We have continued our analysis of assembly and maturation of HIV-1 and murine leukemia virus (MLV) particles. We previously reported that inositol phosphates have profound effects on the assembly properties of HIV-1 Gag protein (the structural protein of HIV-1 particles) in a defined assembly system. We have now localized the sites on Gag to which phosphatidylinositol 4,5-bisphosphate binds, using a mass spectrometric footprinting technique. Interestingly, this small molecule binds to sites near the N-terminus of the protein. This observation implies that this region (the matrix domain) has a major influence on the assembly process. We have studied MLV particles in which the first amino acid of the capsid protein (the protein making up the core structure of the mature virus particle) is altered by mutation. We find that these particles are totally noninfectious and are unable to copy their RNA into DNA upon entering a new host cell. In addition, particles containing a mixture of wild-type and mutant capsid molecules are severely impaired with respect to infectivity and DNA synthesis. These results show that the correct amino acid is required at this position in the capsid protein; even the presence of a minority of mutant capsid molecules is highly detrimental to viral functions. In addition, as a novel approach to analysis of viral particle structure, we analyzed the susceptibility of MLV particles to mechanical deformation using an atomic force microscope (AFM). We found that immature particles, composed of uncleaved Gag proteins, are far more resistant to deformation than mature particles, which contain cleavage products of Gag. However, the results of these experiments show that the outermost protein layer in mature particles contains a lattice or network of these molecules. The structure of this newly discovered lattice will require further analysis. [Corresponds to Rein Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]