Sec62 restricts ER-replicating positive-strand RNA virus infections via UPR-dependent ER-phagy.

Endomembrane remodeling to form a viral replication complex (VRC) is crucial for a virus to establish infection in a host. Although the composition and function of VRCs have been intensively studied, host factors involved in the assembly of VRCs for plant RNA viruses have not been fully explored. TurboID-based proximity labeling (PL) has emerged as a robust tool for probing molecular interactions in planta. However, few studies have employed the TurboID-based PL technique for investigating plant virus replication. Here, we used Beet black scorch virus (BBSV), an endoplasmic reticulum (ER)-replicating virus, as a model and systematically investigated the composition of BBSV VRCs in Nicotiana benthamiana by fusing the TurboID enzyme to viral replication protein p23. Among the 185 identified p23-proximal proteins, the reticulon family of proteins showed high reproducibility in the mass spectrometry data sets. We focused on RETICULON-LIKE PROTEIN B2 (RTNLB2) and demonstrated its proviral functions in BBSV replication. We showed that RTNLB2 binds to p23, induces ER membrane curvature, and constricts ER tubules to facilitate the assembly of BBSV VRCs. Our comprehensive proximal interactome analysis of BBSV VRCs provides a resource for understanding plant viral replication and offers additional insights into the formation of membrane scaffolds for viral RNA synthesis.

All well-characterized positive-strand RNA viruses[(+)RNA viruses] induce the formation of host membrane-bound viral replication complexes (VRCs), yet the underlying mechanism and machinery for VRC formation remain elusive. We report here the biogenesis and topology of the Beet black scorch virus (BBSV) replication complex. Distinct cytopathological changes typical of endoplasmic reticulum (ER) aggregation and vesiculation were observed in BBSV-infected Nicotiana benthamiana cells. Immunogold labeling of the auxiliary replication protein p23 and double-stranded RNA (dsRNA) revealed that the ER-derived membranous spherules provide the site for BBSV replication. Further studies indicated that p23 plays a crucial role in mediating the ER rearrangement. Three-dimensional electron tomographic analysis revealed the formation of multiple ER-originated vesicle packets. Each vesicle packet enclosed a few to hundreds of independent spherules that were invaginations of the ER membranes into the lumen. Strikingly, these vesicle packets were connected to each other via tubules, a rearrangement event that is rare among other virus-induced membrane reorganizations. Fibrillar contents within the spherules were also reconstructed by electron tomography, which showed diverse structures. Our results provide the first, to our knowledge, three-dimensional ultrastructural analysis of membrane-bound VRCs of a plant (+)RNA virus and should help to achieve a better mechanistic understanding of the organization and microenvironment of plant (+)RNA virus replication complexes. Assembly of virus replication complexes for all known positive-strand RNA viruses depends on the extensive remodeling of host intracellular membranes. Beet black scorch virus, a necrovirus in the family Tombusviridae, invaginates the endoplasmic reticulum (ER) membranes to form spherules in infected cells. Double-stranded RNAs, the viral replication intermediate, and the viral auxiliary replication protein p23 are all localized within such viral spherules, indicating that these are the sites for generating progeny viral RNAs. Furthermore, the BBSV p23 protein could to some extent reorganize the ER when transiently expressed in N. benthamiana. Electron tomographic analysis resolves the three-dimensional (3D) architecture of such spherules, which are connected to the cytoplasm via a neck-like structure. Strikingly, different numbers of spherules are enclosed in ER-originated vesicle packets that are connected to each other via tubule-like structures. Our results have significant implications for further understanding the mechanisms underlying the replication of positive-strand RNA viruses.

Positive‐sense RNA viruses remodel cellular cytoplasmic membranes as the membranous sources for the formation of viral replication organelles (VROs) for viral genome replication. In plants, they traffic through plasmodesmata (PD), plasma membrane‐lined pores enabling cytoplasmic connections between cells for intercellular movement and systemic infection. In this study, we employed turnip mosaic virus (TuMV), a plant RNA virus to investigate the involvement of RTNLB3 and RTNLB6, two ER (endoplasmic reticulum) membrane‐bending, PD‐located reticulon‐like (RTNL) non‐metazoan group B proteins (RTNLBs) in viral infection. We show that RTNLB3 interacts with TuMV 6K2 integral membrane protein and RTNLB6 binds to TuMV coat protein (CP). Knockdown of RTNLB3 promoted viral infection, whereas downregulation of RTNLB6 restricted viral infection, suggesting that these two RTNLs play contrasting roles in TuMV infection. We further demonstrate that RTNLB3 targets the α‐helix motif 42LRKSM46 of 6K2 to interrupt 6K2 self‐interactions and compromise 6K2‐induced VRO formation. Moreover, overexpression of AtRTNLB3 apparently promoted the selective degradation of the ER and ER‐associated protein calnexin, but not 6K2. Intriguingly, mutation of the α‐helix motif of 6K2 that is required for induction of VROs severely affected 6K2 stability and abolished TuMV infection. Thus, RTNLB3 attenuates TuMV replication, probably through the suppression of 6K2 function. We also show that RTNLB6 promotes viral intercellular movement but does not affect viral replication. Therefore, the proviral role of RTNLB6 is probably by enhancing viral cell‐to‐cell trafficking. Taken together, our data demonstrate that RTNL family proteins may play diverse complex, even opposite, roles in viral infection in plants.

Beet black scorch virus (BBSV) has been reported as a natural pathogen of sugar beet and distributed all over the world, causing great economic losses to the sugar industry. Research on interactions between BBSV and its host by using model plant Nicotiana benthamiana is significantly important and nessesary for understanding virus infection process and exploring plant resistance mechanism. The results of sequencing the transcriptome of N. benthamiana infected with BBSV as well as gene screening in response to viral infection revealed upregulation of the small heat shock protein 17.6 gene (NbHSP17.6) and the effect of the protein on resistance to the virus. To further examine the involvement of HSP17.6 in defense responses in N. benthamiana, we tested interaction between HSP17.6 and other heat shock proteins such as HSP70 and HSP90 as well as BBSV encoded proteins. The results showed that HSP17.6 interacted with HSP70 and HSP90 but not with BBSV encoded proteins. When combined with other available results, it is possible that HSP17.6 acted as a small molecular chaperone to facilitate proper refolding of the specific proteins HSP70 and HSP90 required for BBSV infection and/or replication.

Positive-strand (+)RNA viruses take advantage of the host cells by subverting a long list of host protein factors and transport vesicles and cellular organelles to build membranous viral replication organelles (VROs) that support robust RNA replication. How RNA viruses accomplish major recruitment tasks of a large number of cellular proteins are intensively studied. In case of tomato bushy stunt virus (TBSV), a single viral replication protein, named p33, carries out most of the recruitment duties. Yet, it is currently unknown how the viral p33 replication protein, which is membrane associated, is capable of the rapid and efficient recruitment of numerous cytosolic host proteins to facilitate the formation of large VROs. In this paper, we show that, TBSV p33 molecules do not recruit each cytosolic host factor one-by-one into VROs, but p33 targets a cytosolic protein interaction hub, namely Rpn11, which interacts with numerous other cytosolic proteins. The highly conserved Rpn11, called POH1 in humans, is the metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates. However, TBSV takes advantage of a noncanonical function of Rpn11 by exploiting Rpn11’s interaction with highly abundant cytosolic proteins and the actin network. We provide supporting evidence that the co-opted Rpn11 in coordination with the subverted actin network is used for delivering cytosolic proteins, such as glycolytic and fermentation enzymes, which are readily subverted into VROs to produce ATP locally in support of VRO formation, viral replicase complex assembly and viral RNA replication. Using several approaches, including knockdown of Rpn11 level, sequestering Rpn11 from the cytosol into the nucleus in plants or temperature-sensitive mutation in Rpn11 in yeast, we show the inhibition of recruitment of glycolytic and fermentation enzymes into VROs. The Rpn11-assisted recruitment of the cytosolic enzymes by p33, however, also requires the combined and coordinated role of the subverted actin network. Accordingly, stabilization of the actin filaments by expression of the Legionella VipA effector in yeast and plant, or via a mutation of ACT1 in yeast resulted in more efficient and rapid recruitment of Rpn11 and the selected glycolytic and fermentation enzymes into VROs. On the contrary, destruction of the actin filaments via expression of the Legionella RavK effector led to poor recruitment of Rpn11 and glycolytic and fermentation enzymes. Finally, we confirmed the key roles of Rpn11 and the actin filaments in situ ATP production within TBSV VROs via using a FRET-based ATP-biosensor. The novel emerging theme is that TBSV targets Rpn11 cytosolic protein interaction hub driven by the p33 replication protein and aided by the subverted actin filaments to deliver several co-opted cytosolic pro-viral factors for robust replication within VROs.

ER stress: Can the liver cope?

Dissecting the complex molecular interplay between the host plant and invading virus improves our understanding of the mechanisms underlying viral pathogenesis. In this study, immunoprecipitation together with the mass spectrometry analysis revealed that the heat shock protein 70 (Hsp70) family homolog, Hsc70-2, was co-purified with beet black scorch virus (BBSV) replication protein p23 and coat protein (CP), respectively. Further experiments demonstrated that Hsc70-2 interacts directly with both p23 and CP, whereas there is no interaction between p23 and CP. Hsc70-2 expression is induced slightly during BBSV infection of Nicotiana benthamiana, and overexpression of Hsc70-2 promotes BBSV accumulation, while knockdown of Hsc70-2 in N. benthamiana leads to drastic reduction of BBSV accumulation. Infection experiments revealed that CP negatively regulates BBSV replication, which can be mitigated by overexpression of Hsc70-2. Further experiments indicate that CP impairs the interaction between Hsc70-2 and p23 in a dose-dependent manner. Altogether, we provide evidence that besides specific functions of Hsp70 family proteins in certain aspects of viral infection, they can serve as a mediator for the orchestration of virus infection by interacting with different viral components. Our results provide new insight into the role of Hsp70 family proteins in virus infection.

Co-infection of none-coding satellite RNAs (sat-RNAs) usually inhibits replication and attenuates disease symptoms of helper viruses. However, we find that the sat-RNA of Beet black scorch virus (BBSV) and low temperature (18°C) additively enhance the systemic infection of BBSV in Nicotiana benthamiana. Northern blotting hybridization revealed a relatively reduced accumulation of BBSV-derived small interfering RNAs (siRNAs) in presence of sat-RNA as compared to that of BBSV alone. Cloning and sequencing of total small RNAs showed that more than 50% of the total small RNAs sequenced from BBSV-infected plants were BBSV-siRNAs, whereas the abundance of sat-siRNAs were higher than BBSV-siRNAs in the sat-RNA co-infected plants, indicating that the sat-RNA occupies most of the silencing components and possibly relieves the RNA silencing-mediated defense against BBSV. Interestingly, the 5′ termini of siRNAs derived from BBSV and sat-RNA were dominated by Uridines (U) and Adenines (A), respectively. Besides, the infection of BBSV alone and with sat-RNA induce down-regulation of miR168 and miR403, respectively, which leads to high accumulation of their targets, Argonaute 1 (AGO1) and AGO2. Our work reveals the profiles of siRNAs of BBSV and sat-RNA and provides an additional clue to investigate the complicated interaction between the helper virus and sat-RNA.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

Cervical spondylotic myelopathy (CSM) refers to a chronic injury of the cervical cord caused by cervical intervertebral disc degeneration. Endoplasmic reticulum (ER) homeostasis is essential to counteract neuronal apoptosis. ER stress, an integral part of ER homeostasis, was observed in a rat model of chronic cervical cord compression in our previous study. However, the correlation between ER homeostasis and CSM remains unknown. The antioxidant melatonin is known to exert therapeutic effects in acute spinal cord injury, but the specific effects and their potential mechanisms in the pathological processes of CSM require further exploration. The present study hypothesized that ER homeostasis is essential for neuronal apoptosis in the CSM and that melatonin maintains this homeostasis. The results showed that ER stress led to neuronal apoptosis in rats with chronic cervical cord compression. Conversely, melatonin attenuates protein kinase R-like ER kinase-eukaryotic initiation factor 2α-C/EBP-homologous protein, inositol-requiring enzyme 1, and transcription factor 6 signaling pathways to release ER stress and prevents Bax translocation to the mitochondrion, thereby promoting motor recovery and protecting neurons in vivo. It also rescued primary rat cortical neurons from ER stress-induced glutamate toxicity in vitro. Moreover, melatonin remodels the ER morphology and restores homeostasis via ER-phagy in injured neurons. FAM134B, CCPG1, RTN3, and Sec. 62 are four known ER-phagy receptors. In this study, Sec. 62 was identified as a key melatonin factor in promoting ER-phagy and restoring ER homeostasis in damaged neurons in vivo and in vitro. In conclusion, melatonin suppresses neuronal apoptosis by reducing ER stress and promoting ER-phagy to restore ER morphology and homeostasis. The current results suggested that melatonin is a promising treatment for CSM owing to its restorative effect on ER homeostasis; however, well-designed randomized controlled trials must be carried out to further investigate its clinical effects.

The Membrane-Bound Transcription Factor CREB3L1 Is Activated in Response to Virus Infection to Inhibit Proliferation of Virus-Infected Cells

The efficient functioning of the endoplasmic reticulum (ER) is essential for most cellular activities and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response (UPR) to restore normal ER function. If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, as ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review, we examine the role of the molecules that are activated during the UPR in order to identify the molecular switch from the adaptive phase to apoptosis. We discuss how the activation of these molecules leads to the commitment of death and the mechanisms that are responsible for the final demise of the cell.

The endoplasmic reticulum (ER) is a multifunctional organelle that constitutes the entry into the secretory pathway. The ER contributes to the maintenance of cellular calcium homeostasis, lipid synthesis and productive secretory, and transmembrane protein folding. Physiological, chemical, and pathological factors that compromise ER homeostasis lead to endoplasmic reticulum stress (ER stress). To cope with this situation, cells activate an adaptive signaling pathway termed the unfolded protein response (UPR) that aims at restoring ER homeostasis. The UPR is transduced through post-translational, translational, post-transcriptional, and transcriptional mechanisms initiated by three ER-resident sensors, inositol-requiring protein 1α, activating transcription factor 6α, and PRKR-like endoplasmic reticulum kinase. Determining the in and out of ER homeostasis control and UPR activation still represents a challenge for the community. Hence, standardized criteria and methodologies need to be proposed for monitoring ER homeostasis and ER stress in different model systems. Here, we summarize the pathways that are activated during ER stress and provide approaches aimed at assess ER homeostasis and stress invitro and invivo mammalian systems that can be used by researchers to plan and interpret experiments. We recommend the use of multiple assays to verify ER stress because no individual assay is guaranteed to be the most appropriate one.

Effects of ER stress on unfolded protein responses, cell survival, and viral replication in primary effusion lymphoma

SKN-1/Nrf, A New Unfolded Protein Response Factor?