The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent.
The highly conserved zinc fingers in retroviral nucleocapsid (NC) proteins have the general structure Cys-(X)2-Cys-(X)4-His-(X)4-Cys. Human immunodeficiency virus type 1 (HIV-1) contains two Zn2+ fingers, and mutants were constructed in which the native sequence of each Zn2+ finger was maintained but their positions in the NC protein were changed. Mutants had either two first-finger sequences (pNC1/1), two second-finger sequences (pNC2/2), or reversed first- and second-finger sequences (pNC2/1). Cells transfected with mutant or wild-type clones produced similar levels of Tat, Gag, Pol, and Env proteins, formed syncytia, and shed viruslike particles that were indistinguishable by electron microscopy. However, the pNC2/1 and pNC2/2 mutants were inefficient in packaging genomic RNA (less than 15% of wild-type levels), whereas the pNC1/1 mutant packaged approximately 70% of wild-type levels of RNA. No infectious virus could be detected with either the pNC2/1 or pNC2/2 mutants, whereas the pNC1/1 mutant appeared to sustain a low level of replication and reverted to a competent wild-type-like viral species after a 2- to 4-week lag period. The data strongly suggest that the two Zn2+ fingers of HIV-1 are not functionally equivalent and that the first Zn2+ finger in the Gag precursor plays a more prominent role in RNA selection and packaging. The data also indicate that both Zn2+ fingers in the mature NC protein play as yet unknown roles in viral assembly or the early stages of the viral infection process.
Gorelick
R J
RJ
AIDS Vaccine Development Program, Program Resources Inc., DynCorp, Frederick, Maryland.
Chabot
D J
DJ
Rein
A
A
Henderson
L E
LE
Arthur
L O
LO
D000595:Amino Acid Sequence; D001483:Base Sequence; D036022:Capsid Proteins; D015683:Gene Products, gag / Q000737:chemistry*; D015497:HIV-1 / Q000737:chemistry* / Q000254:growth & development; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D014162:Transfection; D014764:Viral Proteins; D014779:Virus Replication; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus