RESUMO
A universal taxonomy of viruses is essential for a comprehensive view of the virus world and for communicating the complicated evolutionary relationships among viruses. However, there are major differences in the conceptualisation and approaches to virus classification and nomenclature among virologists, clinicians, agronomists, and other interested parties. Here, we provide recommendations to guide the construction of a coherent and comprehensive virus taxonomy, based on expert scientific consensus. Firstly, assignments of viruses should be congruent with the best attainable reconstruction of their evolutionary histories, i.e., taxa should be monophyletic. This fundamental principle for classification of viruses is currently included in the International Committee on Taxonomy of Viruses (ICTV) code only for the rank of species. Secondly, phenotypic and ecological properties of viruses may inform, but not override, evolutionary relatedness in the placement of ranks. Thirdly, alternative classifications that consider phenotypic attributes, such as being vector-borne (e.g., "arboviruses"), infecting a certain type of host (e.g., "mycoviruses," "bacteriophages") or displaying specific pathogenicity (e.g., "human immunodeficiency viruses"), may serve important clinical and regulatory purposes but often create polyphyletic categories that do not reflect evolutionary relationships. Nevertheless, such classifications ought to be maintained if they serve the needs of specific communities or play a practical clinical or regulatory role. However, they should not be considered or called taxonomies. Finally, while an evolution-based framework enables viruses discovered by metagenomics to be incorporated into the ICTV taxonomy, there are essential requirements for quality control of the sequence data used for these assignments. Combined, these four principles will enable future development and expansion of virus taxonomy as the true evolutionary diversity of viruses becomes apparent.
Assuntos
Bacteriófagos , Vírus , Humanos , Metagenômica , Filogenia , Vírus/genéticaRESUMO
Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations1-8. Because the release of dsRNA into the cytoplasm triggers host defence mechanisms9, dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription10-12. The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10-12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases11-14. However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses.
Assuntos
Bacteriófago phi 6/química , Bacteriófago phi 6/ultraestrutura , Microscopia Crioeletrônica , Empacotamento do DNA , Cristais Líquidos , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/ultraestrutura , RNA Viral/ultraestrutura , Bacteriófago phi 6/genética , Genoma Viral , Modelos Moleculares , RNA de Cadeia Dupla/química , RNA Viral/química , RNA Polimerase Dependente de RNA/metabolismoRESUMO
Herpes simplex virus type 1 (HSV-1) is a common virus of mankind and HSV-1 infections are a significant cause of blindness. The current antiviral treatment of herpes infection relies on acyclovir and related compounds. However, acyclovir resistance emerges especially in the long term prophylactic treatment that is required for prevention of recurrent herpes keratitis. Earlier we have established antiviral siRNA swarms, targeting sequences of essential genes of HSV, as effective means of silencing the replication of HSV in vitro or in vivo. In this study, we show the antiviral efficacy of 2´-fluoro modified antiviral siRNA swarms against HSV-1 in human corneal epithelial cells (HCE). We studied HCE for innate immunity responses to HSV-1, to immunostimulatory cytotoxic double stranded RNA, and to the antiviral siRNA swarms, with or without a viral challenge. The panel of studied innate responses included interferon beta, lambda 1, interferon stimulated gene 54, human myxovirus resistance protein A, human myxovirus resistance protein B, toll-like receptor 3 and interferon kappa. Our results demonstrated that HCE cells are a suitable model to study antiviral RNAi efficacy and safety in vitro. In HCE cells, the antiviral siRNA swarms targeting the HSV UL29 gene and harboring 2´-fluoro modifications, were well tolerated, induced only modest innate immunity responses, and were highly antiviral with more than 99% inhibition of viral release. The antiviral effect of the 2'-fluoro modified swarm was more apparent than that of the unmodified antiviral siRNA swarm. Our results encourage further research in vitro and in vivo on antiviral siRNA swarm therapy of corneal HSV infection, especially with modified siRNA swarms.
Assuntos
Herpes Simples , Herpesvirus Humano 1 , Aciclovir/metabolismo , Aciclovir/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Células Epiteliais/metabolismo , Herpes Simples/genética , Herpes Simples/terapia , Herpesvirus Humano 1/fisiologia , Humanos , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Replicação Viral/genéticaRESUMO
Extracellular vesicles (EVs) are relatively recently discovered biological nanoparticles that mediate intercellular communication. The development of new methods for the isolation and characterization of EVs is crucial to support further studies on these small and structurally heterogenous vesicles. New scalable production methods are also needed to meet the needs of future therapeutic applications. A reliable inline detection method for the EV manufacturing process is needed to ensure reproducibility and to identify any possible variations in real time. Here, we demonstrate the use of an inline Raman detector in conjunction with anion exchange chromatography for the isolation of EVs from human platelets. Anion-exchange chromatography can be easily coupled with multiple inline detectors and provides an alternative to size-based methods for separating EVs from similar-sized impurities, such as lipoprotein particles. Raman spectroscopy enabled us to identify functional groups in EV samples and trace EVs and impurities in different stages of the process. Our results show a notable separation of impurities from the EVs during anion-exchange chromatography and demonstrate the power of inline Raman spectroscopy. Compared to conventional EV analysis methods, the inline Raman approach does not require hands-on work and can provide detailed, real-time information about the sample and the purification process.
Assuntos
Plaquetas , Vesículas Extracelulares , Análise Espectral Raman , Análise Espectral Raman/métodos , Cromatografia por Troca Iônica/métodos , Humanos , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Plaquetas/metabolismo , Plaquetas/química , ÂnionsRESUMO
This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021-March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.
Assuntos
Bacteriófagos , Caudovirales , Siphoviridae , Vírus , Humanos , Vírus/genética , MyoviridaeRESUMO
BACKGROUND & AIMS: Interleukin-26 (IL-26) is a proinflammatory cytokine that has properties atypical for a cytokine, such as direct antibacterial activity and DNA-binding capacity. We previously observed an accumulation of IL-26 in fibrotic and inflammatory lesions in the livers of patients with chronic HCV infection and showed that infiltrating CD3+ lymphocytes were the principal source of IL-26. Surprisingly, IL-26 was also detected in the cytoplasm of hepatocytes from HCV-infected patients, even though these cells do not produce IL-26, even when infected with HCV. Based on this observation and possible interactions between IL-26 and nucleic acids, we investigated the possibility that IL-26 controlled HCV infection independently of the immune system. METHODS: We evaluated the ability of IL-26 to interfere with HCV replication in hepatocytes and investigated the mechanisms by which IL-26 exerts its antiviral activity. RESULTS: We showed that IL-26 penetrated HCV-infected hepatocytes, where it interacted directly with HCV double-stranded RNA replication intermediates, thereby inhibiting viral replication. IL-26 interfered with viral RNA-dependent RNA polymerase activity, preventing the de novo synthesis of viral genomic single-stranded RNA. CONCLUSIONS: These findings reveal a new role for IL-26 in direct protection against HCV infection, independently of the immune system, and increase our understanding of the antiviral defense mechanisms controlling HCV infection. Future studies should evaluate the possible use of IL-26 for treating other chronic disorders caused by RNA viruses, for which few treatments are currently available, or emerging RNA viruses. LAY SUMMARY: This study sheds new light on the body's arsenal for controlling hepatitis C virus (HCV) infection and identifies interleukin-26 (IL-26) as an antiviral molecule capable of blocking HCV replication. IL-26, which has unique biochemical and structural characteristics, penetrates infected hepatocytes and interacts directly with viral RNA, thereby blocking viral replication. IL-26 is, therefore, a new player in antiviral defenses, operating independently of the immune system. It is of considerable potential interest for treating HCV infection and other chronic disorders caused by RNA viruses for which few treatments are currently available, and for combating emerging RNA viruses.
Assuntos
Hepacivirus , Hepatite C , Antivirais/farmacologia , Antivirais/uso terapêutico , Citocinas , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Hepatócitos , Humanos , Interleucinas/farmacologia , Replicação ViralRESUMO
RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1-1000 s duration, of which the short-lived ones (1-10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.
Assuntos
Vírus de RNA/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Temperatura , Replicação Viral , Bacteriófago phi 6/enzimologia , Enterovirus/enzimologia , Ativação Enzimática , Cinética , Microscopia , Poliovirus/enzimologiaRESUMO
In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).
Assuntos
Vírus de Archaea/classificação , Bacteriófagos/classificação , Sociedades Científicas/organização & administração , Archaea/virologia , Bactérias/virologiaRESUMO
Mammalian cells express different types of RNA molecules that can be classified as protein coding RNAs (mRNA) and non-coding RNAs (ncRNAs) the latter of which have housekeeping and regulatory functions in cells. Cellular RNAs are not recognized by cellular pattern recognition receptors (PRRs) and innate immunity is not activated. RNA viruses encode and express RNA molecules that usually differ from cell-specific RNAs and they include for instance 5'capped and 5'mono- and triphosphorylated RNAs, small viral RNAs and viral RNA-protein complexes called vRNPs. These molecules are recognized by certain members of Toll-like receptor (TLR) and RIG-I-like receptor (RLR) families leading to activation of innate immune responses and the production of antiviral cytokines, such as type I and type III interferons (IFNs). Virus-specific ssRNA and dsRNA molecules that mimic the viral genomic RNAs or their replication intermediates can efficiently be produced by bacteriophage T7 DNA-dependent RNA polymerase and bacteriophage phi6 RNA-dependent RNA polymerase, respectively. These molecules can then be delivered into mammalian cells and the mechanisms of activation of innate immune responses can be studied. In addition, synthetic viral dsRNAs can be processed to small interfering RNAs (siRNAs) by a Dicer enzyme to produce a swarm of antiviral siRNAs. Here we describe the biology of RNAs, their in vitro production and delivery into mammalian cells as well as how these molecules can be used to inhibit virus replication and to study the mechanisms of activation of the innate immune system.
Assuntos
Imunidade Inata/genética , Técnicas In Vitro/métodos , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Transfecção/métodos , Animais , Células Cultivadas , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Viral , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Interferons/imunologia , Interferons/metabolismo , Cultura Primária de Células/métodos , Interferência de RNA , Vírus de RNA/genética , Vírus de RNA/imunologia , RNA de Cadeia Dupla , RNA Viral/imunologia , RNA Polimerase Dependente de RNA/metabolismo , Ribonuclease III/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
Finnlakeviridae is a family of icosahedral, internal membrane-containing bacterial viruses with circular, single-stranded DNA genomes. The family includes the genus, Finnlakevirus, with the species, Flavobacterium virus FLiP. Flavobacterium phage FLiP was isolated with its Gram-negative host bacterium from a boreal freshwater habitat in Central Finland in 2010. It is the first described single-stranded DNA virus with an internal membrane and shares minimal sequence similarity with other known viruses. The virion organization (pseudo T=21 dextro) and major capsid protein fold (double-ß-barrel) resemble those of Pseudoalteromonas phage PM2 (family Corticoviridae), which has a double-stranded DNA genome. A similar major capsid protein fold is also found in other double-stranded DNA viruses in the kingdom Bamfordvirae. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Finnlakeviridae, which is available at ictv.global/report/finnlakeviridae.
Assuntos
Bacteriófagos , Vírus de DNA , Flavobacterium/virologia , Bacteriólise , Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/fisiologia , Bacteriófagos/ultraestrutura , Vírus de DNA/classificação , Vírus de DNA/genética , Vírus de DNA/fisiologia , Vírus de DNA/ultraestrutura , DNA de Cadeia Simples/genética , DNA Viral/genética , Genoma Viral , Vírion/química , Vírion/ultraestrutura , Replicação ViralRESUMO
Influenza A viruses (IAVs) are viral pathogens that cause epidemics and occasional pandemics of significant mortality. The generation of efficacious vaccines and antiviral drugs remains a challenge due to the rapid appearance of new influenza virus types and antigenic variants. Consequently, novel strategies for the prevention and treatment of IAV infections are needed, given the limitations of the presently available antivirals. Here, we used enzymatically produced IAV-specific double-stranded RNA (dsRNA) molecules and Giardia intestinalis Dicer for the generation of a swarm of small interfering RNA (siRNA) molecules. The siRNAs target multiple conserved genomic regions of the IAVs. In mammalian cells, the produced 25- to 27-nucleotide-long siRNA molecules are processed by endogenous Dicer into 21-nucleotide siRNAs and are thus designated Dicer-substrate siRNAs (DsiRNAs). We evaluated the efficacy of the above DsiRNA swarm at preventing IAV infections in human primary monocyte-derived macrophages and dendritic cells. The replication of different IAV strains, including avian influenza H5N1 and H7N9 viruses, was significantly inhibited by pretransfection of the cells with the IAV-specific DsiRNA swarm. Up to 7 orders of magnitude inhibition of viral RNA expression was observed, which led to a dramatic inhibition of IAV protein synthesis and virus production. The IAV-specific DsiRNA swarm inhibited virus replication directly through the RNA interference pathway although a weak induction of innate interferon responses was detected. Our results provide direct evidence for the feasibility of the siRNA strategy and the potency of DsiRNA swarms in the prevention and treatment of influenza, including the highly pathogenic avian influenza viruses.IMPORTANCE In spite of the enormous amount of research, influenza virus is still one of the major challenges for medical virology due to its capacity to generate new variants, which potentially lead to severe epidemics and pandemics. We demonstrated here that a swarm of small interfering RNA (siRNA) molecules, including more than 100 different antiviral RNA molecules targeting the most conserved regions of the influenza A virus genome, could efficiently inhibit the replication of all tested avian and seasonal influenza A variants in human primary monocyte-derived macrophages and dendritic cells. The wide antiviral spectrum makes the virus-specific siRNA swarm a potentially efficient treatment modality against both avian and seasonal influenza viruses.
Assuntos
Vírus da Influenza A/genética , Influenza Aviária/genética , RNA Interferente Pequeno/farmacologia , Animais , Antivirais/farmacologia , Aves , Linhagem Celular , RNA Helicases DEAD-box , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/virologia , Cães , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Virus da Influenza A Subtipo H5N1/genética , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/virologia , Interferons/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Células Madin Darby de Rim Canino , Cultura Primária de Células , RNA Interferente Pequeno/genética , Ribonuclease III , Replicação Viral/efeitos dos fármacosRESUMO
This article is a summary of the activities of the ICTV's Bacterial and Archaeal Viruses Subcommittee for the years 2018 and 2019. Highlights include the creation of a new order, 10 families, 22 subfamilies, 424 genera and 964 species. Some of our concerns about the ICTV's ability to adjust to and incorporate new DNA- and protein-based taxonomic tools are discussed.
Assuntos
Vírus de Archaea/classificação , Bacteriófagos/classificação , Classificação/métodos , Archaea/virologia , Bactérias/virologiaRESUMO
Genome packaging of double-stranded RNA (dsRNA) phages has been widely studied using biochemical and molecular biology methods. We adapted the existing in vitro packaging system of one such phage for single-molecule experimentation. To our knowledge, this is the first attempt to study the details of viral RNA packaging using optical tweezers. Pseudomonas phage φ6 is a dsRNA virus with a tripartite genome. Positive-sense (+) single-stranded RNA (ssRNA) genome precursors are packaged into a preformed procapsid (PC), where negative strands are synthesized. We present single-molecule measurements of the viral ssRNA packaging by the φ6 PC. Our data show that packaging proceeds intermittently in slow and fast phases, which likely reflects differences in the unfolding of the RNA secondary structures of the ssRNA being packaged. Although the mean packaging velocity was relatively low (0.07-0.54 nm/sec), packaging could reach 4.62 nm/sec during the fast packaging phase.
Assuntos
Bacteriófago phi 6/fisiologia , RNA Viral/genética , Bacteriófago phi 6/genética , Técnicas In Vitro , Modelos Moleculares , Conformação de Ácido Nucleico , Dobramento de RNA , RNA Viral/química , Montagem de VírusRESUMO
BACKGROUND: Cystoviruses have a phospholipid envelope around their nucleocapsid. Such a feature is unique among bacterial viruses (i.e., bacteriophages) and the mechanisms of virion envelopment within a bacterial host are largely unknown. The cystovirus Pseudomonas phage phi6 has an envelope that harbors five viral membrane proteins and phospholipids derived from the cytoplasmic membrane of its Gram-negative host. The phi6 major envelope protein P9 and the non-structural protein P12 are essential for the envelopment of its virions. Co-expression of P9 and P12 in a Pseudomonas host results in the formation of intracellular vesicles that are potential intermediates in the phi6 virion assembly pathway. This study evaluated the minimum requirements for the formation of phi6-specific vesicles and the possibility to localize P9-tagged heterologous proteins into such structures in Escherichia coli. RESULTS: Using transmission electron microscopy, we detected membranous structures in the cytoplasm of E. coli cells expressing P9. The density of the P9-specific membrane fraction was lower (approximately 1.13 g/cm3 in sucrose) than the densities of the bacterial cytoplasmic and outer membrane fractions. A P9-GFP fusion protein was used to study the targeting of heterologous proteins into P9 vesicles. Production of the GFP-tagged P9 vesicles required P12, which protected the fusion protein against proteolytic cleavage. Isolated vesicles contained predominantly P9-GFP, suggesting selective incorporation of P9-tagged fusion proteins into the vesicles. CONCLUSIONS: Our results demonstrate that the phi6 major envelope protein P9 can trigger formation of cytoplasmic membrane structures in E. coli in the absence of any other viral protein. Intracellular membrane structures are rare in bacteria, thus making them ideal chasses for cell-based vesicle production. The possibility to locate heterologous proteins into the P9-lipid vesicles facilitates the production of vesicular structures with novel properties. Such products have potential use in biotechnology and biomedicine.
Assuntos
Bacteriófago phi 6/química , Escherichia coli/genética , Proteínas da Matriz Viral/genética , Proteínas não Estruturais Virais/genética , Membrana Celular , Fosfolipídeos , Pseudomonas syringae/química , Pseudomonas syringae/genética , Pseudomonas syringae/virologia , VírionRESUMO
Pathogens induce severe damages on cultivated plants and represent a serious threat to global food security. Emerging strategies for crop protection involve the external treatment of plants with double-stranded (ds)RNA to trigger RNA interference. However, applying this technology in greenhouses and fields depends on dsRNA quality, stability and efficient large-scale production. Using components of the bacteriophage phi6, we engineered a stable and accurate in vivo dsRNA production system in Pseudomonas syringae bacteria. Unlike other in vitro or in vivo dsRNA production systems that rely on DNA transcription and postsynthetic alignment of single-stranded RNA molecules, the phi6 system is based on the replication of dsRNA by an RNA-dependent RNA polymerase, thus allowing production of high-quality, long dsRNA molecules. The phi6 replication complex was reprogrammed to multiply dsRNA sequences homologous to tobacco mosaic virus (TMV) by replacing the coding regions within two of the three phi6 genome segments with TMV sequences and introduction of these constructs into P. syringae together with the third phi6 segment, which encodes the components of the phi6 replication complex. The stable production of TMV dsRNA was achieved by combining all the three phi6 genome segments and by maintaining the natural dsRNA sizes and sequence elements required for efficient replication and packaging of the segments. The produced TMV-derived dsRNAs inhibited TMV propagation when applied to infected Nicotiana benthamiana plants. The established dsRNA production system enables the broad application of dsRNA molecules as an efficient, highly flexible, nontransgenic and environmentally friendly approach for protecting crops against viruses and other pathogens.
RESUMO
During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5'-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.
Assuntos
Capsídeo/metabolismo , Genoma Viral/genética , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas do Capsídeo/genética , Humanos , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , Vírion/genética , Vírion/metabolismoRESUMO
Cystoviridae is a family of bacterial viruses (bacteriophages) with a tri-segmented dsRNA genome. It includes a single genus Cystovirus, which has presently only one recognised virus species, Pseudomonas virus phi6. However, a large number of additional dsRNA phages have been isolated from various environmental samples, indicating that such viruses are more widespread and abundant than previously recognised. Six of the additional dsRNA phage isolates (Pseudomonas phages phi8, phi12, phi13, phi2954, phiNN and phiYY) have been fully sequenced. They all infect Pseudomonas species, primarily plant pathogenic Pseudomonas syringae strains. Due to the notable genetic and structural similarities with Pseudomonas phage phi6, we propose that these viruses should be included into the Cystovirus genus (and consequently into the Cystoviridae family). Here, we present an updated taxonomy of the family Cystoviridae and give a short overview of the properties of the type member phi6 as well as the putative new members of the family.
Assuntos
Cystoviridae/genética , Genoma Viral , Filogenia , Pseudomonas/virologia , RNA de Cadeia Dupla/genética , RNA Viral/genética , Sequência de Bases , Cystoviridae/classificação , Cystoviridae/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Homologia de Sequência do Ácido Nucleico , Terminologia como AssuntoRESUMO
Identification of relationships among protein families or superfamilies is a challenge. However, functionally essential protein regions typically retain structural integrity, even when the corresponding protein sequences evolve. Consequently, comparison of protein structures enables deeper phylogenetic analyses than achievable through the use of sequence information only. Here, we focus on a group of distantly related viral and cellular enzymes involved in nucleic acid or nucleotide processing and synthesis. All these enzymes share an apparently similar protein fold at their active site, which resembles the palm subdomain of the right-hand-shaped polymerases. Using a structure-based hierarchical clustering method, we identified a common structural core of 36 equivalent residues for this functionally diverse group of enzymes, representing five protein superfamilies. Based on the properties of these 36 residues, we deduced a structural distance-based tree in which the proteins were accurately clustered according to the established family classification. Within this tree, the enzymes catalyzing genomic nucleic acid replication or transcription were separated from those performing supplementary nucleic acid or nucleotide processing functions. In addition, we found that the family Y DNA polymerases are structurally more closely related to the nucleotide cyclase superfamily members than to the other members of the DNA/RNA polymerase superfamily, and these enzymes share 88 equivalent residues comprising a Β: 1- Α: 1- Α: 2- Β: 2- Β: 3- Α: 3- Β: 4- Α: 4- Β: 5 fold. The results highlight the power of structure-based hierarchical clustering in identifying remote evolutionary relationships. Furthermore, our study implies that a protein substructure of only three-dozen residues can contain a substantial amount of information on the evolutionary history of proteins.
Assuntos
Proteínas/química , Proteínas/genética , Análise de Sequência de Proteína/métodos , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Domínio Catalítico , Análise por Conglomerados , Evolução Molecular , Genômica , Modelos Moleculares , Filogenia , Alinhamento de Sequência/métodos , Relação Estrutura-AtividadeRESUMO
The family Cystoviridae includes enveloped viruses with a tri-segmented dsRNA genome and a double-layered protein capsid. The innermost protein shell is a polymerase complex responsible for genome packaging, replication and transcription. Cystoviruses infect Gram-negative bacteria, primarily plant-pathogenic Pseudomonas syringae strains. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Cystoviridae, which is available at http://www.ictv.global/report/cystoviridae.
Assuntos
Cystoviridae/genética , Cystoviridae/fisiologia , Bactérias Gram-Negativas/virologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Cystoviridae/classificação , Genes Virais , Genoma Viral , RNA Viral/genética , Replicação Viral/fisiologiaRESUMO
Transcription in RNA viruses is highly dynamic, with a variety of pauses interrupting nucleotide addition by RNA-dependent RNA polymerase (RdRp). For example, rare but lengthy pauses (>20 s) have been linked to backtracking for viral single-subunit RdRps. However, while such backtracking has been well characterized for multi-subunit RNA polymerases (RNAPs) from bacteria and yeast, little is known about the details of viral RdRp backtracking and its biological roles. Using high-throughput magnetic tweezers, we quantify the backtracking by RdRp from the double-stranded (ds) RNA bacteriophage Φ6, a model system for RdRps. We characterize the probability of entering long backtracks as a function of force and propose a model in which the bias toward backtracking is determined by the base paring at the dsRNA fork. We further discover that extensive backtracking provides access to a new 3'-end that allows for the de novo initiation of a second RdRp. This previously unidentified behavior provides a new mechanism for rapid RNA synthesis using coupled RdRps and hints at a possible regulatory pathway for gene expression during viral RNA transcription.