The role of nucleocapsid of HIV-1 in virus assembly.
The role of the nucleocapsid protein of HIV-1 Gag in virus assembly was investigated using Gag truncation mutants, a nucleocapsid deletion mutant, and point mutations in the nucleocapsid region of Gag, in transfected COS cells, and in stable T-cell lines. Consistent with previous investigations, a truncation containing only the matrix and capsid regions of Gag was unable to assemble efficiently into particles; also, the pelletable material released was lighter than the density of wild-type HIV-1. A deletion mutant lacking p7 nucleocapsid but containing the C-terminal p6 protein was also inefficient in particle release and released lighter particles, while a truncation containing only the first zinc finger of p7 could assemble more efficiently into virions. These results clearly show that p7 is indispensable for virus assembly and release. Some point mutations in the N-terminal basic domain and in the basic linker region between the two zinc fingers, which had been previously shown to have reduced RNA binding in vitro [Schmalzbauer, E., Strack, B., Dannull, J., Guehmann, S., and Moelling, K. (1996). J. Virol. 70: 771-777], were shown to reduce virus assembly dramatically when expressed in full-length viral clones. A fusion protein consisting of matrix and capsid fused to a heterologous viral protein known to have nonspecific RNA binding activity [Ribas, J. C., Fujimura, T., and Wickner, R. B. (1994) J. Biol. Chem. 269: 28420-28428] released pelletable material slightly more efficiently than matrix and capsid alone, and these particles had density higher than matrix and capsid alone. These results demonstrate the essential role of HIV-1 nucleocapsid in the virus assembly process and show that the positively charged N terminus of p7 is critical for this role.
Dawson
L
L
Department of Molecular Microbiology and Immunology, Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland, 21205, USA.
Yu
X F
XF
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D036022:Capsid Proteins; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004797:Enzyme-Linked Immunosorbent Assay; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015488:HIV Antigens / Q000235:genetics / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000032:analysis; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000378:metabolism; D013601:T-Lymphocytes; D014162:Transfection; D014764:Viral Proteins; D019065:Virus Assembly / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; D015488:HIV Antigens; D016655:HIV Core Protein p24; C071884:NCP7 protein, Human immunodeficiency virus 1; D012367:RNA, Viral; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus