Assembly properties of human immunodeficiency virus type 1 Gag-leucine zipper chimeras: implications for retrovirus assembly.
Expression of the retroviral Gag protein leads to formation of virus-like particles in mammalian cells. In vitro and in vivo experiments show that nucleic acid is also required for particle assembly. However, several studies have demonstrated that chimeric proteins in which the nucleocapsid domain of Gag is replaced by a leucine zipper motif can also assemble efficiently in mammalian cells. We have now analyzed assembly by chimeric proteins in which nucleocapsid of human immunodeficiency virus type 1 (HIV-1) Gag is replaced by either a dimerizing or a trimerizing zipper. Both proteins assemble well in human 293T cells; the released particles lack detectable RNA. The proteins can coassemble into particles together with full-length, wild-type Gag. We purified these proteins from bacterial lysates. These recombinant "Gag-Zipper" proteins are oligomeric in solution and do not assemble unless cofactors are added; either nucleic acid or inositol phosphates (IPs) can promote particle assembly. When mixed with one equivalent of IPs (which do not support assembly of wild-type Gag), the "dimerizing" Gag-Zipper protein misassembles into very small particles, while the "trimerizing" protein assembles correctly. However, addition of both IPs and nucleic acid leads to correct assembly of all three proteins; the "dimerizing" Gag-Zipper protein also assembles correctly if inositol hexakisphosphate is supplemented with other polyanions. We suggest that correct assembly requires both oligomeric association at the C terminus of Gag and neutralization of positive charges near its N terminus.
Crist
Rachael M
RM
HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, Maryland 21702-1201, USA.
Datta
Siddhartha A K
SA
Stephen
Andrew G
AG
Soheilian
Ferri
F
Mirro
Jane
J
Fisher
Robert J
RJ
Nagashima
Kunio
K
Rein
Alan
A
D002478:Cells, Cultured; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D016350:Leucine Zippers; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D012367:RNA, Viral; D011994:Recombinant Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus