Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner

Abstract

There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The 52 amino acid HIV-1 p6 Gag protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two late (L-) domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet. We found that the mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metalloendopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE-insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition. Our cumulative data support a model in which removal of p6 during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1.

The N-Terminus of the HIV-1 p6 Gag Protein Regulates Susceptibility to Degradation by IDE

“…Vice versa, substitution of the proline at position one of the IDE-insensitive p6 variant derived from HIV-1 SG3 by a leucine residue restores the susceptibility towards IDE. In contrast to our previously generated IDE-insensitive p6 mutant, this substitution represents a naturally occurring polymorphism of HIV-1 p6 [16,37].…”

“…Replication assays were performed as reported previously [16]. In short, PBMCs were isolated from buffy coats of several blood donors and stimulated with phytohaemagglutinin (PHA-P) and Interleukin 2 (IL-2) for three days.…”

“…However, the role of free p6, either as part of a mature virion infecting a new host cell or stemming from decayed virions in the extracellular space has not been investigated yet. Recently, we were able to show that the mature HIV-1 p6 protein is a bona fide substrate for a cellular protease, the insulin-degrading enzyme (IDE) [16]. IDE is a 120 kDa highly conserved zinc-metalloprotease with homologs in plants, fungi and even bacteria [17,18,19,20,21].…”

“…Furthermore, IDE is ubiquitously expressed in human tissues and cells, including CD4 + T cells and macrophages [18,24,25,26]. Stabilization of p6 under certain conditions impaired the replication capacity of HIV-1 in an Env-dependent manner, indicating a potential function of p6 that exceeds its hitherto described L-domain function [16].…”

“…We were able to show that this phenomenon appears to be specific for HIV-1, since none of the tested p6 sequences from HIV-2 and SIV, nor the p6 homolog from EIAV, p9, were degraded by IDE [16]. However, the phylogenetic background of the degradation of HIV-1 p6 by IDE has not been investigated thoroughly.…”