MX2 restricts HIV-1 and herpes simplex virus type 1 by forming cytoplasmic biomolecular condensates that mimic nuclear pore complexes

Moschonas, George D.; Delhaye, Louis; Cooreman, Robin; Hüsers, Franziska; Bhat, Anayat; Sutter, Delphine De; Parthoens, Eef; Desmet, Anne-Sophie; Maciejczuk, Aleksandra; Grzesik, Hanna; Lippens, Saskia; Debyser, Zeger; Sodeik, Beate; Eyckerman, Sven; Saelens, Xavier

doi:10.1101/2023.06.22.545931

Cited by 5 publications

(3 citation statements)

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“…The cognate-related human protein MxB is also cytoplasmic and shows antiviral activity against HIV and the herpes virus type 1 [11][12][13]17,22]. MxB, and GFP-MxB, also forms membraneless condensates on the cytosolic side of nuclear pores and thus inhibits the transit of viral RNA into nucleus [22,46] and citations therein. The major murine ortholog of both human MxA and human MxB genes is the murine Mx1 gene; the related protein is murine Mx1 [11][12][13]22].…”

Section: Discussionmentioning

confidence: 99%

“…We recently reported that nuclear murine GFP-Mx1 structures were also LLPS-driven biomolecular condensates which, like cytoplasmic human MxA condensates, disassembled in cells exposed to hypotonic ELB, and reassembled in cells then shifted to isotonic PBS [22]. Thus, not only did the subcellular localization of Mx family members contribute significantly to their respective antiviral spectrum, but also the cytoplasmic human MxB and nuclear murine Mx1 formed biomolecular condensates [22,46]. For our present discussion, we note that GTPase-null mutants of human MxA, which do not have antiviral activity, continue to form cytoplasmic condensates [11][12][13][14]44,45].…”

Section: Discussionmentioning

confidence: 99%

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Oral Antiviral Defense: Saliva- and Beverage-like Hypotonicity Dynamically Regulate Formation of Membraneless Biomolecular Condensates of Antiviral Human MxA in Oral Epithelial Cells

Sehgal,

Yuan,

Centone

et al. 2024

Cells

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The oral mucosa represents a defensive barrier between the external environment and the rest of the body. Oral mucosal cells are constantly bathed in hypotonic saliva (normally one-third tonicity compared to plasma) and are repeatedly exposed to environmental stresses of tonicity, temperature, and pH by the drinks we imbibe (e.g., hypotonic: water, tea, and coffee; hypertonic: assorted fruit juices, and red wines). In the mouth, the broad-spectrum antiviral mediator MxA (a dynamin-family large GTPase) is constitutively expressed in healthy periodontal tissues and induced by Type III interferons (e.g., IFN-λ1/IL-29). Endogenously induced human MxA and exogenously expressed human GFP-MxA formed membraneless biomolecular condensates in the cytoplasm of oral carcinoma cells (OECM1 cell line). These condensates likely represent storage granules in equilibrium with antivirally active dispersed MxA. Remarkably, cytoplasmic MxA condensates were exquisitely sensitive sensors of hypotonicity—the condensates in oral epithelium disassembled within 1–2 min of exposure of cells to saliva-like one-third hypotonicity, and spontaneously reassembled in the next 4–7 min. Water, tea, and coffee enhanced this disassembly. Fluorescence changes in OECM1 cells preloaded with calcein-AM (a reporter of cytosolic “macromolecular crowding”) confirmed that this process involved macromolecular uncrowding and subsequent recrowding secondary to changes in cell volume. However, hypertonicity had little effect on MxA condensates. The spontaneous reassembly of GFP-MxA condensates in oral epithelial cells, even under continuous saliva-like hypotonicity, was slowed by the protein-phosphatase-inhibitor cyclosporin A (CsA) and by the K-channel-blocker tetraethylammonium chloride (TEA); this is suggestive of the involvement of the volume-sensitive WNK kinase-protein phosphatase (PTP)-K-Cl cotransporter (KCC) pathway in the regulated volume decrease (RVD) during condensate reassembly in oral cells. The present study identifies a novel subcellular consequence of hypotonic stress in oral epithelial cells, in terms of the rapid and dynamic changes in the structure of one class of phase-separated biomolecular condensates in the cytoplasm—the antiviral MxA condensates. More generally, the data raise the possibility that hypotonicity-driven stresses likely affect other intracellular functions involving liquid–liquid phase separation (LLPS) in cells of the oral mucosa.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Oral Antiviral Defense: Saliva- and Beverage-like Hypotonicity Dynamically Regulate Formation of Membraneless Biomolecular Condensates of Antiviral Human MxA in Oral Epithelial Cells

Sehgal,

Yuan,

Centone

et al. 2024

Cells

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“…Dimerization is required for MxB antiviral activity against HIV-1 ( 110 , 111 ). Recent experiments suggest that MxB may act as a decoy, luring HIV cores away from nuclear pores and thus impeding nuclear entry ( 112 ).…”

Section: Known Capsid-dependent Restriction Factorsmentioning

confidence: 99%

Capsid-dependent lentiviral restrictions

Twentyman,

Emerman,

Ohainle

2024

J Virol

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Host antiviral proteins inhibit primate lentiviruses and other retroviruses by targeting many features of the viral life cycle. The lentiviral capsid protein and the assembled viral core are known to be inhibited through multiple, directly acting antiviral proteins. Several phenotypes, including those known as Lv1 through Lv5 , have been described as cell type-specific blocks to infection against some but not all primate lentiviruses. Here we review important features of known capsid-targeting blocks to infection together with several blocks to infection for which the genes responsible for the inhibition still remain to be identified. We outline the features of these blocks as well as how current methodologies are now well suited to find these antiviral genes and solve these long-standing mysteries in the HIV and retrovirology fields.

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Interplay between the cyclophilin homology domain of RANBP2 and MX2 regulates HIV-1 capsid dependencies on nucleoporins

Flick,

Venbakkam,

Singh

et al. 2024

Preprint

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Interlinked interactions between the viral capsid (CA), nucleoporins (Nups), the antiviral protein myxovirus resistance 2 (MX2/MXB) influence HIV-1 nuclear entry and the outcome of infection. Although RANBP2/NUP358 has been repeatedly identified as a critical player in HIV-1 nuclear import and MX2 activity, the mechanism by which RANBP2 facilitates HIV-1 infection is not well understood. To explore the interactions between MX2, the viral CA, and RANBP2, we utilized CRISPR-Cas9 to generate cell lines expressing RANBP2 from its endogenous locus but lacking the C-terminal cyclophilin (Cyp) homology domain, and found that both HIV-1 and HIV-2 infection were reduced significantly in RANBP2ΔCypcells. Importantly, although MX2 still localized to the nuclear pore complex in RANBP2ΔCypcells, antiviral activity against HIV-1 was decreased. By generating cells expressing specific point mutations in the RANBP2-Cyp domain we determined that the effect of the RANBP2-Cyp domain on MX2 anti-HIV-1 activity is due to direct interactions between RANBP2 and CA. We further determined that CypA and RANBP2-Cyp have similar effects on HIV-1 integration targeting. Finally, we found that the Nup requirements for HIV infection and MX2 activity were altered in cells lacking the RANBP2-Cyp domain. These findings demonstrate that the RANBP2-Cyp domain affects viral infection and MX2 sensitivity by altering CA-specific interactions with cellular factors that affect nuclear import and integration targeting.Significance StatementHIV-1 entry into the nucleus is an essential step in viral replication that involves complex interactions between the viral capsid and multiple cellular proteins, including nucleoporins such as RANBP2. Nups also mediate the function of the antiviral protein MX2, however determining the precise role of Nups in HIV infection has proved challenging due to the complex nature of the nuclear pore and significant pleiotropic effects elicited by Nup depletion. We have used precise gene editing to assess the role of the Cyp domain of RANBP2 in HIV-1 infection and MX2 activity. We find that this domain affects viral infection, nucleoporin requirements, MX2 sensitivity, and integration targeting in a CA-specific manner, providing detailed insights into how RANBP2 contributes to HIV-1 infection.

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MX2 restricts HIV-1 and herpes simplex virus type 1 by forming cytoplasmic biomolecular condensates that mimic nuclear pore complexes

Cited by 5 publications

References 122 publications

Oral Antiviral Defense: Saliva- and Beverage-like Hypotonicity Dynamically Regulate Formation of Membraneless Biomolecular Condensates of Antiviral Human MxA in Oral Epithelial Cells

Oral Antiviral Defense: Saliva- and Beverage-like Hypotonicity Dynamically Regulate Formation of Membraneless Biomolecular Condensates of Antiviral Human MxA in Oral Epithelial Cells

Capsid-dependent lentiviral restrictions

Interplay between the cyclophilin homology domain of RANBP2 and MX2 regulates HIV-1 capsid dependencies on nucleoporins

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