Host ESCRT machinery orchestrates the assembly of tomato spotted wilt virus ribonucleoproteins

Negative-strand RNA (NSR) viruses contain not only medical important animal pathogens but also agricultural important plant pathogens. These NSR viruses cause deadliest diseases in humans, livestocks and agronomic crops and pose great threat to the health of human being and food security worldwide. The virus reverse genetics system is an important molecular genetic technology to study viral gene functions in virus infection cycle, disease pathology and virus-host interactions. For animal-infecting NSR viruses, the reverse genetics system has been established for more than twenty years. In contrast to the well-established reverse genetics systems for animal-infecting NSR viruses, establishment of such system for the plant-infecting NSR viruses turned out to be an extremely challenge work. Many research groups throughout the world have tried to use the similar strategies used for animal NSR viruses to establish reverse genetics systems for plant-infecting NSR viruses in the last twenty years but none of groups have succeeded. The absence of the reverse genetics system posed a major obstacle to molecular genetic manipulation and subsequent investigation of gene function and disease pathology for plant-infecting NSR viruses. Recently, big breakthroughs have been made in the establishment of the reverse genetics systems for plant NSR viruses by scientists from China and America, respectively. The systems were recently established for both nonsegmented and segmented plant NSR viruses. The reverse genetics system for a nonsegmented plant NSR virus was firstly developed for sonchus yellow net virus (SYNV), a non segmented nucleorhabdovirus NSR. Using the similar strategies as SYNV, barley yellow striate mosaic virus (BYSMV), a nonsegmented cytorhabdovirus NSR, was then succeeded in the establishment of reverse genetics system. The first reverse genetics system for a segmented plant NSR virus was developed using tomato spotted wilt virus (TSWV), a tospovirus with tripartite negative-stranded/ambisense RNA genomes. The reverse genetics system was also recently developed for rose rosette virus (RRV), an emaravirus with seven negative-sense mono-cistronic RNA genomes. The establishment of the reverse genetics system for plant NSR viruses typically involves two steps. The first step requires the construction of a mini-genome replication system. The viral genomic or antigenomic RNA was flanked by hammerhead and ribozyme to generate exact viral 5 ¢ and 3 ¢ end, and driven by 35S promoter. The mini-replicon construct was co-expressed with the constructs expressing nucleocapsid (N) protein, phosphoprotein (P; essential for rhabdovirus) and viral RNA-dependent RNA polymerase (RdRp). For the segmented plant NSR viruses such as TSWV, codon optimization and removal of intron-splicing sites of RdRp sequence are critical for the expression of a functional RdRp from 35S-driven constructs in planta . The second step involves the rescue of virus entirely from full-length infectious cDNA clones in plant. The recombinant virus can carry green fluorescence protein (GFP) gene reporter and infect Nicotiana benthamiana plant systemically. The establishment of reverse genetics for different plant-infecting NSR viruses now provided powerful systems to investigate all aspects of virus infection cycle and disease pathology. The recent breakthroughs of reverse genetics systems have opened a new era for the investigation of plant-infecting NSR viruses in the future.

Endosomal sorting complexes required for transport (ESCRT) play key roles in protein sorting between membrane-bounded compartments of eukaryotic cells. Homologs of many ESCRT components are identifiable in various groups of archaea, especially in Asgardarchaeota, the archaeal phylum that is currently considered to include the closest relatives of eukaryotes, but not in bacteria. We performed a comprehensive search for ESCRT protein homologs in archaea and reconstructed ESCRT evolution using the phylogenetic tree of Vps4 ATPase (ESCRT IV) as a scaffold and using sensitive protein sequence analysis and comparison of structural models to identify previously unknown ESCRT proteins. Several distinct groups of ESCRT systems in archaea outside of Asgard were identified, including proteins structurally similar to ESCRT-I and ESCRT-II, and several other domains involved in protein sorting in eukaryotes, suggesting an early origin of these components. Additionally, distant homologs of CdvA proteins were identified in Thermoproteales which are likely components of the uncharacterized cell division system in these archaea. We propose an evolutionary scenario for the origin of eukaryotic and Asgard ESCRT complexes from ancestral building blocks, namely, the Vps4 ATPase, ESCRT-III components, wH (winged helix-turn-helix fold) and possibly also coiled-coil, and Vps28-like domains. The last archaeal common ancestor likely encompassed a complex ESCRT system that was involved in protein sorting. Subsequent evolution involved either simplification, as in the TACK superphylum, where ESCRT was co-opted for cell division, or complexification as in Asgardarchaeota. In Asgardarchaeota, the connection between ESCRT and the ubiquitin system that was previously considered a eukaryotic signature was already established.IMPORTANCEAll eukaryotic cells possess complex intracellular membrane organization. Endosomal sorting complexes required for transport (ESCRT) play a central role in membrane remodeling which is essential for cellular functionality in eukaryotes. Recently, it has been shown that Asgard archaea, the archaeal phylum that includes the closest known relatives of eukaryotes, encode homologs of many components of the ESCRT systems. We employed protein sequence and structure comparisons to reconstruct the evolution of ESCRT systems in archaea and identified several previously unknown homologs of ESCRT subunits, some of which can be predicted to participate in cell division. The results of this reconstruction indicate that the last archaeal common ancestor already encoded a complex ESCRT system that was involved in protein sorting. In Asgard archaea, ESCRT systems evolved toward greater complexity, and in particular, the connection between ESCRT and the ubiquitin system that was previously considered a eukaryotic signature was established.

The nucleocapsid (N) protein of tomato spotted wilt virus (TSWV) plays key roles in assembling genomic RNA into ribonucleoprotein (RNP), which serves as a template for both viral gene transcription and genome replication. However, little is known about the molecular mechanism of how TSWV N interacts with genomic RNA. In this study, we demonstrated that TSWV N protein forms a range of higher ordered oligomers. Analysis of the RNA binding behavior of N protein revealed that no specific oligomer binds to RNA preferentially, instead each type of N oligomer is able to bind RNA. To better characterize the structure and function of N protein interacting with RNA, we constructed homology models of TSWV N and N-RNA complexes. Based on these homology models, we demonstrated that the positively charged and polar amino acids in its predicted surface cleft of TSWV N are critical for RNA binding. Moreover, by N-RNA homology modeling, we found that the RNA component is deeply embedded in the predicted protein cleft; consistently, TSWV N-RNA complexes are relatively resistant to digestion by RNase. Collectively, using homology modeling, we determined the RNA binding sites on N and found a new protective feature for N protein. Our findings also provide novel insights into the molecular details of the interaction of TSWV N with RNA components.

HighlightsWhat are the main findings?1.In open mitosis, ESCRTs type III are recruited to the anaphase/telophase chromatin core by BAF1 and LEM2; CHMP7 and IST1 bring in VPS4 and Spastin to couple nuclear envelope reformation with spindle microtubule disassembly. Canonical ESCRTs type I and II have not yet been identified as part of this nuclear envelope ESCRT module.2.Beyond mitosis, the same axis operates during interphase nuclear envelope rupture and repair. ESCRT factors also contribute to nuclear pore complex quality control.What are the implication of the main findings?3.Correct ESCRTs type III assembly in mitosis is essential for nuclear reformation and genome integrity. ESCRT dysfunction causes DNA damage, spindle clearance defects, and chromosomal instability, mechanistically linking ESCRTs to disease including cancer and neurodegeneration. 4.ESCRT-dependent nuclear envelope surveillance pathways are emerging as therapeutic entries. Moreover, candidate new factors, such as the ESCRT type I like factor AKTIP, suggest additional druggable nodes. The Endosomal Sorting Complex Required for Transport (ESCRT) is a highly conserved machinery best known for its role in endosomal trafficking and membrane remodeling. Increasing evidence shows that ESCRT components are also key regulators during open mitosis, where precise membrane dynamics are essential for nuclear envelope reformation and spindle disassembly. In this review, we explore how the ESCRT machinery coordinates mitotic processes under physiological conditions and how their dysregulation contributes to genomic instability, altered cell division, and disease. We highlight recent findings on the spatiotemporal control of ESCRT recruitment at mitotic membranes, the interplay with chromatin and nuclear envelope-associated factors, and the consequences of defective ESCRT function in pathological contexts such as cancer and neurodegeneration. By connecting molecular mechanisms with cellular outcomes, we provide an integrated view of how the ESCRT machinery acts as critical guardian of mitotic fidelity and offer some routes for the identification of potential therapeutic targets in human disease.

BackgroundDuring EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain.ResultsMutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation.ConclusionsAs an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation.

β-Coronaviruses assemble at the endoplasmic reticulum-to-Golgi compartment and exit cells through the lysosomal trafficking pathway. However, the molecular mechanisms of virion assembly and egress are largely unknown. Here, we report that β-coronaviruses recruit endosomal sorting complexes required for transport (ESCRTs) to facilitate virion assembly and egress. The viral proteins N and M interacted with ESCRT components TSG101 and VPS28, respectively. Electron microscopy analysis revealed that virion assembly was disrupted by TSG101 knockdown at an early stage and by VPS28 knockdown at a late stage. Knockdown of ESCRT components MVB12A and CHMP6 did not affect virion assembly but inhibited virion egress. Downregulation of these ESCRT factors or VPS4A, or treatment of cells with the TSG101 antagonist tenatoprazole significantly inhibited the production of the virion-like particles of β-coronaviruses and the replication of human coronavirus OC43. These findings indicate that β-coronaviruses exploit ESCRT for virion assembly and egress and suggest that the interaction interface between ESCRT and the viruses could be a target for the development of broad-spectrum anti-coronavirus therapeutics.IMPORTANCEβ-Coronaviruses have caused disastrous pandemics and may cause serious pandemics in the future. Virion assembly and egress are critical steps in the life cycle of coronaviruses. However, despite extensive studies in the past few years, the molecular mechanisms for virion assembly and egress are still largely unknown. Here we show that β-coronaviruses recruit ESCRT components TSG101 and VPS28 for virion assembly and that ESCRT components MVB12A and CHMP6 are required for virion egress. Treatment of cells with the TSG101 antagonist inhibited the assembly of multiple β-coronaviruses. These findings indicate that ESCRT participates in β-coronavirus assembly and egress and might be a potential target for the development of broad-spectrum anti-coronavirus therapeutics.

Crystallographic and Functional Analysis of the ESCRT-I /HIV-1 Gag PTAP Interaction

The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley’s L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.

BackgroundComparing a parasitic lineage to its free-living relatives is a powerful way to understand how that evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia’s pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis, and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species.ResultsWe used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36 and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the endoplasmic reticulum and, for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago.ConclusionsOur findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, associates to multiple cellular locations, and presents changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.

The endosomal sorting complex required for transport (ESCRT) is a protein machine mediating membrane scission. In intraluminal vesicle (ILV) formation, ESCRT-0 targets cargoes and recruits ESCRT-I/-II to create membrane invagination, whereas ESCRT-III coordinates with the AAA ATPase Vps4 and its cofactor Vta1 to catalyze the membrane fission. Recently, ESCRT-I/-III and Vps4 were found to be involved in the entry of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, the necessity of other ESCRT components and the interplay of viral proteins and ESCRTs in regulating the virus entry remain elusive. Here, we identified ESCRT-0 (Hse1 and Vps27), ESCRT-II (Vps22, Vps25, and Vps36), and Vta1 of Spodoptera frugiperda. RNAi depletion of Vta1 but not the components of ESCRT-0 or ESCRT-II in Sf9 cells significantly reduced budded virus (BV) production. Quantitative PCR together with confocal microscopy analyses indicated that Vta1 was required for internalization and endosomal trafficking of BV. In the late phase of infection, although Vps4 and Vta1 were both distributed to the nucleus and at the plasma membrane, depletion of Vta1 did not affect BV release. Further analysis revealed that 7 of 14 BV envelope proteins (Ac75, Ac93, E25, F-like, P33, P48, and vUbiquitin) interacted with Vps4 and Vta1. Intriguingly, Ac93 adopted a similar mode as ESCRT-III proteins to interact with the microtubule-interacting and transport (MIT) domains of Vps4 and Vta1 via its C-terminal MIT-interacting motifs (MIM1), and the interactions were necessary for BV internalization. Together, our studies highlight the coordination of Vps4-Vta1 and Ac93, and probably other BV envelope proteins, in facilitating entry of AcMNPV.IMPORTANCEThe endosomal sorting complex required for transport (ESCRT) system is involved in the entry of diverse DNA and RNA viruses. However, the interplay of viral proteins and ESCRTs in promoting virus endocytosis remains largely unknown. Here, we found that the ESCRT early acting factors ESCRT-0/-II were not necessary for infectious budded virus (BV) production of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In contrast, the Vps4 cofactor Vta1 was required for entry but not egress of BV. Several core or essential BV envelope proteins were identified to interact with Vps4 and Vta1. Among them, Ac93 plays a central role in connecting other viral proteins and mimics ESCRT-III proteins to interact with Vps4-Vta1, facilitating entry of BV virions. These studies provide evidence for the coordination of viral proteins and ESCRTs in regulating entry of large enveloped DNA viruses.

Virus interactions between Tomato spotted wilt virus (TSWV) and Potato virus X (PVX) containing the nucleocapsid protein (N) gene sequences were examined to evaluate the capacity of the N gene sequences from TSWV to promote RNA-mediated cross-protection. Plants simultaneously inoculated with TSWV and PVX containing the 3′ 96 bp of the N gene were highly resistant to TSWV infection, whereas no such resistance was observed in plants inoculated with TSWV and PVX containing the 5′ 96 bp. These results suggest that the 3′ portion of the N gene has a higher capacity for promoting RNA-mediated cross-protection of TSWV.

Negative-sense single-stranded RNA virus (NSRV) is featured by their ribonucleoprotein (RNP) complex composed by viral polymerase and genomic RNA enwrapped by nucleocapsid protein (NP). The RNP is packaged in virions and plays a central role throughout virus lifecycle. In the past decade, structural biology presents molecular insights into NPs encoded by most representative NSRVs, helping to understand the mechanism of RNP formation. Interestingly, works initiated from structural biology also reveal unexpected biological functions of virus NP beyond a structural protein. All these further the knowledge of virus NP and provide great potential for the discovery of antiviral agents to target virus RNP formation. In this chapter, we will summarize the structures and functions of viral NPs, as well as the attempt of NP-targeted antiviral development.

The endosomal compartment performs extensive sorting functions in most eukaryotes, some of which are accomplished with the help of the multivesicular body (MVB) sorting pathway. This pathway depends on the sequential action of complexes, termed the endosomal sorting complex required for transport (ESCRT). After successful sorting, the crucial step of recycling of the ESCRT complex components requires the activation of the AAA ATPase Vps4, and Did2/Vps46 plays an important role in this activation event. The endolysosomal system of the protozoan parasite Giardia lamblia appears to lack complexity, for instead of having distinct early endosomes, late endosomes and lysosomes, there are only peripheral vesicles (PVs) that are located close to the cell periphery. Additionally, comparative genomics studies predict the presence of only a subset of the ESCRT components in G. lamblia. Thus, it is possible that the MVB pathway is not functional in G. lamblia. To address this issue, the present study focused on the two putative orthologues of Did2/Vps46 of G. lamblia as their function is likely to be pivotal for a functional MVB sorting pathway. In spite of considerable sequence divergence, compared to other eukaryotic orthologues, the proteins encoded by both these genes have the ability to function as Did2/Vps46 in the context of the yeast ESCRT pathway. Furthermore, they also localized to the cellular periphery, where PVs are also located. Thus, this report is the first to provide experimental evidence indicating the presence of a functional ESCRT component in G. lamblia by characterizing the putative Did2/Vps46 orthologues.

Endosomal “sort” of signaling control: The role of ESCRT machinery in regulation of receptor-mediated signaling pathways

HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation. Recruitment of ESCRT by late-domain motifs in the viral Gag polyprotein and a role of ESCRT in HIV release are firmly established, but the order of events, their kinetics and the mechanism of action of individual ESCRT components in HIV budding are unclear at present. Using live-cell imaging, we show late-domain-dependent recruitment of VPS4A to nascent HIV particles at the host cell plasma membrane. Recruitment of VPS4A was transient, resulting in a single or a few bursts of at least two to five VPS4 dodecamers assembling at HIV budding sites. Bursts lasted for ∼35 s and appeared with variable delay before particle release. These results indicate that VPS4A has a direct role in membrane scission leading to HIV-1 release.