Remodeling nuclear architecture allows efficient transport of herpesvirus capsids by diffusion

Abstract

The nuclear chromatin structure confines the movement of large macromolecular complexes to interchromatin corrals. Herpesvirus capsids of approximately 125 nm assemble in the nucleoplasm and must reach the nuclear membranes for egress. Previous studies concluded that nuclear herpesvirus capsid motility is active, directed, and based on nuclear filamentous actin, suggesting that large nuclear complexes need metabolic energy to escape nuclear entrapment. However, this hypothesis has recently been challenged. Commonly used microscopy techniques do not allow the imaging of rapid nuclear particle motility with sufficient spatiotemporal resolution. Here, we use a rotating, oblique light sheet, which we dubbed a ring-sheet, to image and track viral capsids with high temporal and spatial resolution. We do not find any evidence for directed transport. Instead, infection with different herpesviruses induced an enlargement of interchromatin domains and allowed particles to diffuse unrestricted over longer distances, thereby facilitating nuclear egress for a larger fraction of capsids.he nucleus is structured into chromatin and interchromatin compartments, giving it a "sponge-like" appearance, with the chromatin representing the actual sponge material and the interchromatin regions representing the enclosed pores, tubes, tunnels, and cisterna. This conceptual model has important implications for how differently sized molecules could move inside the nucleus. Although small molecules like GFP, streptavidin, fluorescent dextrans, or mRNA can mostly freely diffuse throughout the nucleus (1-3), large macromolecular assemblies, such as 100-nm fluorescent beads and promyelocytic leukemia (PML) or Cajal bodies, are trapped in interchromatin spaces called corrals. These corrals slowly move as a result of chromatin dynamics, and particles can also escape over long time scales (4, 5). Herpesvirus capsids of 125-nm diameter are assembled in the nucleus and must move to the nuclear periphery to exit the nucleus by budding through the nuclear membranes (6). Similarly, large host cargo like ribonucleoprotein (RNP) particles may need to move through the nucleus before they exit the nucleus in the same way (7). Earlier, we and others provided data that suggested that herpesvirus capsids use an active, directed mechanism based on F-actin to transport through the nucleoplasm (8, 9) and/or that nuclear F-actin is involved in capsid assembly. However, we recently showed that herpesviruses do not induce nuclear F-actin in most cells, and, more importantly, that nuclear capsid motility is not dependent on nuclear F-actin (10). This led us to reinvestigate nuclear capsid motility by single particle tracking to address whether molecular motors power it.During herpesvirus infection, the interchromatin domains enlarge and the nuclear volume increases as much as twofold (11). At the same time, viral replication compartments expand, move, and coalesce, but do not mix (12-16). By using a light-sheet modality we dubbed a ring-sheet, we were able to trace...

Visualizing Herpesvirus Procapsids in Living Cells

“…Consistent with expectations, green-fluorescent diffraction-limited intranuclear punctae became prevalent in the nuclei of infected cells, and these structures failed to acquire red fluorescence. These particles exhibited random motion, similar to a report of angularized capsids (45). In contrast, the pUL25/mCherry signal was acquired by green-fluorescent particles in the presence of a wild-type protease.…”

“…Occasional dual-fluorescent punctae detected at the nuclear periphery that were consistent with the input capsids that initiated the infection were observed. The GFP-VP24 particles displayed random motion in the nucleus similar to that described for angularized capsids labeled with GFP-VP26 fusions (see Movie S1 in the supplemental material) (45).…”

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

“…To date, there are no FDA-approved antiviral approaches that perturb HIV-1 gene expression or the stages upstream of immature virus particle assembly, although several strategies have been pursued, including disruption of Tat or Rev function, virus-specific miRNAs, and transdominant proteins (reviewed in reference 122). Indeed, the regulation RNP complex formation is of increasing interest in other diseases, where malformation of aberrant RNA/protein aggregates or disruption of normal fluid-phase dynamics is evident (4,107,(123)(124)(125). Our results suggest that strategies to successfully disrupt viral mRNA subcellular distribution, gRNP complex formation, or fluid-phase transitions using small-molecule inhibitors or alternative strategies (e.g., provision of trans-acting synthetic "restriction" factors via gene therapy) merit further exploration.…”

Human Cytomegalovirus pUL93 Links Nucleocapsid Maturation and Nuclear Egress

“…The factors involved in the movement of nucleocapsids from the site of viral DNA packaging to the INM are not well understood, but in a recent study it was proposed that this movement occurs by diffusion of nucleocapsids rather than by directed movement (32). Given that the homologs of pUL93 and pUL77 in HSV-1 and PRV are known to interact to form the CVSC and that they individually interact with a component of the NEC in HSV-1, it has been hypothesized that the CVSC is responsible for the selection of capsids at the INM for nuclear egress (5,12).…”