly, albeit at reduced kinetics compared to M-PMV Gag. The ability of the ISD to induce assembly of HIV Gag, which normally assembles at the plasma membrane, suggests a common requirement for a concentrating factor in retrovirus assembly. Despite the dramatic effect of the ISD on chimera assembly, the function of HIV Gag domains in that process was found to remain essential, since an assembly-defective mutant of HIV CA, M185A, abolished assembly when introduced into the chimera. This continued requirement for HIV Gag domain function in the assembly of chimeric molecules will allow this in vitro system to be used for the analysis of potential inhibitors of HIV immature particle assembly.