Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages

Inlora, Jingga; Chukkapalli, Vineela; Bedi, Sukhmani; Ono, Akira

doi:10.1128/jvi.01004-16

Cited by 11 publications

(8 citation statements)

References 106 publications

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“…Previous studies have shown that NC deletion abrogates Gag localization to specific PM domains, such as uropods in polarized T cells or the virus-containing compartments (VCCs) in macrophages, whereas insertion of LZ restores such localization (71,72). In contrast, we do not see a difference in the subcellular localization of Gag between full-length Gag and delNC Gag for any of the MA mutants examined (Fig.…”

Section: Discussioncontrasting

confidence: 76%

Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes

Thornhill

Olety

Ono

2019

J Virol

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The HIV-1 Gag matrix (MA) domain mediates the localization of Gag to the plasma membrane (PM), the site for infectious virion assembly. The MA highly basic region (MA-HBR) interacts with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific acidic lipid. The MA-HBR also binds RNAs. To test whether acidic lipids alone determine PM-specific localization of Gag or whether MA-RNA binding also plays a role, we compared a panel of MA-HBR mutants that contain two types of substitutions at MA residues 25 and 26 or residues 29 and 31: Lys→Arg (KR) (25/26KR and 29/31KR) and Lys→Thr (KT) (25/26KT and 29/31KT). Consistent with the importance of the HBR charge in RNA binding, both KT mutants failed to bind RNA via MA efficiently, unlike the corresponding KR mutants. Both 25/26KT Gag-yellow fluorescent protein (YFP) and 29/31KT Gag-YFP bound nonspecifically to the PM and intracellular membranes, presumably via the myristoyl moiety and remaining MA basic residues. In contrast, 25/26KR Gag-YFP bound specifically to the PM, suggesting a role for the total positive charge and/or MA-bound RNA in navigating Gag to the PM. Unlike 29/31KT Gag-YFP, 29/31KR Gag-YFP was predominantly cytosolic and showed little intracellular membrane binding despite having a higher HBR charge. Therefore, it is likely that MA-RNA binding blocks promiscuous Gag membrane binding in cells. Notably, the introduction of a heterologous multimerization domain restored PI(4,5)P2-dependent PM-specific localization for 29/31KR Gag-YFP, suggesting that the blocking of PM binding is more readily reversed than that of intracellular membrane binding. Altogether, these cell-based data support a model in which MA-RNA binding ensures PM-specific localization of Gag via suppression of nonspecific membrane binding. IMPORTANCE The PM-specific localization of HIV-1 Gag is a crucial early step in infectious progeny production. The interaction between the MA highly basic region (MA-HBR) of Gag and the PM-specific lipid PI(4,5)P2 is critical for Gag localization to the PM. Additionally, in vitro evidence has indicated that MA-RNA binding prevents nonspecific binding of Gag to non-PI(4,5)P2-containing membranes. However, cell-based evidence supporting a role for HIV-1 MA-RNA binding in PM-specific subcellular localization has been scarce; thus, it remained possible that in cells, just the high basic charge or the PI(4,5)P2 binding ability is sufficient for MA to direct Gag specifically to the PM. The present study reveals for the first time an excellent correlation between RNA binding of the MA-HBR and inhibition of promiscuous Gag localization, both within the cells, and thereby provides cell-based evidence supporting a mechanism in which HIV-1 MA binding to RNA ensures the specific localization of Gag to the PM.

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Section: Discussioncontrasting

confidence: 76%

Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes

Thornhill

Olety

Ono

2019

J Virol

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“…Thus, the combination of RNA labeling techniques with the HIV-1 Gag-iFRET system would provide a unique method in this context to elucidate the dynamics of Gag-viral RNA release from the budding site into progeny virions. Moreover, viral assembly appears to be cell-type dependent ( Ono and Freed, 2004 ), and virions are assembled and released in viral-containing compartments (VCCs) in macrophages, beyond the reach of antivirals or antibodies, and from where cell-to-cell infection can occur unhindered ( Pelchen-Matthews et al, 2003 ; Sharova et al, 2005 ; Gousset et al, 2008 ; Groot et al, 2008 ; Chu et al, 2012 ; Inlora et al, 2016 ). HIV-1 Gag-iFRET could be used in live-cell imaging to localize and visualize maturation in various cellular compartments in different cell-type settings.…”

Section: Discussionmentioning

confidence: 99%

FRET-Based Detection and Quantification of HIV-1 Virion Maturation

et al. 2021

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HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.

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“…Aiming to elucidate the role of the viral factors dictating the assembly site of HIV in macrophages, Inlora and colleagues evaluated the impact of targeted deletions or mutations in the different Gag domains, on virion assembly at the VCC ( 118 ). Viral mutants unable of high-order multimerization, due to NC substitutions, distributed equally between the VCC and the cell surface ( 118 ). This indicates that Gag initially binds the membrane arbitrarily at the surface membrane or the VCC and initiates low-order multimerization.…”

Section: Cell Biology Of Hiv Assembly In Macrophages: From Gag Synthementioning

confidence: 99%

Myeloid Cell Interaction with HIV: A Complex Relationship

et al. 2017

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Cells of the myeloid lineage, particularly macrophages, serve as primary hosts for HIV in vivo, along with CD4 T lymphocytes. Macrophages are present in virtually every tissue of the organism, including locations with negligible T cell colonization, such as the brain, where HIV-mediated inflammation may lead to pathological sequelae. Moreover, infected macrophages are present in multiple other tissues. Recent evidence obtained in humanized mice and macaque models highlighted the capacity of macrophages to sustain HIV replication in vivo in the absence of T cells. Combined with the known resistance of the macrophage to the cytopathic effects of HIV infection, such data bring a renewed interest in this cell type both as a vehicle for viral spread as well as a viral reservoir. While our understanding of key processes of HIV infection of macrophages is far from complete, recent years have nevertheless brought important insight into the uniqueness of the macrophage infection. Productive infection of macrophages by HIV can occur by different routes including from phagocytosis of infected T cells. In macrophages, HIV assembles and buds into a peculiar plasma membrane-connected compartment that preexists to the infection. While the function of such compartment remains elusive, it supposedly allows for the persistence of infectious viral particles over extended periods of time and may play a role on viral transmission. As cells of the innate immune system, macrophages have the capacity to detect and respond to viral components. Recent data suggest that such sensing may occur at multiple steps of the viral cycle and impact subsequent viral spread. We aim to provide an overview of the HIV–macrophage interaction along the multiple stages of the viral life cycle, extending when pertinent such observations to additional myeloid cell types such as dendritic cells or blood monocytes.

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Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages

Cited by 11 publications

References 106 publications

Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes

Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes

FRET-Based Detection and Quantification of HIV-1 Virion Maturation

Myeloid Cell Interaction with HIV: A Complex Relationship

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