Rous sarcoma virus Gag protein-oligonucleotide interaction suggests a critical role for protein dimer formation in assembly.
The structural protein Gag is the only viral product required for retrovirus assembly. Purified Gag proteins or fragments of Gag are able in vitro to spontaneously form particles resembling immature virions, but this process requires nucleic acid, as well as the nucleocapsid domain of Gag. To examine the role of nucleic acid in the assembly in vitro, we used a purified, slightly truncated version of the Rous sarcoma virus Gag protein, Delta MBD Delta PR, and DNA oligonucleotides composed of the simple repeating sequence GT. Apparent binding constants were determined for oligonucleotides of different lengths, and from these values the binding site size of the protein on the DNA was calculated. The ability of the oligonucleotides to promote assembly in vitro was assessed with a quantitative assay based on electron microscopy. We found that excess zinc or magnesium ion inhibited the formation of virus-like particles without interfering with protein-DNA binding, implying that interaction with nucleic acid is necessary but not sufficient for assembly in vitro. The binding site size of the Delta MBD Delta PR protein, purified in the presence of EDTA to remove zinc ions at the two cysteine-histidine motifs, was estimated to be 11 nucleotides (nt). This value decreased to 8 nt when the protein was purified in the presence of low concentrations of zinc ions. The minimum length of DNA oligonucleotide that promoted efficient assembly in vitro was 22 nt for the zinc-free form of the protein and 16 nt for the zinc-bound form. To account for this striking 1:2 ratio between binding site size and oligonucleotide length requirement, we propose a model in which the role of nucleic acid in assembly is to promote formation of a species of Gag dimer, which itself is a critical intermediate in the polymerizaton of Gag to form the protein shell of the immature virion.
Ma
Yu May
YM
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
Vogt
Volker M
VM
D001358:Avian Sarcoma Viruses / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D001665:Binding Sites; D002413:Cations, Divalent; D004279:DNA, Viral / Q000378:metabolism*; D019281:Dimerization; D015683:Gene Products, gag / Q000378:metabolism* / Q000502:physiology; D008274:Magnesium; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology*; D015032:Zinc
D002413:Cations, Divalent; D004279:DNA, Viral; D015683:Gene Products, gag; D009838:Oligodeoxyribonucleotides; D008274:Magnesium; D015032:Zinc