Role of inositol phosphates in retrovirus assembly
Retroviruses like HIV encode a single internal structural protein, Gag, which
forms a curved protein lattice underneath the plasma membrane, eventually leading
to budding of an immature virus particle. The viral protease then causes maturation,
cleaving Gag into its three main constituent domains. One of those liberated domains,
CA, re-assembles into the mature capsid that encloses the viral genome. Both the
immature Gag lattice and the mature CA lattice are built of hexamers. For the immature
lattice, a short segment of polypeptide just C-terminal to CA folds into a critical six-helix
bundle that stabilizes the hexamer.
We have identified a group of small molecules called inositol phosphates (IPs) as
potent stimulators of both immature and mature assembly. Inositol hexaphosphate
(IP6) is the most abundant IP in cells, and also the most potent assembly stimulator.
We generated high resolution structures by cryoEM subtomogram averaging and X-ray
crystallography, which showed that IP6 binds in the immature lattice to two rings of
highly conserved lysine residues formed by Gag hexamerization. Mutation of either
lysine results in a loss of viral infectivity. Molecular dynamic simulations showed that
IP6 acts by stabilizing the critical six-helix bundle. Surprisingly, we found that IP6 also
strongly promotes formation of the mature lattice. X-ray crystallography and mutational
analyses revealed that a ring of six Arginine residues, positioned around the mature CA
hexamer pore, interacts with IP6. A charged residue at this position is highly conserved
among different retroviral genera including the alpha-retrovirus Rous Sarcoma Virus
(RSV), and as predicted, IP6 profoundly stimulates RSV mature core assembly. In
summary, this is the first molecular characterization of retrovirus assembly promoted
by a small molecule.