Inositol phosphates promote HIV-1 assembly and maturation to facilitate viral spread in human CD4+ T cells

Abstract

Gag polymerization with viral RNA at the plasma membrane initiates HIV-1 assembly. Assembly processes are inefficient in vitro but are stimulated by inositol (1,3,4,5,6) pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) metabolites. Previous studies have shown that depletion of these inositol phosphate species from HEK293T cells reduced HIV-1 particle production but did not alter the infectivity of the resulting progeny virions. Moreover, HIV-1 substitutions bearing Gag/CA mutations ablating IP6 binding are noninfectious with destabilized viral cores. In this study, we analyzed the effects of cellular depletion of IP5 and IP6 on HIV-1 replication in T cells in which we disrupted the genes encoding the kinases required for IP6 generation, IP5 2-kinase (IPPK) and Inositol Polyphosphate Multikinase (IPMK). Knockout (KO) of IPPK from CEM and MT-4 cells depleted cellular IP6 in both T cell lines, and IPMK disruption reduced the levels of both IP5 and IP6. In the KO lines, HIV-1 spread was delayed relative to parental wild-type (WT) cells and was rescued by complementation. Virus release was decreased in all IPPK or IPMK KO lines relative to WT cells. Infected IPMK KO cells exhibited elevated levels of intracellular Gag protein, indicative of impaired particle assembly. IPMK KO compromised virus production to a greater extent than IPPK KO suggesting that IP5 promotes HIV-1 particle assembly in IPPK KO cells. HIV-1 particles released from infected IPPK or IPMK KO cells were less infectious than those from WT cells. These viruses exhibited partially cleaved Gag proteins, decreased virion-associated p24, and higher frequencies of aberrant particles, indicative of a maturation defect. Our data demonstrate that IP6 enhances the quantity and quality of virions produced from T cells, thereby preventing defects in HIV-1 replication.

The HIV-1 capsid and reverse transcription

“…During HIV-1 particle formation, the cellular metabolite inositol hexakisphosphate (IP6) binds to the assembling (i.e., immature) Gag lattice, stabilizing it and leading to IP6 incorporation within virus particles [ 4 , 5 ]. In cells depleted of IP6 and its precursor inositol pentakisphosphate (IP5), HIV-1 assembly is impaired [ 6 , 7 ]. During maturation, IP6 is thought to dissociate from the disassembling immature lattice and to subsequently promote capsid assembly by coordinating a conserved arginine side chain in the center of the CA hexamer as the mature capsid is formed [ 4 ] (Fig.…”

“…The results support a role of virion associated IP6 in capsid assembly and a possible function in target cells. Although it appears that the major role of IP6 in the HIV-1 life cycle is at the stage of virion assembly [ 6 , 7 ], an effect in target cells also seems plausible given the strong effects of IP6 on the ERT reaction, as described below. One interesting possibility is that stabilization of the capsid by IP5 and IP6 modulates the effects of capsid-targeting antiviral compounds, such as PF74 and GS-CA1 [ 30 , 53 ].…”

“…Infection of the knockout cells was not substantially reduced, indicating that IPMK (which converts IP3 into IP5, the precursor to IP6) is not essential for HIV-1 reverse transcription in this cell line. Sowd and Aiken further showed that knockout of the IPMK gene resulted in a negligible effect on HIV-1 infection of T cells [ 7 ]. Because IP6 is incorporated into HIV-1 particles during assembly, it remains possible that virion-associated IP6 is sufficient for capsid stabilization during infection.…”

A Structural Perspective of the Role of IP6 in Immature and Mature Retroviral Assembly

“…As already outlined in the above sections, mutations of residues involved in IP6 coordination have a significant impact on viral replication and spread. An alternative approach taken by multiple research groups has been to modify IP6 (and IP5) levels in cells by knocking out the enzymes that convert IPs to IP6, or to transiently express enzymes that ablate IP6 [ 32 , 37 , 44 , 52 , 53 ], or to carry out recovery assays in which kinases required for IP6 synthesis are added back to KO cell lines [ 52 ].…”

Derivation and characterization of an HIV-1 mutant that rescues IP6 binding deficiency

“…The importance of each of the IP 6 -coordinating residues has been established, as mutagenesis of any such residue to an alanine (HIV-1 R18A, HIV-1 K290A , or HIV-1 K359A ) significantly impairs infectivity in either single cycle or spreading infection assays [ 9 , 13 ]. Additionally, yield of infectious virions is also substantially reduced in cells lacking key enzymes in the IP 6 biosynthetic pathway (IPPK or IPMK) or in cells overexpressing MINPP1, a phosphatase that dephosphorylates IP 6 [ 9 , 13 – 15 ]. The IP 6 -coordinating amino acids are conserved among diverse lentiviruses, suggesting a general requirement for IP 6 [ 16 ].…”