A short, 14-amino-acid segment called SP1, located in the Gag structural protein1, has a critical role during the formation of the HIV-1 virus particle. During virus assembly, the SP1 peptide and seven preceding residues fold into a six-helix bundle, which holds together the Gag hexamer and facilitates the formation of a curved immature hexagonal lattice underneath the viral membrane2,3. Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the six-helix bundle are crucial rate-limiting steps of both Gag assembly and disassembly, and the six-helix bundle is an established target of HIV-1 inhibitors4,5. Here, using a combination of structural and functional analyses, we show that inositol hexakisphosphate (InsP6, also known as IP6) facilitates the formation of the six-helix bundle and assembly of the immature HIV-1 Gag lattice. IP6 makes ionic contacts with two rings of lysine residues at the centre of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP6 interaction promotes the assembly of the mature capsid lattice. These studies identify IP6 as a naturally occurring small molecule that promotes both assembly and maturation of HIV-1.

Previous studies showed that lithium, beginning at therapeutic plasma concentrations in the treatment of manic depression, increased the accumulation of second-messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in cerebral cortex slices of guinea pig and rhesus monkey [Lee, Dixon, Reichman, Moummi, Los and Hokin (1992) Biochem. J. 282, 377-385; Dixon, Lee, Los and Hokin (1992) J. Neurochem. 59, 2332-2335; Dixon, Los and Hokin (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 8358-8362]. These studies have now been extended to a peripheral tissue, mouse pancreatic minilobules. In the presence of carbachol, concentrations of lithium from 1 to 20 mM sharply and progressively increased the accumulation of Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate, followed by a decrease. Assay of these inositol polyphosphates by either the prelabelling technique or mass assay gave similar results. Atropine quenching of cholinergically stimulated pancreatic minilobules led to a rapid disappearance of Ins(1,4,5)P3. This disappearance was impeded by lithium. This suggested that the lithium-induced elevation in Ins(1,4,5)P3 was due to inhibition of the 5-phosphatase and, on the basis of the markedly elevated concentrations of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and inositol 1,4-bisphosphate in the presence of lithium, probably by feedback inhibition by these latter two compounds. An additional mechanism, i.e. a stimulatory effect of lithium on phospholipase C, cannot, however, be ruled out. The other reaction product of phospholipase C, inositol cyclic 1:2,4,5-trisphosphate, also increased in the presence of lithium. This may also be due to inhibition of the 5-phosphatase, which is the exclusive mechanism for removal of this compound. The effects of lithium on the accumulation of other inositol phosphates paralleled that of Ins(1,4,5)P3, with the exception of inositol 3,4-bisphosphate, which decreased. This was presumably due to the inhibition of Ins(1,3,4)P3 1-phosphatase by lithium. Unlike mouse cerebral cortex slices [Lee, Dixon, Reichman, Moummi, Los and Hokin (1992) Biochem. J. 282, 377-385], inositol supplementation was not required to demonstrate lithium-stimulated Ins(1,4,5)P3 accumulation in mouse pancreatic minilobules. This indicates that inositol depletion sufficient to impair lithium-stimulated Ins(1,4,5)P3 accumulation does not occur in mouse pancreatic minilobules, even though an elevation of cytidine diphosphodiacylglycerol occurred, indicating some inositol depletion due to lithium. Elevation of Ins(1,4,5)P3 by lithium may be a general phenomenon in the central nervous system and peripheral tissues under non-rate-limiting concentrations of inositol.

Previous studies demonstrated a remarkable increase of urinary InsP6 by topical administration. However, the methodology used for InsP6 analysis was not specific. The aim of this paper is to measure urinary inositol phosphates InsPs using more advanced methodologies and to compare the results with those obtained by the non-specific method.

Tumour evolution and efficacy of treatments are controlled by the microenvironment, the composition of which is primarily dependent on the angiogenic reaction to hypoxic stress. Tumour angiogenesis normalization is a challenge for adjuvant therapy strategies to chemo-, radio- and immunotherapeutics. Myo-inositol trispyrophosphate (ITPP) appears to provide the means to alleviate hypoxia in the tumour site by a double molecular mechanism. First, it modifies the properties of red blood cells (RBC) to release oxygen (O2 ) in the hypoxic sites more easily, leading to a rapid and stable increase in the partial pressure of oxygen (pO2 ). And second, it activates the endothelial phosphatase and tensin homologue deleted on Chromosome 10 (PTEN). The hypothesis that stable normalization of the vascular system is due to the PTEN, a tumour suppressor and phosphatase which controls the proper angiogenic reaction was ascertained. Here, by direct biochemical measurements of PTEN competitive activity in relation to PIP2 production, we show that the kinetics are complex in terms of the activation/inhibition effects of ITPP with an inverted consequence towards the kinase PI3K. The use of the surface plasmon resonance (SPR) technique allowed us to demonstrate that PTEN binds inositol derivatives differently but weakly. This method permitted us to reveal that PTEN is highly sensitive to the local concentration conditions, especially that ITPP increases the PTEN activity towards PIP3, and importantly, that PTEN affinity for ITPP is considerably increased by the presence of PIP3, as occurs in vivo. Our approach demonstrates the validity of using ITPP to activate PTEN for stable vessel normalization strategies.

Class I histone deacetylases (HDAC1, HDAC2, and HDAC3) are recruited by cognate corepressor proteins into specific transcriptional repression complexes that target HDAC activity to chromatin resulting in chromatin condensation and transcriptional silencing. We previously reported the structure of HDAC3 in complex with the SMRT corepressor. This structure revealed the presence of inositol-tetraphosphate [Ins(1,4,5,6)P4] at the interface of the two proteins. It was previously unclear whether the role of Ins(1,4,5,6)P4 is to act as a structural cofactor or a regulator of HDAC3 activity. Here we report the structure of HDAC1 in complex with MTA1 from the NuRD complex. The ELM2-SANT domains from MTA1 wrap completely around HDAC1 occupying both sides of the active site such that the adjacent BAH domain is ideally positioned to recruit nucleosomes to the active site of the enzyme. Functional assays of both the HDAC1 and HDAC3 complexes reveal that Ins(1,4,5,6)P4 is a bona fide conserved regulator of class I HDAC complexes.

Inositol phosphates (IPs) comprise a network of phosphorylated molecules that play multiple signaling roles in eukaryotes. IPs synthesis is believed to originate with IP3 generated from PIP2 by phospholipase C (PLC). Here, we report that in mammalian cells PLC-generated IPs are rapidly recycled to inositol, and uncover the enzymology behind an alternative "soluble" route to synthesis of IPs. Inositol tetrakisphosphate 1-kinase 1 (ITPK1)-found in Asgard archaea, social amoeba, plants, and animals-phosphorylates I(3)P1 originating from glucose-6-phosphate, and I(1)P1 generated from sphingolipids, to enable synthesis of IP6 We also found using PAGE mass assay that metabolic blockage by phosphate starvation surprisingly increased IP6 levels in a ITPK1-dependent manner, establishing a route to IP6 controlled by cellular metabolic status, that is not detectable by traditional [3H]-inositol labeling. The presence of ITPK1 in archaeal clades thought to define eukaryogenesis indicates that IPs had functional roles before the appearance of the eukaryote.

Myo-inositol hexakisphosphate (IP6) is a natural product known to inhibit vascular calcification (VC), but with limited potency and low plasma exposure following bolus administration. Here we report the design of a series of inositol phosphate analogs as crystallization inhibitors, among which 4,6-di-O-(methoxy-diethyleneglycol)-myo-inositol-1,2,3,5-tetrakis(phosphate), (OEG2)2-IP4, displays increased in vitro activity, as well as more favorable pharmacokinetic and safety profiles than IP6 after subcutaneous injection. (OEG2)2-IP4 potently stabilizes calciprotein particle (CPP) growth, consistently demonstrates low micromolar activity in different in vitro models of VC (i.e., human serum, primary cell cultures, and tissue explants), and largely abolishes the development of VC in rodent models, while not causing toxicity related to serum calcium chelation. The data suggest a mechanism of action independent of the etiology of VC, whereby (OEG2)2-IP4 disrupts the nucleation and growth of pathological calcification.

Histone deacetylases (HDACs) 1, 2 and 3 form the catalytic subunit of several large transcriptional repression complexes. Unexpectedly, the enzymatic activity of HDACs in these complexes has been shown to be regulated by inositol phosphates, which bind in a pocket sandwiched between the HDAC and co-repressor proteins. However, the actual mechanism of activation remains poorly understood. Here we have elucidated the stereochemical requirements for binding and activation by inositol phosphates, demonstrating that activation requires three adjacent phosphate groups and that other positions on the inositol ring can tolerate bulky substituents. We also demonstrate that there is allosteric communication between the inositol-binding site and the active site. The crystal structure of the HDAC1:MTA1 complex bound to a novel peptide-based inhibitor and to inositol hexaphosphate suggests a molecular basis of substrate recognition, and an entropically driven allosteric mechanism of activation.

To evaluate the urinary levels of inositol phosphates (InsPs) in rats that received different salts of myo-inositol hexaphosphate (InsP6) by gavage or by oral administration.

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.

Auxilin-1 is a neuron-specific membrane-binding protein involved in a late stage of clathrin-mediated endocytosis. It recruits Hsc70, thus initiating uncoating of the clathrin-coated vesicles. Interactions of auxilin-1 with the vesicle membrane are crucial for this function and are mediated via an N-terminal PTEN-like domain. We have used multiscale molecular dynamics simulations to probe the interactions of the auxilin-1 PTEN-like domain with lipid bilayers containing differing phospholipid composition, including bilayers containing phosphatidyl inositol phosphates. Our results suggest a novel, to our knowledge, model for the auxilin/membrane encounter and subsequent interactions. Negatively charged lipids (especially PIP2) enhance binding of auxilin to lipid bilayers and facilitate its correct orientation relative to the membrane. Mutations in three basic residues (R301E/R307E/K311E) of the C2 subdomain of the PTEN-like domain perturbed its interaction with the bilayer, changing its orientation. The interaction of membrane-bound auxilin-1 PTEN-like domain with negatively charged lipid headgroups results in nanoclustering of PIP2 molecules in the adjacent bilayer leaflet.

[This corrects the article DOI: 10.1371/journal.ppat.1009190.].

Inositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO2) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP6 (phytate), its pyrophosphate derivatives InsP7 and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO2 bead purification also enabled us to quantify InsP6 in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid. These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130 kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130 kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.

We recently reported on the successful generation of immortalized (CEPI-17-CL4) cells from primary human corneal epithelial (P-CEPI) cells which exhibited phenotypic, immunohistochemical and metabolic characteristics akin to the P-CEPI cells. The aims of the present studies were to investigate the ligand binding and functional coupling of the histamine receptors to various biochemical and physiological systems in the P-CEPI and CEPI-17-CL4 cells and to relate these findings to the normal and/or pathophysiological role of histamine on the human ocular surface. Specific [3H]-pyrilamine binding to CEPI-17-CL4 cell homogenates comprised >93% of the total binding and represented interaction with an apparent single population of high affinity (Kd=3.76+/-0.78 nM; n=4) and saturable (Bmax = 1582+/-161 fmol g(-1) tissue) number of histamine-1 (H1) receptor binding sites on CEPI-17-CL4 cell homogenates. The H1-receptor selective antagonists, pyrilamine (Ki=3.6+/-0.84 nM, n=4) and triprolidine (Ki = 7.7+/-2.6 nM, n=3), potently displaced [3H]-pyrilamine binding, while the H2- and H3-receptor selective antagonists, ranitidine and clobenpropit, were weak inhibitors (K(i)s>13 microM). Histamine induced phosphoinositide (PI) hydrolysis 2.7-4.4 fold above basal levels and with a potency of 14.9+/-4.9 microM (n=9) and 4.7+/-0.2 microM (n=9) in P-CEPI and CEPI-17-CL4 cells, respectively. Histamine-induced PI turnover was antagonized by H1-receptor selective antagonist, triprolidine, with a potency (Ki) of 3.2+/-0.66 nM (n=10) and 3.03+/-0.8 nM (n=4) in P-CEPI and CEPI-17-CL4 cells, respectively, but weakly effected by 10 microM cimetidine and clobenpropit, H2- and H3-receptor antagonists. The PI turnover response was attenuated by pre-treatment of the cells with the selective phospholipase C inhibitor, U73122 (1-(6-((17beta-3-methoxyestra- 1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC50=4.8+/-2.4 microM, n = 3). Histamine stimulated intracellular Ca2+ ([Ca2+]i) mobilization in CEPI-17-CL4 cells with a potency of 6.3+/-1.5 microM (n=4). The histamine-induced [Ca2+]i mobilization was reduced by about 28% following pre-incubation of the cells with 4 mM EGTA. While triprolidine completely inhibited histamine-induced [Ca2+]i mobilization, it did not influence the bradykinin-induced [Ca2+]i mobilization response. Histamine (EC50s = 1.28-2.77 microM, n=3-4) concentration-dependently stimulated the release of interleukin-6 (IL-6), IL-8 and granulocyte macrophage colony-stimulating factor, but it did not significantly alter release of tumour necrosis factor-alpha, PGE2 or collagenase-1 (matrix metalloproteinase-1; MMP-1) from CEPI cells. However, IL-1 (10 ng ml(-1)), foetal bovine serum (10%) and phorbol-12-myristate-13-acetate (3 microg ml(-1)) were effective positive control secretagogues of all the cytokines, PGE2 and MMP-1, respectively, from these cells. It is concluded that the CEPI cells express H1-histamine receptors which are positively coupled to PI turnover and [Ca2+]i mobilization which may be directly or indirectly responsible for the release of various cytokines from these cells at physiologically and/or pathologically relevant concentrations.

We describe a multifunctional inositol polyphosphate kinase/phosphotransferase from Solanum tuberosum, StITPKalpha (GenBank accession number: EF362784), hereafter called StITPK1. StITPK1 displays inositol 3,4,5,6-tetrakisphosphate 1-kinase activity: K(m) = 27 microM, and V(max) = 19 nmol min(-1) mg(-1). The enzyme displays inositol 1,3,4,5,6-pentakisphosphate 1-phosphatase activity in the absence of a nucleotide acceptor and inositol 1,3,4,5,6-pentakisphosphate-ADP phosphotransferase activity in the presence of physiological concentrations of ADP. Additionally, StITPK1 shows inositol phosphate-inositol phosphate phosphotransferase activity. Homology modelling provides a structural rationale of the catalytic abilities of StITPK1. Inter-substrate transfer of phosphate groups between inositol phosphates is an evolutionarily conserved function of enzymes of this class.

Iron chelates of inositol 1,2,3-trisphosphate and inositol 1,2,3,6-tetrakisphosphate lacked free coordination sites and prevented the iron-catalyzed oxidation of ascorbic acid and peroxidation of arachidonic acid. In contrast, iron chelates of inositol 1,2,6-trisphosphate and inositol 1,2,5,6-tetrakisphosphate contained available coordination sites, permitted iron-catalyzed ascorbic acid oxidation, and enhanced arachidonic acid peroxidation. It was concluded that the 1,2,3-trisphosphate grouping of inositol hexakisphosphate was responsible for the inhibition of iron-catalyzed hydroxyl radical formation. The structure of the chelate with the phosphates in an axial-equatorial-axial configuration appeared to be the only possible inositol trisphosphate that could form bonds between six oxygen atoms and the six coordination sites on iron. Km values for cleavage by Escherichia coli alkaline phosphatase were as follows: inositol 1,2,3-trisphosphate, 56 microM; inositol 1,2,6-trisphosphate, 35 microM; inositol 1,2,3,6-tetrakisphosphate, 139 microM; and inositol 1,2,5,6-tetrakisphosphate, 100 microM. The initial hydrolysis rates of 200 microM solutions of the latter three isomers by E. coli alkaline phosphatase were not affected by an equimolar concentration of iron, whereas the rate for inositol 1,2,3-trisphosphate decreased in the presence of iron to 50% of the control. Therefore, the antioxidant potential of inositol 1,2,3-trisphosphate and inositol 1,2,3,6-tetrakisphosphate in cells and other biological systems may be fortified by the resistance of their iron chelates to enzymatic hydrolysis of the functional 1,2,3-trisphosphate array.

The combinatorial phosphorylation of myo-inositol results in the generation of different inositol phosphates (InsPs), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 is also an important precursor of the higher phosphorylated inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6 kinases. Recent work has shown that Arabidopsis (Arabidopsis thaliana) INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1 (ITPK1) and ITPK2 display in vitro InsP6 kinase activity and that, in planta, ITPK1 stimulates 5-InsP7 and InsP8 synthesis and regulates phosphate starvation responses. Here we report a critical role of ITPK1 in auxin-related processes that is independent of the ITPK1-controlled regulation of phosphate starvation responses. Those processes include primary root elongation, root hair development, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity to exogenously applied auxin. We found that the recombinant auxin receptor complex, consisting of the F-Box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1), ARABIDOPSIS SKP1 HOMOLOG 1 (ASK1), and the transcriptional repressor INDOLE-3-ACETIC ACID INDUCIBLE 7 (IAA7), binds to anionic inositol polyphosphates with high affinity. We further identified a physical interaction between ITPK1 and TIR1, suggesting a localized production of 5-InsP7, or another ITPK1-dependent InsP/PP-InsP isomer, to activate the auxin receptor complex. Finally, we demonstrate that ITPK1 and ITPK2 function redundantly to control auxin responses, as deduced from the auxin-insensitive phenotypes of itpk1 itpk2 double mutant plants. Our findings expand the mechanistic understanding of auxin perception and suggest that distinct inositol polyphosphates generated near auxin receptors help to fine-tune auxin sensitivity in plants.

Inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (IP(7)), are water-soluble inositol phosphates that contain high energy diphosphate moieties on the inositol ring. Inositol hexakisphosphate kinase 1 (IP6K1) participates in inositol pyrophosphate synthesis, converting inositol hexakisphosphate (IP(6)) to IP(7). In the present study, we show that mouse embryonic fibroblasts (MEFs) lacking IP6K1 exhibit impaired DNA damage repair via homologous recombination (HR). IP6K1 knock-out MEFs show decreased viability and reduced recovery after induction of DNA damage by the replication stress inducer, hydroxyurea, or the radiomimetic antibiotic, neocarzinostatin. Cells lacking IP6K1 arrest after genotoxic stress, and markers associated with DNA repair are recruited to DNA damage sites, indicating that HR repair is initiated in these cells. However, repair does not proceed to completion because these markers persist as nuclear foci long after drug removal. A fraction of IP6K1-deficient MEFs continues to proliferate despite the persistence of DNA damage, rendering the cells more susceptible to chromosomal aberrations. Expression of catalytically active but not inactive IP6K1 can restore the repair process in knock-out MEFs, implying that inositol pyrophosphates are required for HR-mediated repair. Our study therefore highlights inositol pyrophosphates as novel small molecule regulators of HR signaling in mammals.

Inositol hexakisphosphate (IP6) potently stimulates HIV-1 particle assembly in vitro and infectious particle production in vivo. However, knockout cells lacking inositol-pentakisphosphate 2-kinase (IPPK-KO), the enzyme that produces IP6 by phosphorylation of inositol pentakisphosphate (IP5), were still able to produce infectious HIV-1 particles at a greatly reduced rate. HIV-1 in vitro assembly can also be stimulated to a lesser extent with IP5, but until recently, it was not known if IP5 could also function in promoting assembly in vivo. Here we addressed whether there is an absolute requirement for IP6 or IP5 in the production of infectious HIV-1 particles. IPPK-KO cells expressed no detectable IP6 but elevated IP5 levels and displayed a 20-100-fold reduction in infectious particle production, correlating with lost virus release. Transient transfection of an IPPK expression vector stimulated infectious particle production and release in IPPK-KO but not wildtype cells. Several attempts to make IP6/IP5 deficient stable cells were not successful, but transient expression of the enzyme multiple inositol polyphosphate phosphatase-1 (MINPP1) into IPPK-KOs resulted in near ablation of IP6 and IP5. Under these conditions, we found that HIV-1 infectious particle production and virus release were essentially abolished (1000-fold reduction) demonstrating an IP6/IP5 requirement. However, other retroviruses including a Gammaretrovirus, a Betaretrovirus, and two non-primate Lentiviruses displayed only a modest (3-fold) reduction in infectious particle production from IPPK-KOs and were not significantly altered by expression of IPPK or MINPP1. The only other retrovirus found to show a clear IP6/IP5 dependence was the primate (macaque) Lentivirus Simian Immunodeficiency Virus, which displayed similar sensitivity as HIV-1. We were not able to determine if producer cell IP6/IP5 is required at additional steps beyond assembly because viral particles devoid of both molecules could not be generated. Finally, we found that loss of IP6/IP5 in viral target cells had no effect on permissivity to HIV-1 infection.