HIV needs the small molecule IP6 to build its capsid

Abstract

The retrovirus Human Immunodeficiency Virus Type-1 (HIV-1) infects cells of the immune system causing their depletion and resulting in the Acquired Immunodeficiency Syndrome (AIDS). The viral capsid protein (CA) is essential for replication. Upon budding of a new HIV particle, CA is part of the Gag polyprotein which assembles into an immature virus lattice. During maturation, the viral protease cleaves Gag into single components and CA assembles into a mature capsid. The capsid protects the viral RNA genome which is reverse-transcribed into DNA, trafficked to the nucleus, and integrated into the host genome. Both immature and mature HIV capsid lattices need to maintain a delicate balance of stability to allow efficient assembly without compromising maturation or disassembly. The work described in this thesis shows that coordination of the small molecule inositol hexakisphosphate (IP6), which can bind to both the immature (via residues K158 and K227) and mature (R18 and K25) CA lattice is crucial. The data herein demonstrate the importance of these charged rings for viral replication and infection. In the mature capsid, R18 and K25 are thought to have a dual role. Both residues are important IP6 binders, and their mutation interferes with mature capsid formation. In addition, they are proposed to recruit dNTPs to facilitate encapsidated DNA synthesis. Here I show that infection and conical capsid formation of K25A can be rescued by compensating mutants without restoring the charged K25 ring. This suggests that K25 is necessary for assembly but not for nucleotide import. In contrast, maintaining the positively charged R18 appears to be indispensable. In the immature lattice, lack of either of the positively charged rings is strongly destabilizing and results in aberrant assembly. However, simultaneous mutation of both lysine residues (K158 and K227) allows immature lattice formation, indicating that HIV immature assembly can become independent of IP6. Moreover, the immature lattice is more tolerant to changes in inositol phosphate levels compared to the mature lattice. Nevertheless, HIV-1 with a disabled IP6-enrichment mechanism is non-infectious and my data suggest that this is because IP6 is important in the formation of mature cores that support productive infection