The nuclear export factor CRM1 controls juxta-nuclear microtubule-dependent virus transport

Abstract

Transport of large cargo through the cytoplasm requires motor proteins and polarized filaments. Viruses that replicate in the nucleus of post-mitotic cells use microtubules and the dynein-dynactin motor to traffic to the nuclear membrane and deliver their genome through nuclear pore complexes (NPCs) into the nucleus. How virus particles (virions) or cellular cargo are transferred from microtubules to the NPC is unknown. Here, we analyzed trafficking of incoming cytoplasmic adenoviruses by single-particle tracking and superresolution microscopy. We provide evidence for a regulatory role of CRM1 (chromosome-region-maintenance-1; also known as XPO1, exportin-1) in juxta-nuclear microtubule-dependent adenovirus transport. Leptomycin B (LMB) abolishes nuclear targeting of adenovirus. It binds to CRM1, precludes CRM1-cargo binding and blocks signal-dependent nuclear export. LMB-inhibited CRM1 did not compete with adenovirus for binding to the nucleoporin Nup214 at the NPC. Instead, CRM1 inhibition selectively enhanced virion association with microtubules, and boosted virion motions on microtubules less than ∼2 µm from the nuclear membrane. The data show that the nucleus provides positional information for incoming virions to detach from microtubules, engage a slower microtubule-independent motility to the NPC and enhance infection.

Single Virion Tracking Microscopy for the Study of Virus Entry Processes in Live Cells and Biomimetic Platforms

“…In addition to investigating movement towards the nucleus, live-cell single virion tracking has been used to monitor movement within the nuclear region. Adenovirus utilizes microtubules for movement within the cell, but detaches from them when the virions reach close proximity to the nucleus [119]. HIV cores move toward the nucleus with a microtuble-and actin-dependent motion; within the nucleus, the motion is slow and diffuse [100,116,120].…”

OMEGA: a software tool for the management, analysis, and dissemination of intracellular trafficking data that incorporates motion type classification and quality control

“…The time-resolved visualization of individual heterogeneous intracellular particles by fluorescence-microscopy, coupled with feature point tracking techniques -referred to as Single-Particle Tracking (SPT) (De Brabander et al, 1985) or Multiple-Particle Tracking (MPT) -and mathematical analysis of motion, is ideally suited to follow the fate of particles as they progress within the cell, to map fleeting interactions with other cellular components, and to dissect individual transport steps. For example, single viral imaging experiments coupled with SPT have improved understanding of the early phases of viral entry and revealed previously un-recognized entry stages (Brandenburg and Zhuang, 2007;Flatt and Greber, 2017;Sun et al, 2013;Greber and Way, 2006;Wang et al, 2017;Ewers et al, 2005;Helmuth et al, 2007;Yamauchi et al, 2011).…”

“…This manual method can be used in conjunction with an iterative analysis process to obtain homogeneous trajectory segments (see below and Figure 6). In subsequent releases, automated methods for trajectory segmentation will be integrated in OMEGA (Helmuth et al, 2007;Wagner et al, 2017;Huet et al, 2006;Persson et al, 2013;Wang et al, 2017).…”

Cell-to-cell and genome-to-genome variability of Adenovirus transcription tuned by the cell cycle

“…Studies with AdV show further cell-to-cell heterogeneity, namely in virion binding to the cells [20], endosomal and cytoplasmic trafficking [21,22], and virion uncoating [23 -28]. Single-cell, single-particle analyses show that different steps of entry occur 5 with different efficiencies in individual infected cells and thus the number of vDNAs delivered into the nucleus varies between cells [21,25,27].…”