Visualizing Nudivirus Assembly and Egress

Abstract

Enveloped viruses hijack cellular membranes in order to provide the necessary material for virion assembly. In particular, viruses that replicate and assemble inside the nucleus have developed special approaches to modify the nuclear landscape for their advantage. We used electron microscopy to investigate cellular changes occurring during nudivirus infection and we characterized a unique mechanism for assembly, packaging, and transport of new virions across the nuclear membrane and through the cytoplasm. Our three-dimensional reconstructions describe the complex remodeling of the nuclear membrane necessary to release vesicle-associated viruses into the cytoplasm. This is the first report of nuclear morphological reconfigurations that occur during nudiviral infection.IMPORTANCE The dynamics of nuclear envelope has a critical role in multiple cellular processes. However, little is known regarding the structural changes occurring inside the nucleus or at the inner and outer nuclear membranes. For viruses assembling inside the nucleus, remodeling of the intranuclear membrane plays an important role in the process of virion assembly. Here, we monitored the changes associated with viral infection in the case of nudiviruses. Our data revealed dramatic membrane remodeling inside the nuclear compartment during infection with Oryctes rhinoceros nudivirus, an important biocontrol agent against coconut rhinoceros beetle, a devastating pest for coconut and oil palm trees. Based on these findings, we propose a model for nudivirus assembly in which nuclear packaging occurs in fully enveloped virions.

Keywords: electron microscopy; enveloped viruses; nudiviruses; viral assembly.

FIG 1

Infection of insect cells with OrNV. (A) Stages of infection. (A1) Uninfected cell with intact nucleus. (A2) At midinfection, few virions (white arrows) surrounded by newly evolved vesicles (arrowheads) are observed inside the nucleus. (A3) In late infection the nuclear membrane which appears slightly distorted undergoes major changes to assist the assembly and the transport of virions. (A4) Cell lysis occurs around 72 h postinfection (hpi). Scale bar, 1,000 nm. (B) Comparison of nucleus and cytoplasmic organelles in OrNV-infected cells to uninfected cells. (B1) Low magnification of uninfected HA cell. High magnification of the inset (B5) shows that mitochondria, Golgi bodies, and ER remain intact in uninfected HA cells. (B2) HA cells infected with (low dose) OrNV at 16 hpi. While normal Golgi and ER are seen at a higher magnification (B6), the mitochondrial structures appear to be affected. Large MMV are also observed in the cytoplasm when virion replication and budding from plasma membrane peaked. (B3) OrNV-infected cells at 72 hpi (high dose). At a higher magnification (B7), the cytoplasm seems to be less electron dense, and organelles such as Golgi bodies and the ER seem to be disrupted. (B4) Infected HA cells resembling the late infection phase. Inset, at a high magnification (B8), shows no evident intact Golgi or ER structures. The scale bar of low-magnification micrographs (B1 to B4) represents 1,000 nm. The scale bar of images from inset (B5 to B8) represents 500 nm. (C) Time course of OrNV infection showing the evolution of viral titers and the concomitant decrease in viable cell density postinfection. (D) Cryo-EM of OrNV shed into the extracellular space showing the capsid surrounded by a thick envelope and its segmentation (right panel). Scale bar, 50 nm.

FIG 2

Intranuclear assembly of OrNV virions. (A) A slice from FIB-SEM reconstruction of an infected cell. Scale bar, 2 μm. (A1) The nucleus is filled with long tubules (Tu) extending from chromatin-like structures (CLS). Clusters of new virions associated with Tu migrate toward the inner nuclear membrane (INM). The INM invaginates inside the nucleus (A2), engulfing fully assembled virions (A3). Scale bar, 500 nm. (B) TEM micrograph of an OrNV-infected cell displaying the mechanism of virion assembly and virion enwrapping in vesicles (scale bar, 1 μm). Tomographic reconstruction of the region shown in the inset. (B1) A 11-nm-thick section showing long filled tubules (fTu) and virion clusters being enveloped in membranes. Scale bar, 500 nm. (B2) Segmentation of the tomogram displaying viral capsids (red) inside filled tubules (gray) and membrane structures (blue).

FIG 3

Virion trafficking inside the cell. (A) Micrograph of an OrNV-infected cell showing virions encapsulated into a vesicle inside the nucleus. Scale bar, 1 μm. (A1) A 11-nm slice through the tomographic reconstruction indicated in inset showing virions enclosed in a double membrane vesicle. Scale bar, 500 nm. (A2) Segmentation of the tomogram. (B) Micrograph displaying expanded NE. Scale bar, 1 μm. (B1) A 11-nm slice through the tomographic reconstruction indicated in inset shows virions inside an expanded NE lumen. Scale bar, 500 nm. (B2) Segmentation of B1 highlights vesicle wrapped virions inside the NE lumen. (C) Micrograph shows cytoplasm completely traversing the nucleus. Scale bar, 1 μm. (C1) A 11-nm slice through the tomographic reconstruction of the area indicated in inset displaying virions encapsulated into multimembrane vesicles (MMVs) in the cytoplasm. Scale bar, 500 nm. (C2) Segmentation showing virions inside complex MMVs, and vesicle-free virions close to the cellular membrane. (D) An infected cell showing vesicles being expelled into the extracellular space. Scale bar, 1 μm. (D1) A slice through the tomographic reconstruction of the area indicated in inset showing vesicles containing fully enveloped virions. Scale bar, 500 nm. (D2) Segmentation showing virions within vesicles.

FIG 4

Proposed model for nudivirus assembly and trafficking. (Step 1) Membrane material is accumulated at the replication sites as small vesicles and membrane fragments. Cytoplasmic invaginations (CI) facilitate membrane presence deep inside nucleus. (Step 2) Virion assembling sites occur adjacent to virogenic stroma (VS) and are populated with Tu and fTu, from which the new virions are seen budding out. Inner nuclear membrane (INM) forms finger-like structures (step 3) into the nucleus and growing larger (step 4), eventually engulfing fully formed virions (step 5). (Step 6) Virion filled vesicles are transported into the NE lumen. Budding at the outer nuclear membrane (ONM) (step 7) releases multimembrane vesicles inside the cytoplasm (step 8). (Step 9) Inside the cytoplasm virions can be encapsulated into multivesicular bodies released into the extracellular space in an exosome-like fashion. (Step 10) Membrane fusion in this compartment can occasionally release virions inside the cytoplasm. (Step 11) Fusion on the plasma membrane can release virion filled vesicles into the extracellular space.