Analysis of Borna Disease Virus Trafficking in Live Infected Cells by Using a Virus Encoding a Tetracysteine-Tagged P Protein

Abstract

f Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus characterized by noncytolytic persistent infection and replication in the nuclei of infected cells. To gain further insight on the intracellular trafficking of BDV components during infection, we sought to generate recombinant BDV (rBDV) encoding fluorescent fusion viral proteins. We successfully rescued a virus bearing a tetracysteine tag fused to BDV-P protein, which allowed assessment of the intracellular distribution and dynamics of BDV using real-time live imaging. In persistently infected cells, viral nuclear inclusions, representing viral factories tethered to chromatin, appeared to be extremely static and stable, contrasting with a very rapid and active trafficking of BDV components in the cytoplasm. Photobleaching (fluorescence recovery after photobleaching [FRAP] and fluorescence loss in photobleaching [FLIP]) imaging approaches revealed that BDV components were permanently and actively exchanged between cellular compartments, including within viral inclusions, albeit with a fraction of BDV-P protein not mobile in these structures, presumably due to its association with viral and/or cellular proteins. We also obtained evidence for transfer of viral material between persistently infected cells, with routing of the transferred components toward the cell nucleus. Finally, coculture experiments with noninfected cells allowed visualization of cell-to-cell BDV transmission and movement of the incoming viral material toward the nucleus. Our data demonstrate the potential of tetracysteine-tagged recombinant BDV for virus tracking during infection, which may provide novel information on the BDV life cycle and on the modalities of its interaction with the nuclear environment during viral persistence. Borna disease virus (BDV) is an enveloped virus with a nonsegmented, negative-strand RNA genome (1, 2), which represents the prototypic member of the family Bornaviridae. In contrast to other Mononegavirales members, BDV replicates in the nuclei of infected cells (3) and uses the host cell splicing machinery for maturation of viral transcripts (4, 5). The BDV compact genome encodes six proteins, namely, the nucleoprotein (N), phosphoprotein (P), protein X, matrix protein (M), glycoprotein (G), and polymerase (L). Whereas M and G are involved in particle formation, P, N, and L are components of the ribonucleoprotein complex (RNP). The small X protein has been shown to contribute to apoptosis resistance during infection (6) and may also have interferon antagonist activities (7). BDV persistently infects a wide range of mammalian and avian species (8-11), resulting in a large spectrum of neurological disorders ranging from immune-mediated diseases to behavioral syndromes without inflammation (8, 12, 13). BDV is highly neurotropic but can also replicate in other cells of the central nervous system (CNS), as well as in many established cell lines in vitro (9).To date, the BDV cell cycle remains poorly characterized. It is known that BDV enters ce...

Borna Disease Virus Phosphoprotein Modulates Epigenetic Signaling in Neurons To Control Viral Replication

“…Interestingly, the impact of BDV P on histone acetylation appears to be dissociated from its interaction with chromatin, in particular, from its binding to HMGB-1, as revealed by the use of the BDV P E84N mutant, although we did not formally test whether this was true for all lysine residues affected by BDV and used only H2B K20 as a readout. This fact is consistent with our recent results determined using real-time live-cell imaging, which showed that BDV P is actually highly mobile in vSPOTs, suggesting a labile and transitory interaction with chromatin that may not be sufficient to directly interfere with chromatin dynamics (8).…”

“…It was shown that vSPOTs interact in a dynamic manner with host chromosomes, using high-mobility protein group B-1 (HMGB-1) as a scaffold protein between RNP and chromatin (1,8). Based on these findings, it was tempting to hypothesize that BDV may modify the host cell chromatin or interact with factors controlling chromatin dynamics.…”

Borna Disease Virus Assembles Porous Cage-like Viral Factories in the Nucleus

“…Such mesh-like shells might play an important role in the shuttling of viral and host factors in and out of vSPOTs. Indeed, BDV P was shown to shuttle in and out of vSPOTs, whereas N was immobilized within them (13,24). Furthermore, we demonstrated that certain host factors, which advantageously play a role in viral replication, such as high mobility group box protein 1 (HMGB1), can enter vSPOTs, thus indicating that the pores might allow the selective permeability of these viral factories.…”

Trafficking of Bluetongue Virus Visualized by Recovery of Tetracysteine-Tagged Virion Particles

“…Whereas enveloped viruses have been successfully labeled with genetically encoded fluorescent proteins (21)(22)(23)(24)43), the structural constraints of nonenveloped virus capsid structures have made the insertion of tags difficult. An alternate strategy involving nonspecific labeling of nonenveloped virus capsid structures with fluorescent dyes has helped to elucidate the entry pathway of viruses with naked capsids.…”

“…In addition, the differential labeling of the tagged proteins with two fluorescent biarsenical dyes, FlAsH (green) and ReAsH (red) (18,19), makes this technology a powerful tool for the real-time visualization of nascent protein synthesis and trafficking in cells. To date, this technology has been used successfully in enveloped viruses (21)(22)(23)(24) but not for any complex capsid virus such as BTV.…”