Biology subjects

Burrone, O. R.

Publications and source records attributed to Burrone, O. R..

8 recordsLinked to original sources

bioRxiv2026

ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

Misfolded proteins are eliminated from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD), a process requiring their exit from the ER and delivery to cytosolic proteasomes via the key retrotranslocation step. Conventional assays detect fully extracted substrates, overlooking early initiation events. Here, we use an Escherichia coli-derived Biotin Acceptor Peptide (BAP)-biotin ligase BirA system to detect ERAD substrates upon cytosolic exposure. Using the model ERAD substrate H2a, we show that cytosolic BirA selectively biotinylates luminal BAP tags during early cytosolic exposure, enabling detection of substrates engaging the mammalian retrotranslocation machinery independently of subsequent extraction. Combined with high-resolution expansion fluorescence microscopy, this approach reveals active initiation of ERAD at the specialized ER-derived quality control compartment (ERQC). Substrates become transiently exposed to the cytosol while remaining membrane-associated, followed by tight coupling of polyubiquitination, transmembrane segment extraction, deglycosylation and degradation. Functional perturbations indicate that mannose trimming enables initiation, whereas p97 and HRD1 support later extraction and ubiquitination.

cell biology↗

bioRxiv2022

Recruitment of TRiC chaperonin in rotavirus viroplasms directly associates with virus replication.

Rotavirus replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which is the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. To investigate the relationship between rotavirus proteins and host components of the viroplasms, we performed a pull-down assay of lysates from rotavirus-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the TRiC complex, a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our validated results show that TRiC is recruited in viroplasms and specifically surrounds newly formed double-layered particles (DLPs). Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Interestingly, TRiC-inhibited RV-infected cells lacked triple-layered particles (TLPs) but harbored empty DLPs. Through sequence-specific direct RNA nanopore sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly (-)ssRNA. Moreover, TRiC associates and regulates the folding of VP2, a cofactor allowing dsRNA synthesis. This study provides in-cell culture evidence of the regulatory mechanism by which dsRNA genome segment replication is controlled and coordinated in the rotavirus viroplasms. ImportanceThe replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the eleven double-stranded RNA genome segments are synthesized and packaged individually into the newly generated virus particles. In this study, we show for the first time that the TRiC complex, a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral (-)ssRNA. Specifically, TRiC interacts and assists in folding VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited in viroplasm to assist replication.

microbiology↗

bioRxiv2022

Rotavirus spike protein VP4 mediates viroplasm assembly by association to actin filaments

The formation of viroplasms is a well-conserved step in the rotavirus (RV) life cycle. In these structures, both virus genome replication and progeny assembly take place. A stabilized microtubule cytoskeleton and lipid droplets are required for the viroplasm formation, which involves several virus proteins. The viral spike protein VP4 has not previously been shown to have a direct role in viroplasm formation. However, it is involved with virus-cell attachment, endocytic internalization, and virion morphogenesis. Moreover, VP4 interacts with actin cytoskeleton components, mainly in processes involving virus entrance and egress, and thereby may have an indirect role in viroplasm formation. In this study, we used reverse genetics to construct a recombinant RV, rRV/VP4-BAP, which contains a biotin acceptor peptide (BAP) in the K145-G150 loop of the VP4 lectin domain, permitting live monitoring. The recombinant virus was replication competent but showed a reduced fitness. We demonstrate that rRV/VP4-BAP infection, as opposed to rRV/wt infection, did not lead to a reorganized actin cytoskeleton as viroplasms formed were insensitive to drugs that depolymerize actin and inhibit myosin. Moreover, wt VP4, but not VP4-BAP, appeared to associate with actin filaments. Similarly, VP4 in co-expression with NSP5 and NSP2 induced a significant increase in the number of viroplasm-like structures. Interestingly, a small peptide mimicking loop K145-G150 rescued the phenotype of rRV/VP4-BAP by increasing its ability to form viroplasms and hence, improve virus progeny formation. Collectively, these results provide a direct link between VP4 and the actin cytoskeleton to catalyze viroplasm assembly. IMPORTANCEThe spike protein VP4 participates in diverse steps of the rotavirus (RV) life cycle, including virus-cell attachment, internalization, modulation of endocytosis, virion morphogenesis, and virus egress. Using reverse genetics, we constructed for the first time a recombinant RV, rRV/VP4-BAP, harboring a heterologous peptide in the lectin domain (loop K145-G150) of VP4. The rRV/VP4-BAP was replication-competent but with reduced fitness due to a defect in the ability to reorganize the actin cytoskeleton, which affected the efficiency of viroplasm assembly. This defect was rescued by adding a permeable small-peptide mimicking the wild-type VP4 loop K145-G150. In addition to revealing a new role of VP4, our findings suggest that rRV harboring an engineered VP4 could be used as a new dual vaccination platform providing immunity against RV and additional heterologous antigens.

microbiology↗

bioRxiv2021

A recombinant rotavirus harboring a spike protein with a heterologous peptide reveals a novel role of VP4 in viroplasm stability

The rotavirus (RV) VP4 spike protrudes as a trimeric structure from the five-fold axes of the virion triple-layer. Infectious RV particles need to be proteolytically cleaved in VP4 into two subunits, VP8* and VP5*, constituting both the distal part and central body of the virus spike. Modification of VP4 has been challenging as it is involved in biological process including the interaction with sialic acid and integrins, cell tropism and hemagglutinin activity. Here, we engineered a loop at position K145-G150 in the lectin domain of the VP8* subunit to harbor a small biotin acceptor peptide (BAP) tag and rescued viable viral particles using RV reverse genetics system. This rRV/VP4-BAP internalizes, replicates, and generates virus progeny, demonstrating that the VP4 spike of RV particles can be genetically manipulated by the incorporation of at least 15 exogenous amino acids. Although, VP4-BAP had a similar distribution as VP4 in infected cells by localizing in the cytoskeleton and surrounding viroplasms. However, compared to wild-type RV, rRV/VP4-BAP featured a reduced replication fitness and impaired viroplasm stability. Upon treatment of viroplasms with 1,6-hexanediol, a drug disrupting liquid-liquid phase-separated condensates, the kinetic of rRV/VP4-BAP viroplasm recovery was delayed, and their size and numbers reduced when compared to viroplasms of wild type RV. Moreover, siRNA silencing of VP4 expression in RV strain SA11 showed similar recovery patterns as rRV/VP4-BAP, revealing a novel function of VP4 in viroplasm stability. IMPORTANCEThe rotavirus (RV) spike protein, VP4, has a relevant role in several steps involving virion internalization. The strategic position of VP4 in the virion resulted in a challenge for the addition of an exogenous peptide producing infectious particles. The identification of a specific loop in position K145-G150 in the VP8* subunit of VP4 allowed the rescue by RV reverse genetics of a recombinant RV harboring VP4 containing a 15 amino acids tag. This study demonstrates this recombinant virus has similar replication properties as a wild-type virus. Moreover, we also discovered that VP4 is necessary for the assembly and stabilization of the cytosolic replication compartments, the viroplasms, demonstrating a novel role of this protein in the RV life cycle.

microbiology↗

bioRxiv2020

Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

RNA viruses induce formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation. We demonstrate that rotavirus proteins NSP5 and NSP2 undergo phase separation in vitro and form RNA-rich condensates in vivo that can be reversibly dissolved by aliphatic diols. During infection, these RNA-protein condensates became less dynamic and impervious to aliphatic diols, indicating a transition from a liquid to solid state. Some aspects of assembly of rotavirus replication factories mirror the formation of cytoplasmic ribonucleoprotein granules, while the selective enrichment of viral transcripts appears to be a unique feature of these condensates. Such complex RNA-protein condensates that underlie replication of RNA viruses represent an attractive target for developing novel therapeutic approaches.

biophysics↗

bioRxiv2020

CRISPR-Csy4-mediated editing of rotavirus double-stranded RNA genome

CRISPR-nucleases have been widely applied for editing cellular and viral genomes, but nuclease-mediated genome editing of double-stranded RNA (dsRNA) viruses has not yet been reported. Here, by engineering CRISPR-Csy4 nuclease to localise to rotavirus viral factories, we achieved the first nuclease-mediated genome editing of rotavirus, an important human and livestock pathogen with a multi-segmented dsRNA genome. Rotavirus replication intermediates cleaved by Csy4 were repaired through the formation of defined deletions in the targeted genome segments in a single replication cycle. Using CRISPR-Csy4-mediated editing of rotavirus genome, we labelled for the first time the products of rotavirus secondary transcription made by newly assembled viral particles during rotavirus replication, demonstrating that this step largely contributes to the overall production of viral proteins. We anticipate that the nuclease-mediated cleavage of dsRNA virus genomes will promote a new level of understanding of viral replication and host-pathogen interactions, offering the opportunity to develop new therapeutics.

microbiology↗

bioRxiv2019

Recombinant rotaviruses rescued by reverse genetics reveal the role of NSP5 hyperphosphorylation in the assembly of viral factories

Rotavirus (RV) replicates in round-shaped cytoplasmic viral factories although how they assemble remains unknown.\n\nDuring RV infection, NSP5 undergoes hyperphosphorylation, which is primed by the phosphorylation of a single serine residue. The role of this post-translational modification in the formation of viroplasms and its impact on the virus replication remains obscure. Here we investigated the role of NSP5 during RV infection by taking advantage of a modified fully tractable reverse genetics system. An NSP5 trans-complementing cell line was used to generate and characterise several recombinant rotaviruses (rRVs) with mutations in NSP5. We demonstrate that a rRV lacking NSP5, was completely unable to assemble viroplasms and to replicate, confirming its pivotal role in rotavirus replication.\n\nA number of mutants with impaired NSP5 phosphorylation were generated to further interrogate the function of this post-translational modification in the assembly of replication-competent viroplasms. We showed that the rRV mutant strains exhibit impaired viral replication and the ability to assemble round-shaped viroplasms in MA104 cells. Furthermore, we have investigated the mechanism of NSP5 hyper-phosphorylation during RV infection using NSP5 phosphorylation-negative rRV strains, as well as MA104-derived stable transfectant cell lines expressing either wt NSP5 or selected NSP5 deletion mutants. Our results indicate that NSP5 hyper-phosphorylation is a crucial step for the assembly of round-shaped viroplasms, highlighting the key role of the C-terminal tail of NSP5 in the formation of replication-competent viral factories. Such a complex NSP5 phosphorylation cascade may serve as a paradigm for the assembly of functional viral factories in other RNA viruses.\n\nIMPORTANCERotavirus (RV) double-stranded RNA genome is replicated and packaged into virus progeny in cytoplasmic structures termed viroplasms. The non-structural protein NSP5, which undergoes a complex hyperphosphorylation process during RV infection, is required for the formation of these virus-induced organelles. However, its roles in viroplasm formation and RV replication have never been directly assessed due to the lack of a fully tractable reverse genetics (RG) system for rotaviruses. Here we show a novel application of a recently developed RG system by establishing a stable trans-complementing NSP5-producing cell line required to rescue rotaviruses with mutations in NSP5. This approach allowed us to provide the first direct evidence of the pivotal role of this protein during RV replication. Furthermore, using recombinant RV mutants we shed light on the molecular mechanism of NSP5 hyperphosphorylation during infection and its involvement in the assembly and maturation of replication-competent viroplasms.

microbiology↗

bioRxiv2019

The guanine nucleotide exchange factor GBF1 participates in rotavirus replication

Cellular and viral factors participate in the replication cycle of rotavirus. We report that the guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference, or inhibiting its activity by treatment with Brefeldin A (BFA) or Golgicide A (GCA) significantly reduce the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly since in the presence of either BFA or GCA the assembly of infectious mature triple-layered virions was significantly prevented and only doubled layered-particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface VP7, a step required for its incorporation into virus particles. Although a post-translational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking-down its expression reduces VP7 trimerizarion. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when co-transfected with NSP4.\n\nIMPORTANCERotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in children and young animals worldwide. Despite the significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine nucleotide exchange factor GBF1, relevant for the COPI/Arf1-mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4, and the assembly of the virus surface proteins VP7 and VP4.

microbiology↗