RESUMO
RNA viruses induce the 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 of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches.
Assuntos
Grânulos de Ribonucleoproteínas Citoplasmáticas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Rotavirus/genética , Proteínas não Estruturais Virais/metabolismo , Animais , Bovinos , Linhagem Celular , Grânulos de Ribonucleoproteínas Citoplasmáticas/efeitos dos fármacos , Grânulos de Ribonucleoproteínas Citoplasmáticas/ultraestrutura , Grânulos de Ribonucleoproteínas Citoplasmáticas/virologia , Regulação Viral da Expressão Gênica , Genes Reporter , Glicóis/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Haplorrinos , Interações Hospedeiro-Patógeno/genética , Humanos , Concentração Osmolar , Fosforilação , Propilenoglicol/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Rotavirus/efeitos dos fármacos , Rotavirus/crescimento & desenvolvimento , Rotavirus/ultraestrutura , Transdução de Sinais , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Many viruses sequester the materials needed for their replication into discrete subcellular factories. For rotaviruses (RVs), these factories are called viroplasms, and they are formed in the host cell cytosol via the process of liquid-liquid phase separation (LLPS). The nonstructural protein 2 (NSP2) and its binding partner, nonstructural protein 5 (NSP5), are critical for viroplasm biogenesis. Yet it is not fully understood how NSP2 and NSP5 cooperate to form factories. The C-terminal region (CTR) of NSP2 (residues 291 to 317) is flexible, allowing it to participate in domain-swapping interactions that promote interoctamer interactions and, presumably, viroplasm formation. Molecular dynamics simulations showed that a lysine-to-glutamic acid change at position 294 (K294E) reduces NSP2 CTR flexibility in silico. To test the impact of reduced NSP2 CTR flexibility during infection, we engineered a mutant RV bearing this change (rRV-NSP2K294E). Single-cycle growth assays revealed a >1.2-log reduction in endpoint titers for rRV-NSP2K294E versus the wild-type control (rRV-WT). Using immunofluorescence assays, we found that rRV-NSP2K294E formed smaller, more numerous viroplasms than rRV-WT. Live-cell imaging experiments confirmed these results and revealed that rRV-NSP2K294E factories had delayed fusion kinetics. Moreover, NSP2K294E and several other CTR mutants formed fewer viroplasm-like structures in NSP5 coexpressing cells than did control NSP2WT. Finally, NSP2K294E exhibited defects in its capacity to induce LLPS droplet formation in vitro when incubated alongside NSP5. These results underscore the importance of NSP2 CTR flexibility in supporting the biogenesis of RV factories. IMPORTANCE Viruses often condense the materials needed for their replication into discrete intracellular factories. For rotaviruses, agents of severe gastroenteritis in children, factory formation is mediated in part by an octameric protein called NSP2. A flexible C-terminal region of NSP2 has been proposed to link several NSP2 octamers together, a feature that might be important for factory formation. Here, we created a change in NSP2 that reduced C-terminal flexibility and analyzed the impact on rotavirus factories. We found that the change caused the formation of smaller and more numerous factories that could not readily fuse together like those of the wild-type virus. The altered NSP2 protein also had a reduced capacity to form factory-like condensates in a test tube. Together, these results add to our growing understanding of how NSP2 supports rotavirus factory formation-a key step of viral replication.
Assuntos
Rotavirus , Proteínas não Estruturais Virais , Replicação Viral , Fosforilação , Rotavirus/química , Rotavirus/fisiologia , Proteínas não Estruturais Virais/químicaRESUMO
Due to genome segmentation, rotaviruses must co-package eleven distinct genomic RNAs. The packaging is mediated by virus-encoded RNA chaperones, such as the rotavirus NSP2 protein. While the activities of distinct RNA chaperones are well studied on smaller RNAs, little is known about their global effect on the entire viral transcriptome. Here, we used Selective 2'-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to examine the secondary structure of the rotavirus transcriptome in the presence of increasing amounts of NSP2. SHAPE-MaP data reveals that despite the well-documented helix-unwinding activity of NSP2 in vitro, its incubation with cognate rotavirus transcripts does not induce a significant change in the SHAPE reactivities. However, a quantitative analysis of mutation rates measured by mutational profiling reveals a global 5-fold rate increase in the presence of NSP2. We demonstrate that the normalization procedure used in deriving SHAPE reactivities from mutation rates can mask an important global effect of an RNA chaperone. Analysis of the mutation rates reveals a larger effect on stems rather than loops. Together, these data provide the first experimentally derived secondary structure model of the rotavirus transcriptome and reveal that NSP2 acts by globally increasing RNA backbone flexibility in a concentration-dependent manner.
Assuntos
Rotavirus , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estrutura Secundária de Proteína , RNA Viral/genética , RNA Viral/metabolismo , Rotavirus/genética , Transcriptoma/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.
Assuntos
Genoma Viral/genética , Dobramento de RNA/genética , RNA Viral/genética , Rotavirus/crescimento & desenvolvimento , Empacotamento do Genoma Viral/genética , Proteínas não Estruturais Virais/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Rotavirus/genética , Rotavirus/metabolismoRESUMO
Species A rotavirus (RVA) vaccines based on live attenuated viruses are used worldwide in humans. The recent establishment of a reverse genetics system for rotoviruses (RVs) has opened the possibility of engineering chimeric viruses expressing heterologous peptides from other viral or microbial species in order to develop polyvalent vaccines. We tested the feasibility of this concept by two approaches. First, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian RV SA11 strain viral protein (VP) 4. Second, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C terminus of nonstructural protein (NSP) 3 of the bovine RV RF strain, with or without an intervening Thosea asigna virus 2A (T2A) peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants, no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RV RF strain. Except for the RBD mutant that demonstrated a rescue defect, all NSP3 mutants delivered endpoint infectivity titers and exhibited replication kinetics comparable to that of the wild-type virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross-reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants, but cross-reactivity with SARS-CoV-2 RBD antibody was only detected for the RBM mutant. The tolerance of large SARS-CoV-2 peptide insertions at the C terminus of NSP3 in the presence of T2A element highlights the potential of this approach for the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCE We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an example. Small SARS-CoV-2 peptide insertions (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titer and replication, demonstrating a limited tolerance for peptide insertions at this site. To test the RV RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids in length. With a T2A-separated 193 amino acid tag on NSP3, there was no significant effect on the viral rescue efficiency, endpoint titer, and replication kinetics. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.
Assuntos
Genética Reversa , Rotavirus , Glicoproteína da Espícula de Coronavírus , Desenvolvimento de Vacinas , Aminoácidos/metabolismo , Animais , Anticorpos Antivirais/metabolismo , COVID-19/virologia , Epitopos/genética , Epitopos/metabolismo , Humanos , Microrganismos Geneticamente Modificados , Rotavirus/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Desenvolvimento de Vacinas/métodosRESUMO
The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid-19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single-fluorophore-containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential off-target binding effects that create background noise. Here, we used click chemistry and alkyne-modified DNA oligonucleotides to prepare multiple-fluorophore-containing probes. We found that these multiple-dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5-10) of probe strands. The new method enabled the inâ situ detection of viral transcripts as early as 4 hours after infection.
Assuntos
Química Click/métodos , Diagnóstico Precoce , Hibridização in Situ Fluorescente/métodos , Sondas de Oligonucleotídeos/química , RNA Mensageiro/análise , RNA Viral/análise , Alcinos/química , Betacoronavirus/genética , COVID-19 , Infecções por Coronavirus/diagnóstico , Humanos , Oligodesoxirribonucleotídeos/química , Pandemias , Pneumonia Viral/diagnóstico , SARS-CoV-2RESUMO
Rotavirus (RV) replicates in round-shaped cytoplasmic viral factories, although how they assemble remains unknown. During RV infection, NSP5 undergoes hyperphosphorylation, which is primed by the phosphorylation of a single serine residue. The role of this posttranslational modification in the formation of viroplasms and its impact on virus replication remain obscure. Here, we investigated the role of NSP5 during RV infection by taking advantage of a modified fully tractable reverse-genetics system. A trans-complementing cell line stably producing NSP5 was used to generate and characterize several recombinant rotaviruses (rRVs) with mutations in NSP5. We demonstrate that an rRV lacking NSP5 was completely unable to assemble viroplasms and to replicate, confirming its pivotal role in rotavirus replication. A number of mutants with impaired NSP5 phosphorylation were generated to further interrogate the function of this posttranslational modification in the assembly of replication-competent viroplasms. We showed that the rRV mutant strains exhibited impaired viral replication and the ability to assemble round-shaped viroplasms in MA104 cells. Furthermore, we investigated the mechanism of NSP5 hyperphosphorylation during RV infection using NSP5 phosphorylation-negative rRV strains, as well as MA104-derived stable transfectant cell lines expressing either wild-type NSP5 or selected NSP5 deletion mutants. Our results indicate that NSP5 hyperphosphorylation 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.IMPORTANCE The rotavirus (RV) double-stranded RNA genome is replicated and packaged into virus progeny in cytoplasmic structures termed viroplasms. The nonstructural 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.
Assuntos
Genética Reversa/métodos , Rotavirus/genética , Rotavirus/fisiologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus/fisiologia , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Citoplasma/virologia , Regulação Viral da Expressão Gênica , Técnicas de Inativação de Genes , Mutação , Organelas , Fosforilação , RNA Viral/isolamento & purificação , Infecções por Rotavirus/virologia , Deleção de Sequência , Transfecção , Proteínas não Estruturais Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.
Assuntos
Chaperonas Moleculares/metabolismo , Orthoreovirus Aviário/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Bases , Genoma Viral/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Conformação de Ácido Nucleico , Orthoreovirus Aviário/genética , Ligação Proteica , Estrutura Secundária de Proteína , RNA Viral/química , RNA Viral/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Reoviruses are important human, animal and plant pathogens having 10-12 segments of double-stranded genomic RNA. The mechanisms controlling the assortment and packaging of genomic segments in these viruses, remain poorly understood. RNA-protein and RNA-RNA interactions between viral genomic segment precursors have been implicated in the process. While non-structural viral RNA-binding proteins, such as avian reovirus σNS, are essential for virus replication, the mechanism by which they assist packaging is unclear. Here we demonstrate that σNS assembles into stable elongated hexamers in vitro, which bind single-stranded nucleic acids with high affinity, but little sequence specificity. Using ensemble and single molecule fluorescence spectroscopy, we show that σNS also binds to a partially double-stranded RNA, resulting in gradual helix unwinding. The hexamer can bind multiple RNA molecules and exhibits strand-annealing activity, thus mediating conversion of metastable, intramolecular stem-loops into more stable heteroduplexes. We demonstrate that the ARV σNS acts as an RNA chaperone facilitating specific RNA-RNA interactions between genomic precursors during segment assortment and packaging.
Assuntos
Orthoreovirus Aviário/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas Virais/metabolismo , Animais , Embrião de Galinha , Genoma Viral , Conformação de Ácido Nucleico , Multimerização Proteica , RNA/química , Proteínas de Ligação a RNA/química , Proteínas Virais/químicaRESUMO
Long RNA molecules are at the core of gene regulation across all kingdoms of life, while also serving as genomes in RNA viruses. Few studies have addressed the basic physical properties of long single-stranded RNAs. Long RNAs with nonrepeating sequences usually adopt highly ramified secondary structures and are better described as branched polymers. To test whether a branched polymer model can estimate the overall sizes of large RNAs, we employed fluorescence correlation spectroscopy to examine the hydrodynamic radii of a broad spectrum of biologically important RNAs, ranging from viral genomes to long noncoding regulatory RNAs. The relative sizes of long RNAs measured at low ionic strength correspond well to those predicted by two theoretical approaches that treat the effective branching associated with secondary structure formation-one employing the Kramers theorem for calculating radii of gyration, and the other featuring the metric of maximum ladder distance. Upon addition of multivalent cations, most RNAs are found to be compacted as compared with their original, low ionic-strength sizes. These results suggest that sizes of long RNA molecules are determined by the branching pattern of their secondary structures. We also experimentally validate the proposed computational approaches for estimating hydrodynamic radii of single-stranded RNAs, which use generic RNA structure prediction tools and thus can be universally applied to a wide range of long RNAs.
Assuntos
Conformação de Ácido Nucleico , RNA/química , Sequência de Bases , Hidrodinâmica , Modelos Moleculares , RNA/genéticaRESUMO
Genome packaging is an essential step in virus replication and a potential drug target. Single-stranded RNA viruses have been thought to encapsidate their genomes by gradual co-assembly with capsid subunits. In contrast, using a single molecule fluorescence assay to monitor RNA conformation and virus assembly in real time, with two viruses from differing structural families, we have discovered that packaging is a two-stage process. Initially, the genomic RNAs undergo rapid and dramatic (approximately 20-30%) collapse of their solution conformations upon addition of cognate coat proteins. The collapse occurs with a substoichiometric ratio of coat protein subunits and is followed by a gradual increase in particle size, consistent with the recruitment of additional subunits to complete a growing capsid. Equivalently sized nonviral RNAs, including high copy potential in vivo competitor mRNAs, do not collapse. They do support particle assembly, however, but yield many aberrant structures in contrast to viral RNAs that make only capsids of the correct size. The collapse is specific to viral RNA fragments, implying that it depends on a series of specific RNA-protein interactions. For bacteriophage MS2, we have shown that collapse is driven by subsequent protein-protein interactions, consistent with the RNA-protein contacts occurring in defined spatial locations. Conformational collapse appears to be a distinct feature of viral RNA that has evolved to facilitate assembly. Aspects of this process mimic those seen in ribosome assembly.
Assuntos
Genoma Viral/fisiologia , Levivirus/fisiologia , RNA Viral/metabolismo , Montagem de Vírus/fisiologia , Levivirus/química , Levivirus/ultraestrutura , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genéticaRESUMO
Rotaviruses (RVs) are 11-segmented, double-stranded (ds) RNA viruses and important causes of acute gastroenteritis in humans and other animal species. Early RV particle assembly is a multi-step process that includes the assortment, packaging and replication of the 11 genome segments in close connection with capsid morphogenesis. This process occurs inside virally induced, cytosolic, membrane-less organelles called viroplasms. While many viral and cellular proteins play roles during early RV assembly, the octameric nonstructural protein 2 (NSP2) has emerged as a master orchestrator of this key stage of the viral replication cycle. NSP2 is critical for viroplasm biogenesis as well as for the selective RNA-RNA interactions that underpin the assortment of 11 viral genome segments. Moreover, NSP2's associated enzymatic activities might serve to maintain nucleotide pools for use during viral genome replication, a process that is concurrent with early particle assembly. The goal of this review article is to summarize the available data about the structures, functions and interactions of RV NSP2 while also drawing attention to important unanswered questions in the field.
Assuntos
Genoma Viral , Rotavirus , Proteínas não Estruturais Virais , Montagem de Vírus , Replicação Viral , Rotavirus/genética , Rotavirus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Humanos , Animais , RNA Viral/genética , RNA Viral/metabolismo , Capsídeo/metabolismo , Proteínas de Ligação a RNARESUMO
Long RNAs often exist as multiple conformers in equilibrium. For the genomes of single-stranded RNA viruses, one of these conformers must include a compacted state allowing the RNA to be confined within the virion. We have used single molecule fluorescence correlation spectroscopy to monitor the conformations of viral genomes and sub-fragments in the absence and presence of coat proteins. Cognate RNA-coat protein interactions in two model viruses cause a rapid collapse in the hydrodynamic radii of their respective RNAs. This is caused by protein binding at multiple sites on the RNA that facilitate additional protein-protein contacts. The collapsed species recruit further coat proteins to complete capsid assembly with great efficiency and fidelity. The specificity in RNA-coat protein interactions seen at single-molecule concentrations reflects the packaging selectivity seen for such viruses in vivo. This contrasts with many in vitro reassembly measurements performed at much higher concentrations. RNA compaction by coat protein or polycation binding are distinct processes, implying that defined RNA-coat protein contacts are required for assembly.
Assuntos
Proteínas do Capsídeo/metabolismo , Levivirus/química , Dobramento de RNA/genética , RNA Viral/química , Espectrometria de Fluorescência/métodos , Vírus Satélite da Necrose do Tabaco/química , Montagem de Vírus/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Cátions/química , Cátions/metabolismo , Genoma Viral , Levivirus/genética , Levivirus/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica/genética , Dobramento de RNA/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Vírus Satélite da Necrose do Tabaco/genética , Vírus Satélite da Necrose do Tabaco/metabolismo , Vírion/genética , Vírion/metabolismo , Montagem de Vírus/fisiologiaRESUMO
Phase separation of viral biopolymers is a key factor in the formation of cytoplasmic viral inclusions, known as sites of virus replication and assembly. This review describes the mechanisms and factors that affect phase separation in viral replication and identifies potential areas for future research. Drawing inspiration from studies on cellular RNA-rich condensates, we compare the hierarchical coassembly of ribosomal RNAs and proteins in the nucleolus to the coordinated coassembly of viral RNAs and proteins taking place within viral factories in viruses containing segmented RNA genomes. We highlight the common characteristics of biomolecular condensates in viral replication and how this new understanding is reshaping our views of virus assembly mechanisms. Such studies have the potential to uncover unexplored antiviral strategies targeting these phase-separated states.
Assuntos
Condensados Biomoleculares , Vírus , Linhagem Celular , Vírus/genética , Replicação Viral , RNARESUMO
Rotaviruses transcribe 11 distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that support segmented genome assembly and replication via a poorly understood mechanism. Here, we analysed the RV transcriptome by combining DNA-barcoded smFISH of rotavirus-infected cells. Rotavirus RNA stoichiometry in viroplasms appears to be distinct from the cytoplasmic transcript distribution, with the largest transcript being the most enriched in viroplasms, suggesting a selective RNA enrichment mechanism. While all 11 types of transcripts accumulate in viroplasms, their stoichiometry significantly varied between individual viroplasms. Accumulation of transcripts requires the presence of 3' untranslated terminal regions and viroplasmic localisation of the viral polymerase VP1, consistent with the observed lack of polyadenylated transcripts in viroplasms. Our observations reveal similarities between viroplasms and other cytoplasmic RNP granules and identify viroplasmic proteins as drivers of viral RNA assembly during viroplasm formation.
Assuntos
Rotavirus , Replicação Viral , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas não Estruturais Virais/genética , Linhagem Celular , Rotavirus/genética , RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Avian (ortho)reovirus (ARV), which belongs to Reoviridae family, is a major domestic fowl pathogen and is the causative agent of viral tenosynovitis and chronic respiratory disease in chicken. ARV replicates within cytoplasmic inclusions, so-called viral factories, that form by phase separation and thus belong to a wider class of biological condensates. Here, we evaluate different optical imaging methods that have been developed or adapted to follow formation, fluidity and composition of viral factories and compare them with the complementary structural information obtained by well-established transmission electron microscopy and electron tomography. The molecular and cellular biology aspects for setting up and following virus infection in cells by imaging are described first. We then demonstrate that a wide-field version of fluorescence recovery after photobleaching is an effective tool to measure fluidity of mobile viral factories. A new technique, holotomographic phase microscopy, is then used for imaging of viral factory formation in live cells in three dimensions. Confocal Raman microscopy of infected cells provides "chemical" contrast for label-free segmentation of images and addresses important questions about biomolecular concentrations within viral factories and other biological condensates. Optical imaging is complemented by electron microscopy and tomography which supply higher resolution structural detail, including visualization of individual virions within the three-dimensional cellular context.
Assuntos
Reoviridae , Compartimentos de Replicação Viral , Linhagem Celular , Corpos de Inclusão Viral , Microscopia Eletrônica , Imagem Multimodal , Replicação ViralRESUMO
Antimicrobial peptides (AMPs), which combat bacterial infections by disrupting the bacterial cell membrane or interacting with intracellular targets, are naturally produced by a number of different organisms, and are increasingly also explored as therapeutics. However, the mechanisms by which AMPs act on intracellular targets are not well understood. Using machine learning-based sequence analysis, we identified a significant number of AMPs that have a strong tendency to form liquid-like condensates in the presence of nucleic acids through phase separation. We demonstrate that this phase separation propensity is linked to the effectiveness of the AMPs in inhibiting transcription and translation in vitro, as well as their ability to compact nucleic acids and form clusters with bacterial nucleic acids in bacterial cells. These results suggest that the AMP-driven compaction of nucleic acids and modulation of their phase transitions constitute a previously unrecognised mechanism by which AMPs exert their antibacterial effects. The development of antimicrobials that target nucleic acid phase transitions may become an attractive route to finding effective and long-lasting antibiotics.
Assuntos
Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Antimicrobianos , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Bactérias/metabolismoRESUMO
Viruses are vulnerable as they transmit between hosts, and we aimed to exploit this critical window. We found that the ubiquitous, safe, inexpensive and biodegradable small molecule propylene glycol (PG) has robust virucidal activity. Propylene glycol rapidly inactivates a broad range of viruses including influenza A, SARS-CoV-2 and rotavirus and reduces disease burden in mice when administered intranasally at concentrations commonly found in nasal sprays. Most critically, vaporised PG efficiently abolishes influenza A virus and SARS-CoV-2 infectivity within airborne droplets, potently preventing infection at levels well below those tolerated by mammals. We present PG vapour as a first-in-class non-toxic airborne virucide that can prevent transmission of existing and emergent viral pathogens, with clear and immediate implications for public health.
Assuntos
COVID-19 , Vírus da Influenza A , Influenza Humana , Animais , Camundongos , Humanos , Aerossóis e Gotículas Respiratórios , COVID-19/prevenção & controle , Propilenoglicóis , MamíferosRESUMO
The assembly of biomolecules into condensates is a fundamental process underlying the organisation of the intracellular space and the regulation of many cellular functions. Mapping and characterising phase behaviour of biomolecules is essential to understand the mechanisms of condensate assembly, and to develop therapeutic strategies targeting biomolecular condensate systems. A central concept for characterising phase-separating systems is the phase diagram. Phase diagrams are typically built from numerous individual measurements sampling different parts of the parameter space. However, even when performed in microwell plate format, this process is slow, low throughput and requires significant sample consumption. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for rapid and high-resolution acquisition of multidimensional biomolecular phase diagrams. Using this platform, we characterise the phase behaviour of a wide range of systems under a variety of conditions and demonstrate that this approach allows the quantitative characterisation of the effect of small molecules on biomolecular phase transitions.
Assuntos
Condensados Biomoleculares , Microfluídica , Espaço Intracelular , Transição de FaseRESUMO
Rotaviruses are major causes of acute gastroenteritis in infants and young children worldwide and also cause disease in the young of many other mammalian and of avian species. During the recent 5-6 years rotavirus research has benefitted in a major way from the establishment of plasmid only-based reverse genetics systems, the creation of human and other mammalian intestinal enteroids, and from the wide application of structural biology (cryo-electron microscopy, cryo-EM tomography) and complementary biophysical approaches. All of these have permitted to gain new insights into structure-function relationships of rotaviruses and their interactions with the host. This review follows different stages of the viral replication cycle and summarizes highlights of structure-function studies of rotavirus-encoded proteins (both structural and non-structural), molecular mechanisms of viral replication including involvement of cellular proteins and lipids, the spectrum of viral genomic and antigenic diversity, progress in understanding of innate and acquired immune responses, and further developments of prevention of rotavirus-associated disease.