A single herpesvirus protein can mediate vesicle formation in the nuclear envelope

Abstract

Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission.

Keywords: Herpesvirus; Membrane Reconstitution; Membrane Trafficking; Nuclear Envelope; Nuclear Translocation.

FIGURE 1.

The pUL31-pUL34 complex is sufficient to induce intralumenal vesicles. A, recombinant Alexa Fluor 546-labeled pUL34 (red) was reconstituted into GUVs. The GUV membrane was stained with the lipophilic dye DiDC₁₈. B, Alexa Fluor 546-labeled pUL34 or the inner nuclear membrane protein SCL1 were reconstituted into GUVs (red). Addition of EGFP-pUL31 (green in overlay) induced formation of intra-GUV vesicles on pUL34 but not SCL1 GUVs. C, recombinant proteins employed were separated on 12% SDS-PAGE and stained with Coomassie Blue. D, quantitation of the number of GUVs with intralumenal vesicles (ILVs) shows the mean ± S.E. of three independent experiments, each including at least 70 GUVs/condition and experiment. E, the number of ILVs per GUV was quantified from three independent experiments, each including at least 20 GUVs/condition and experiment. The mean ± S.E. is shown. F, the size distribution of ILVs formed in pUL34-GUVs after EGFP-pUL31 addition was analyzed from three independent experiments, each including at least 20 GUVs/condition and experiment. G, cascade blue-labeled neutravidin (fluid phase marker) was added together with EGFP-pUL31 (top row) or 20 min after EGFP-pUL31 addition to GUVs (bottom row) with reconstituted Alexa Fluor 546-labeled pUL34 or SCL1. H, three-dimensional reconstruction of an EGFP-pUL31-treated (green) pUL34 GUV (red). I, higher magnification of a budding spot on a pUL34 GUV after EGFP-pUL31 addition. EGFP-UL31 and Alexa Fluor 546-pUL34 was analyzed along the limiting GUV membrane. Scale bars = 5 μm (1 μm in I and insets).

FIGURE 2.

Membrane tethering of pUL31 is sufficient to induce intralumenal vesicles. A, an Alexa Fluor 546-labeled N- or C-terminally His₆-tagged pUL34 fragment (top and bottom rows, respectively) comprising the soluble domain (amino acids 1–240) was bound to 1% Ni-NTA-DGS-containing GUVs. Addition of EGFP-pUL31 induced intra-GUV vesicles. B, His₆-tagged-EGFP or His₆-tagged-EGFP-pUL31 was directly bound to Ni-NTA-DGS-containing GUVs. Membranes were stained with DiDC₁₈. Cascade blue-labeled neutravidin as a fluid phase marker was added together with His₆-tagged EGFP-pUL31 or His₆-tagged-EGFP or 20 min after His₆-EGFP-pUL31 addition to GUVs. The purity of the employed recombinant proteins is shown by SDS-PAGE and Coomassie staining. Quantitation shows the mean ± S.E. of three independent experiments, each including at least 80 GUVs/condition and experiment. C, the number of ILVs per GUV was quantified from three independent experiments, each including at least 20 GUVs/condition and experiment. The mean ± S.E. is shown. D, the size distribution of ILVs formed in Ni-NTA-DGS-containing GUVs after His₆-EGFP-pUL31 addition was analyzed from three independent experiments, each including at least 20 GUVs/condition and experiment. E, higher magnification visualizing budding spots on His₆-EGFP-pUL31-tethered Ni-NTA-DGS GUVs. F, three-dimensional reconstruction of a His₆-EGFP-pUL31-treated (green) GUV containing 1% Ni-NTA-DGS. Membranes were stained with DiDC₁₈ (gray). Scale bars = 5 μm (1 μm in E and insets).

FIGURE 3.

Cholesterol and sphingomyelin are required for pUL31-mediated vesicle formation. A, recombinant Alexa Fluor 546-labeled pUL34 was reconstituted into GUVs containing the nuclear envelope lipid mix (complete lipid mix) or the same lipid mix lacking either cholesterol (chol), sphingomyelin (SM), the negatively charged phospholipids phosphoinositol (PI) or phosphatidylserine (PS), or both (PI+PS). Formation of intralumenal vesicles was induced and quantified after EGFP-pUL31 addition (mean ± S.E. of three independent experiments, each including at least 50 GUVs/experiment and condition). B, His₆-tagged-EGFP-pUL31 was bound directly to Ni-NTA-DGS-containing GUVs with the same lipid compositions as in A, and ILV numbers were quantified (mean ± S.E. of three independent experiments, each including at least 60 GUVs/experiment and condition). C, pUL31 was added to pUL34-GUVs loaded with the membrane dyes naphthopyrene and DiDC₁₈, which label the liquid ordered (L_O) or liquid disordered phase (L_D), respectively. Although we cannot exclude an enrichment of specific lipids in internal vesicles, there no induction and separation of liquid-ordered and -disordered phases was detectable. D, as a control for the functionality of the membrane dyes, their segregation was tested on phase-separating membrane GUVs (33 mol% cholesterol, 33 mol% sphingomyelin, 33 mol% phosphatidylcholine). Scale bars = 5 μm (1 μm in insets).

FIGURE 4.

pUL31 self-interacts on membranes but not in solution. A, where indicated, 60 nm His₆-tagged-pUL31 was bound directly to Ni-NTA-DGS-containing GUVs. Increasing amounts of EGFP-pUL31 were added to the GUVs showing a pUL31-mediated membrane recruitment of EGFP-pUL31 and formation of intra-GUV vesicles. Where indicated, cascade blue-labeled neutravidin was added as a fluid phase marker 20 min after protein addition to GUVs to confirm scission of ILVs from the limiting GUV membrane. Quantitation shows the mean ± S.E. of three independent experiments, each including at least 60 GUVs/condition and experiment. Scale bars are 10 μm (1 μm in insets). B, GST pulldown using GST (control), GST-pUL31, and GST-pUL34ΔTMR (amino acids 1–240, i.e. lacking the transmembrane region) as bait and EGFP-pUL31 as prey. GST-bound EGFP-pUL31 was eluted with precision protease-containing buffer, which cleaves the GST fusions C-terminal of the GST tag, as analyzed by Western blotting using anti-EGFP-antibodies. C, GST pulldown as in B, but with His₆-pUL31 as prey, detected using anti-His₆ antibodies. D, size exclusion chromatography on a Superdex 75/300 GL column followed by multiangle static laser light scattering of EGFP-UL31 shows that it is monomeric in solution (calculated mass, 58.7 kDa). The red dots relate to the secondary axis and show the molecular weight of the eluting particle.

FIGURE 5.

pUL31 forms patches on supported lipid bilayers. A, supported lipid bilayers (SLB) mimicking the nuclear envelope lipid composition (supplemented with Ni-NTA-DGS) show a flat topography before protein addition. Graphs below the image are profiles of the yellow line in the image. B, upon addition of His₆-EGFP-pUL31, patches form with aggregated structures. The right figure is a 3 × 3 μm enlarged image of the yellow box within the left panel. C, supported lipid bilayers before addition of His₆-EGFP. D, supported lipid bilayers after addition of His₆-EGFP with no change in topography detectable. E, diameter and roughness (average height of the patches) distributions of the patches observed upon addition of His₆-EGFP-pUL31. Gaussian fitting of the histograms shows a peak 1.0 ± 0.5 μm in diameter (mean ± S.D.) and 2.0 ± 0.1 nm in roughness. Scale bars = 10 μm for A and 2 μm for B–D.

FIGURE 6.

pUL31 disrupts supported lipid bilayers. A, supported lipid bilayers (SLB) mimicking the nuclear envelope with rhodamine-PE (red) form continuous fluid phase. Bilayers are disrupted upon addition of His6-EGFP-pUL31 (green). A few seconds after addition, pUL31 forms 1.2 ± 0.2 μm patches that grow over time until they cover the whole area (around 90 s). The bilayer is destroyed, and lipid aggregation is seen over this period of time. B, addition of His₆-EGFP did not change the bilayer. C, particle analysis of the patches showing the growth of the particles over time. After 40 s, particles fuse and can no longer be analyzed. Scale bars = 50 μm.