Tracking the assembly pathway of human immunodeficiency virus type 1 Gag deletion mutants by immunogold labeling

The Pr55^gag gene product of human immunodeficiency virus type 1 (HIV-1) is sufficient to direct the formation of retrovirus-like particles (RVLPs). Recent biochemical evidence has indicated the presence of Gag intermediates in the cytoplasm; however, the Gag assembly process into RVLPs remains incompletely defined. The authors present here the subcellular localization of Gag mutant proteins in BSC40 and Jurkat cells by immunoelectron microscopy (IEM). The full Gag/Pol and Gag precursors, a C-terminal deletion mutant lacking a portion of nucleocapsid (NC), and all p6^Gag gave rise to similar levels of RVLPs at the cell surface. A C-terminal deletion of all NC and p6^Gag abrogated particle formation, whereas p24 was found in patches at the cell surface. Deletion of matrix (MA) sequences from Gag resulted in intracellular particles, and myristylation was not required for particle formation in the context of the MA deletion. Matrix expression was enhanced with Gag/Pol or Env coexpression as determined by semiquantitative IEM. p24 protein was targeted at vacuolar and mitochondrial membranes, but not at Golgi cisternae. In addition, aggregations of Gag intermediates and RVLPs in the cytoplasm, rough endoplasmic reticulum, cisternae, and mitochondria were noted. These results provide defined in situ evidence that HIV-1 particle assembly occurs in the cytosol in addition to budding at most intracellular membranes.