Korboukh, V. | Search bioRxiv

bioRxiv2024

Non-lytic spread of poliovirus requires the nonstructural protein 3CD

Non-enveloped viruses like poliovirus (PV) have evolved the capacity to spread by non-lytic mechanisms. For PV, this mechanism exploits the host secretory autophagy pathway. Virions are selectively incorporated into autophagosomes, double-membrane vesicles that travel to the plasma membrane, fuse, and release single-membrane vesicles containing virions. Loading of cellular cargo into autophagosomes relies on direct or indirect interactions with microtubule-associated protein 1B-light chain 3 (LC3) that are mediated by motifs referred to as LC3-interaction regions (LIRs). We have identified a PV mutant with a severe defect in non-lytic spread. An F-to-Y substitution in a putative LIR of the nonstructural protein 3CD prevented virion incorporation into LC3-positive autophagosomes and virion trafficking to the plasma membrane for release. Using high-angle annular dark-field scanning transmission electron microscopy to monitor PV-induced autophagosome biogenesis, for the first time, we show that virus-induced autophagic signals yield normal autophagosomes, even in the absence of virions. The F-to-Y derivative of PV 3CD was unable to support normal autophagosome biogenesis. Together, these studies make a compelling case for a direct role of a viral nonstructural protein in the formation and loading of the vesicular carriers used for non-lytic spread that may depend on the proper structure, accessibility, and/or dynamics of its LIR. The studies of PV 3CD protein reported here will hopefully provoke a more deliberate look at the presence and function of LIR motifs in viral proteins of viruses known to use autophagy as the basis for non-lytic spread. IMPORTANCEPV and other enteroviruses hijack the cellular secretory autophagy pathway for non-lytic virus transmission. While much is known about the cellular factors required for non-lytic transmission, much less is known about viral factors contributing to transmission. We have discovered a PV nonstructural protein required for multiple steps of the pathway leading to vesicle-enclosed virions. This discovery should facilitate identification of the specific steps of the cellular secretory autophagy pathway and corresponding factors commandeered by the virus and may uncover novel targets for antiviral therapy.

microbiology↗

bioRxiv2018

Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

USP14 is a cysteine-protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A changed the level of TDP-43, tau or -synuclein in HEK293T cells. However, USP14 C114A led to an accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated {beta}-catenin was accumulated in the cells expressing USP14 C114A with biochemistry and molecular biology experiments. The proteasome binding of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine-protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect {beta}-catenin. Using ubiquitin immunoprecipitation and mass spectrometry, we identified the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those accumulated in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function.

molecular biology↗