THE ROLE OF NUCLEOCAPSID OF HIV-1 IN VIRUS ASSEMBLY

The role of the nucleocapsid protein of HIV-I Gag in virus assembly wa s investigated using Gag truncation mutants, a nucleocapsid deletion m utant, and point mutations in the nucleocapsid region of Gag, in trans fected COS cells, and in stable T-cell lines. Consistent with previous investigations, a truncation containing only the matrix and capsid re gions of Gag was unable to assemble efficiently into particles; also, the pelletable material released was lighter than the density of wild- type HIV-I. A deletion mutant lacking p7 nucleocapsid but containing t he C-terminal p6 protein was also inefficient in particle release and released lighter particles, while a truncation containing only the fir st zinc finger of p7 could assemble more efficiently into virions. The se results clearly show that p7 is indispensable for virus assembly an d release. Some point mutations in the N-terminal basic domain and in the basic linker region between the two zinc fingers, which had been p reviously shown to have reduced RNA binding in vitro [Schmalzbauer, E. , Strack, B., Dannull, J., Guehmann, S., and Moelling, K. (1996). J. V irol. 70: 771-777], were shown to reduce virus assembly dramatically w hen expressed in full-length viral clones. A fusion protein consisting of matrix and capsid fused to a heterologous viral protein known to h ave nonspecific RNA binding activity [Ribas, J. C., Fujimura, T., and Wickner, R. B. (1994) J. Biol. Chem. 269: 28420-28428] released pellet able material slightly more efficiently than matrix and capsid alone, and these particles had density higher than matrix and capsid alone. T hese results demonstrate the essential role of HIV-I nucleocapsid in t he virus assembly process and show that the positively charged N termi nus of p7 is critical for this role. (C) 1998 Academic Press.