Biochemical Reconstitution of HIV-1 Assembly and Maturation

Kucharska, Iga; Ding, Pengfei; Zadrozny, Kaneil K.; Dick, Robert A.; Summers, Michael F.; Ganser‐Pornillos, Barbie K.; Pornillos, Owen

doi:10.1128/jvi.01844-19

Cited by 32 publications

(54 citation statements)

References 70 publications

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“…IP6 interacts with Gag in the region of the SP1 six-helix bundle, thereby promoting lattice assembly and stabilizing it [ 11 , 13 ]. Consistently, addition of the related metabolite inositol (1,3,4,5,6) pentakisphosphate (IP5) or IP6 to recombinant Gag stimulates immature particle formation in vitro [ 11 , 14 , 15 ], an effect that has also been observed with BIV, FIV, EIAV, SIV, and HIV-2 Gag proteins [ 16 ] implying that this is a general mechanism of assembly employed by lentiviruses.…”

Section: Introductionmentioning

confidence: 92%

“…IP6 and IP5 can bind to CA hexamers in vitro [ 17 ] and promote CA mature cone-like particle formation in vitro [ 11 ]. Moreover, IP6 promotes the conversion of immature virus-like particles assembled from Gag and viral Ψ RNA into mature-like capsid lattices upon cleavage by PR in vitro [ 15 ]. CA mutants that are impaired in IP6 binding exhibit lower capsid stabilities than wild-type (WT) HIV-1 [ 17 , 18 ], and addition of exogenously added IP6 to HIV-1 cores prevents core disassembly [ 12 ].…”

Section: Introductionmentioning

confidence: 99%

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

Sowd

Aiken

2021

PLoS Pathog

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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.

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Section: Introductionmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

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

Sowd

Aiken

2021

PLoS Pathog

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“…Confocal studies indicate that genomes are sequestered at plasma membrane assembly sites by a small number of Gag proteins, probably a dozen or fewer, and that this initial complex nucleates further Gag recruitment and virus assembly (43,44). In vitro assembly of immature virus-like particles can also be stimulated by the presence of the dimeric [Ψ CES ] 2 RNA (45).…”

Section: Significancementioning

confidence: 99%

Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal

Ding

Kharytonchyk

Waller

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5ʹ-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5′-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 μM, and that all high-affinity sites (Kd ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles’ heel to which HIV therapeutics may be targeted.

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“…As mentioned above, IP6 interacts with two lysine rings (formed by CA-K158 and K227) just above the six-helix bundle that serves as the binding site for MIs [ 49 ] ( Figure 4 ). In vitro, IP6 dramatically increases the kinetics of Gag assembly [ 89 ] and promotes the assembly of immature VLPs under conditions that favor the assembly of the mature lattice [ 49 ]. Furthermore, IP6 inhibits CA-SP1 proteolysis in a manner that is additive or synergistic with BVM in vitro [ 89 ].…”

Section: Mis That Block Ca-sp1 Processingmentioning

confidence: 99%

“…In vitro, IP6 dramatically increases the kinetics of Gag assembly [ 89 ] and promotes the assembly of immature VLPs under conditions that favor the assembly of the mature lattice [ 49 ]. Furthermore, IP6 inhibits CA-SP1 proteolysis in a manner that is additive or synergistic with BVM in vitro [ 89 ]. These findings suggest that IP6 promotes assembly of the immature Gag lattice by stabilizing the six-helix bundle in a BVM-independent manner, indicating that IP6 does not compete with BVM for binding.…”

Section: Mis That Block Ca-sp1 Processingmentioning

confidence: 99%

HIV-1 Maturation: Lessons Learned from Inhibitors

Kleinpeter

Freed

2020

Viruses

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Since the emergence of HIV and AIDS in the early 1980s, the development of safe and effective therapies has accompanied a massive increase in our understanding of the fundamental processes that drive HIV biology. As basic HIV research has informed the development of novel therapies, HIV inhibitors have been used as probes for investigating basic mechanisms of HIV-1 replication, transmission, and pathogenesis. This positive feedback cycle has led to the development of highly effective combination antiretroviral therapy (cART), which has helped stall the progression to AIDS, prolong lives, and reduce transmission of the virus. However, to combat the growing rates of virologic failure and toxicity associated with long-term therapy, it is important to diversify our repertoire of HIV-1 treatments by identifying compounds that block additional steps not targeted by current drugs. Most of the available therapeutics disrupt early events in the replication cycle, with the exception of the protease (PR) inhibitors, which act at the virus maturation step. HIV-1 maturation consists of a series of biochemical changes that facilitate the conversion of an immature, noninfectious particle to a mature infectious virion. These changes include proteolytic processing of the Gag polyprotein by the viral protease (PR), structural rearrangement of the capsid (CA) protein, and assembly of individual CA monomers into hexamers and pentamers that ultimately form the capsid. Here, we review the development and therapeutic potential of maturation inhibitors (MIs), an experimental class of anti-HIV-1 compounds with mechanisms of action distinct from those of the PR inhibitors. We emphasize the key insights into HIV-1 biology and structure that the study of MIs has provided. We will focus on three distinct groups of inhibitors that block HIV-1 maturation: (1) compounds that block the processing of the CA-spacer peptide 1 (SP1) cleavage intermediate, the original class of compounds to which the term MI was applied; (2) CA-binding inhibitors that disrupt capsid condensation; and (3) allosteric integrase inhibitors (ALLINIs) that block the packaging of the viral RNA genome into the condensing capsid during maturation. Although these three classes of compounds have distinct structures and mechanisms of action, they share the ability to block the formation of the condensed conical capsid, thereby blocking particle infectivity.

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Biochemical Reconstitution of HIV-1 Assembly and Maturation

Cited by 32 publications

References 70 publications

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

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

Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal

HIV-1 Maturation: Lessons Learned from Inhibitors

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