Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

Abstract

We have previously demonstrated that infection by coxsackievirus B3 (CVB3), a positive-stranded RNA enterovirus, results in the accumulation of insoluble ubiquitin-protein aggregates, which resembles the common feature of neurodegenerative diseases. The importance of protein aggregation in viral pathogenesis has been recognized; however, the underlying regulatory mechanisms remain ill-defined. Transactive response DNA-binding protein-43 (TDP-43) is an RNA-binding protein that has an essential role in regulating RNA metabolism at multiple levels. Cleavage and cytoplasmic aggregation of TDP-43 serves as a major molecular marker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration and contributes significantly to disease progression. In this study, we reported that TDP-43 is translocated from the nucleus to the cytoplasm during CVB3 infection through the activity of viral protease 2A, followed by the cleavage mediated by viral protease 3C. Cytoplasmic translocation of TDP-43 is accompanied by reduced solubility and increased formation of protein aggregates. The cleavage takes place at aminoacid 327 between glutamine and alanine, resulting in the generation of an N-and C-terminal cleavage fragment of~35 and~8 kDa, respectively. The C-terminal product of TDP-43 is unstable and quickly degraded through the proteasome degradation pathway, whereas the N-terminal truncation of TDP-43 acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Lastly, we demonstrated that knockdown of TDP-43 results in an increase in viral titers, suggesting a protective role for TDP-43 in CVB3 infection. Taken together, our findings suggest a novel model by which cytoplasmic redistribution and cleavage of TDP-43 as a consequence of CVB3 infection disrupts the solubility and transcriptional activity of TDP-43. Our results also reveal a mechanism evolved by enteroviruses to support efficient viral infection. Coxsackievirus B3 (CVB3) is a small, positive-stranded RNA enterovirus. 1 The single open reading frame of CVB3 is translated into a viral polypeptide that is subsequently cleaved by two virus-encoded proteases 2A and 3C to generate structural and non-structural proteins. 2 In addition to processing viral polyprotein, 2A and 3C target host proteins important for maintenance of protein translation and transcription, antiviral activity, and cellular architecture and signaling, contributing to virus-induced pathogenesis. [3][4][5] Although enteroviral replication takes place exclusively in the cytoplasm, viral infection has been demonstrated to lead to cytoplasmic translocation of nuclear proteins. 6 For example, heterogeneous ribonucleoprotein D (hnRNP D) has been shown to translocate from the nucleus to the cytoplasm during enteroviral infection. 5,7,8 Moreover, hnRNP D is cleaved by 3C and has an antiviral function against enteroviral infection. 5,7,8 Cytoplasmic translocation after enteroviral infection has also been demonstrated for several other hnRNPs (A1, C, and K)...

Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

“…Our conclusion is supported by three lines of evidence. First, EV71 and 2A still blocked tSG formation in cells expressing 3C cleavage-resistant G3BP (G3BP Q326E ) ( Fig 3D and 3E , S4D, S4E , S5D and S5E Figs), which contradicts previous findings [ 21 , 38 , 49 ]. We speculate that most of the viruses failed to replicate in cells expressing G3BP Q326E , because G3BP exhibits antiviral activity against several picornaviruses [ 53 ], thus resulting in an eight-fold reduction in viral titer and tSG formation rescue [ 21 ].…”

Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells

“…TDP-43 is then cleaved by 3C pro at Q327, generating two fragments. The C -terminal fragment of TDP-43 is rapidly degraded, whereas the N -terminal product negatively regulates the function of native TDP-43 [ 123 ]. These processes benefit viral replication.…”

Different aggregation states of a nuclear localization signal‐tagged 25‐kDa C‐terminal fragment of TAR RNA/DNA‐binding protein 43 kDa

“…Thus, TDP-43 is subject to nuclear-cytoplasmic shuttling and mediates RNA-associated functions, mRNA splicing, microRNA processing and mRNA transport to the cytoplasm. It has been shown that not only intact TDP-43 but also several CTFs of TDP-43 are incorporated into cytoplasmic IBs (Neumann et al 2006;Fung et al 2015). The 25-kDa TDP-43 CTF ) is also known as TDP25 (Zhang et al 2009).…”