RESUMO
CrAss-like phages are a recently described expansive group of viruses that includes the most abundant virus in the human gut1-3. The genomes of all crAss-like phages encode a large virion-packaged protein2,4 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)5. Here, using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited state, which probably occurs before virion packaging. This conformation is attained with the help of a cleft-blocking domain that interacts with the active site and occupies the cavity in which the RNA-DNA hybrid binds. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs that are involved in RNA interference6,7, although most of the phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of the protein fold space. Considering this structural similarity, we propose that eukaryal RNA interference polymerases have their origins in phage, which parallels the emergence of the mitochondrial transcription apparatus8.
Assuntos
Bacteriófagos/classificação , Bacteriófagos/enzimologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Flavobacteriaceae/virologia , Bacteriófagos/genética , Domínio Catalítico , Sistema Livre de Células , Cristalografia por Raios X , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/genética , RNA Polimerases Dirigidas por DNA/genética , Evolução Molecular , Regulação Viral da Expressão Gênica , Genes Virais/genética , Modelos Biológicos , Modelos Moleculares , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Interferência de RNA , Transcrição GênicaRESUMO
TarBase is a reference database dedicated to produce, curate and deliver high quality experimentally-supported microRNA (miRNA) targets on protein-coding transcripts. In its latest version (v9.0, https://dianalab.e-ce.uth.gr/tarbasev9), it pushes the envelope by introducing virally-encoded miRNAs, interactions leading to target-directed miRNA degradation (TDMD) events and the largest collection of miRNA-gene interactions to date in a plethora of experimental settings, tissues and cell-types. It catalogues â¼6 million entries, comprising â¼2 million unique miRNA-gene pairs, supported by 37 experimental (high- and low-yield) protocols in 172 tissues and cell-types. Interactions are annotated with rich metadata including information on genes/transcripts, miRNAs, samples, experimental contexts and publications, while millions of miRNA-binding locations are also provided at cell-type resolution. A completely re-designed interface with state-of-the-art web technologies, incorporates more features, and allows flexible and ingenious use. The new interface provides the capability to design sophisticated queries with numerous filtering criteria including cell lines, experimental conditions, cell types, experimental methods, species and/or tissues of interest. Additionally, a plethora of fine-tuning capacities have been integrated to the platform, offering the refinement of the returned interactions based on miRNA confidence and expression levels, while boundless local retrieval of the offered interactions and metadata is enabled.
Assuntos
Bases de Dados de Ácidos Nucleicos , MicroRNAs , Genes Virais/genética , Internet , MicroRNAs/genética , MicroRNAs/metabolismo , AnimaisRESUMO
Viral genomes frequently harbor overlapping genes, complicating the development of virus-vectored vaccines and gene therapies. This study introduces a novel conditional splicing system to precisely control the expression of such overlapping genes through recombinase-mediated conditional splicing. We refined site-specific recombinase (SSR) conditional splicing systems and explored their mechanisms. The systems demonstrated exceptional inducibility (116,700-fold increase) with negligible background expression, facilitating the conditional expression of overlapping genes in adenovirus-associated virus (AAV) and human immunodeficiency virus type 1. Notably, this approach enabled the establishment of stable AAV producer cell lines, encapsulating all necessary packaging genes. Our findings underscore the potential of the SSR-conditional splicing system to significantly advance vector engineering, enhancing the efficacy and scalability of viral-vector-based therapies and vaccines. IMPORTANCE: Regulating overlapping genes is vital for gene therapy and vaccine development using viral vectors. The regulation of overlapping genes presents challenges, including cytotoxicity and impacts on vector capacity and genome stability, which restrict stable packaging cell line development and broad application. To address these challenges, we present a "loxp-splice-loxp"-based conditional splicing system, offering a novel solution for conditional expression of overlapping genes and stable cell line establishment. This system may also regulate other cytotoxic genes, representing a significant advancement in cell engineering and gene therapy as well as biomass production.
Assuntos
Dependovirus , Homologia de Genes , Genes Virais , Engenharia Genética , HIV-1 , Splicing de RNA , Humanos , Linhagem Celular , Dependovirus/genética , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Regulação Viral da Expressão Gênica , Homologia de Genes/genética , Genes Virais/genética , Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos/genética , HIV-1/genética , Splicing de RNA/genética , Vacinas/biossíntese , Vacinas/genética , Empacotamento do Genoma Viral/genéticaRESUMO
Adeno-associated viruses (AAVs) package a single-stranded (ss) DNA genome of 4.7 kb in their capsid of ~20 nm in diameter. AAV replication requires co-infection of a helper virus, such as adenovirus. During the optimization of recombinant AAV production, a small viral nonstructural protein, membrane-associated accessory protein (MAAP), was identified. However, the function of the MAAP in the context of AAV infection remains unknown. Here, we investigated the expression strategy and function of the MAAP during infection of both AAV2 and AAV5 in human embryonic kidney (HEK)293 cells. We found that AAV2 MAAP2 and AAV5 MAAP5 are expressed from the capsid gene (cap)-transcribing mRNA spliced from the donor to the second splice site that encodes VP2 and VP3. Thus, this AAV cap gene transcribes a multicistronic mRNA that can be translated to four viral proteins, MAAP, VP2, AAP, and VP3 in order. In AAV2 infection, MAAP2 predominantly localized in the cytoplasm, alongside the capsid, near the nuclear and plasma membranes, but a fraction of MAAP2 exhibited nuclear localization. In AAV5 infection, MAAP5 revealed a distinct pattern, predominantly localizing within the nucleus. In the cells infected with an MAAP knockout mutant of AAV2 or AAV5, both viral DNA replication and virus replication increased, whereas virus egress decreased, and the decrease in virus egress can be restored by providing MAAP in trans. In summary, MAAP, a novel AAV nonstructural protein translated from a multicistronic viral cap mRNA, not only facilitates cellular egress of AAV but also likely negatively affects viral DNA replication during infection. IMPORTANCE: Recombinant adeno-associated virus (rAAV) has been used as a gene delivery vector in clinical gene therapy. In current gene therapies employing rAAV, a high dose of the vector is required. Consequently, there is a high demand for efficient and high-purity vector production systems. In this study, we demonstrated that membrane-associated accessory protein (MAAP), a small viral nonstructural protein, is translated from the same viral mRNA transcript encoding VP2 and VP3. In AAV-infected cells, apart from its prevalent expression in the cytoplasm with localization near the plasma and nuclear membranes, the MAAP also exhibits notable localization within the nucleus. During AAV infection, MAAP expression increases the cellular egress of progeny virions and decreases viral DNA replication and progeny virion production. Thus, the choice of MAAP expression has pros and cons during AAV infection, which could provide a guide to rAAV production.
Assuntos
Dependovirus , Infecções por Parvoviridae , Proteínas não Estruturais Virais , Humanos , Proteínas do Capsídeo/genética , Dependovirus/genética , Dependovirus/metabolismo , Dependovirus/fisiologia , Células HEK293 , Infecções por Parvoviridae/virologia , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Replicação Viral , Genes Virais/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.
Assuntos
Genoma Viral , Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Mutação , RNA Polimerase II , Transcrição Viral , Animais , Humanos , Linhagem Celular , Chlorocebus aethiops , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Genes Virais/genética , Genoma Viral/genética , Herpes Simples/virologia , Herpes Simples/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/genética , Poli A/genética , Poli A/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Células Vero , Transcrição Viral/efeitos dos fármacos , Transcrição Viral/genética , Replicação Viral/genéticaRESUMO
Cytosolic DNA triggers innate immune responses through the activation of cyclic GMP-AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2',3'-cyclic GMP-AMP (cGAMP)1-4. 2',3'-cGAMP is a potent inducer of immune signalling; however, no intracellular nucleases are known to cleave 2',3'-cGAMP and prevent the activation of the receptor stimulator of interferon genes (STING)5-7. Here we develop a biochemical screen to analyse 24 mammalian viruses, and identify poxvirus immune nucleases (poxins) as a family of 2',3'-cGAMP-degrading enzymes. Poxins cleave 2',3'-cGAMP to restrict STING-dependent signalling and deletion of the poxin gene (B2R) attenuates vaccinia virus replication in vivo. Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2',3'-cGAMP degradation through metal-independent cleavage of the 3'-5' bond, converting 2',3'-cGAMP into linear Gp[2'-5']Ap[3']. Poxins are conserved in mammalian poxviruses. In addition, we identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses that infect these insects. Baculovirus and insect host poxin homologues retain selective 2',3'-cGAMP degradation activity, suggesting an ancient role for poxins in cGAS-STING regulation. Our results define poxins as a family of 2',3'-cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity.
Assuntos
Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais/imunologia , Vaccinia virus/enzimologia , Animais , Baculoviridae/enzimologia , Borboletas/enzimologia , Linhagem Celular , Sequência Conservada , Cristalografia por Raios X , DNA Viral/imunologia , Feminino , Genes Virais/genética , Humanos , Evasão da Resposta Imune , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mariposas/enzimologia , Sistemas do Segundo Mensageiro , Vaccinia virus/genética , Vaccinia virus/crescimento & desenvolvimento , Vaccinia virus/imunologia , Replicação Viral/genéticaRESUMO
BACKGROUND: Human cytomegalovirus (HCMV) is the most common and serious opportunistic infection after solid organ and hematopoietic stem cell transplantation. In this study, we used whole-genome HCMV data to investigate viral factors associated with the clinical outcome. METHODS: We sequenced HCMV samples from 16 immunocompromised pediatric patients with persistent viremia. Eight of the 16 patients died of complications due to HCMV infection. We also sequenced samples from 35 infected solid organ adult recipients, of whom 1 died with HCMV infection. RESULTS: We showed that samples from both groups have fixed variants at resistance sites and mixed infections. Next-generation sequencing also revealed nonfixed variants at resistance sites in most of the patients who died (6/9). A machine learning approach identified 10 genes with nonfixed variants in these patients. These genes formed a viral signature that discriminated patients with HCMV infection who died from those who survived with high accuracy (area under the curve = 0.96). Lymphocyte numbers for a subset of patients showed no recovery posttransplant in the patients who died. CONCLUSIONS: We hypothesize that the viral signature identified in this study may be a useful biomarker for poor response to antiviral drug treatment and indirectly for poor T-cell function, potentially identifying early those patients requiring nonpharmacological interventions.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Hospedeiro Imunocomprometido , Humanos , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/mortalidade , Citomegalovirus/genética , Criança , Masculino , Feminino , Adulto , Pré-Escolar , Antivirais/uso terapêutico , Adolescente , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Lactente , Transplante de Órgãos/efeitos adversos , Sequenciamento de Nucleotídeos em Larga Escala , Viremia , Genes Virais/genética , Variação Genética , Farmacorresistência Viral/genética , Adulto Jovem , Pessoa de Meia-IdadeRESUMO
IMPORTANCE: Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Transcrição Gênica , Humanos , Genes Virais/genética , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/metabolismo , Iniciação da Transcrição Genética , Elongação da Transcrição Genética , Terminação da Transcrição GenéticaRESUMO
The African swine fever virus (ASFV) has caused a devastating pandemic in domestic and wild swine, causing economic losses to the global swine industry. Recombinant live attenuated vaccines are an attractive option for ASFV treatment. However, safe and effective vaccines against ASFV are still scarce, and more high-quality experimental vaccine strains need to be developed. In this study, we revealed that deletion of the ASFV genes DP148R, DP71L, and DP96R from the highly virulent isolate ASFV CN/GS/2018 (ASFV-GS) substantially attenuated virulence in swine. Pigs infected with 104 50% hemadsorbing doses of the virus with these gene deletions remained healthy during the 19-day observation period. No ASFV infection was detected in contact pigs under the experimental conditions. Importantly, the inoculated pigs were protected against homologous challenges. Additionally, RNA sequence analysis showed that deletion of these viral genes induced significant upregulation of the host histone H3.1 gene (H3.1) and downregulation of the ASFV MGF110-7L gene. Knocking down the expression of H3.1 resulted in high levels of ASFV replication in primary porcine macrophages in vitro. These findings indicate that the deletion mutant virus ASFV-GS-Δ18R/NL/UK is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce full protection against the highly virulent ASFV-GS virus strain. IMPORTANCE Ongoing outbreaks of African swine fever (ASF) have considerably damaged the pig industry in affected countries. Thus, a safe and effective vaccine is important to control African swine fever spread. Here, an ASFV strain with three gene deletions was developed by knocking out the viral genes DP148R (MGF360-18R), NL (DP71L), and UK (DP96R). The results showed that the recombinant virus was completely attenuated in pigs and provided strong protection against parental virus challenge. Additionally, no viral genomes were detected in the sera of pigs housed with animals infected with the deletion mutant. Furthermore, transcriptome sequencing (RNA-seq) analysis revealed significant upregulation of histone H3.1 in virus-infected macrophage cultures and downregulation of the ASFV MGF110-7L gene after viral DP148R, UK, and NL deletion. Our study provides a valuable live attenuated vaccine candidate and potential gene targets for developing strategies for anti-ASFV treatment.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Deleção de Genes , Genes Virais , Vacinas Virais , Fatores de Virulência , Animais , Febre Suína Africana/imunologia , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/imunologia , Vírus da Febre Suína Africana/patogenicidade , Células Cultivadas , Genes Virais/genética , Histonas/genética , Suínos , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Fatores de Virulência/genéticaRESUMO
IMPORTANCE: African swine fever (ASF) caused by ASF virus (ASFV) is a highly contagious and acute hemorrhagic viral disease in domestic pigs. Until now, no effective commercial vaccine and antiviral drugs are available for ASF control. Here, we generated a new live-attenuated vaccine candidate (ASFV-ΔH240R-Δ7R) by deleting H240R and MGF505-7R genes from the highly pathogenic ASFV HLJ/18 genome. Piglets immunized with ASFV-ΔH240R-Δ7R were safe without any ASF-related signs and produced specific antibodies against p30. Challenged with a virulent ASFV HLJ/18, the piglets immunized with high-dose group (105 HAD50) exhibited 100% protection without clinical symptoms, showing that low levels of virus replication with no observed pathogenicity by postmortem and histological analysis. Overall, our results provided a new strategy by designing live-attenuated vaccine candidate, resulting in protection against ASFV infection.
Assuntos
Vírus da Febre Suína Africana , Deleção de Genes , Genes Virais , Vacinas Atenuadas , Vacinas Virais , Animais , Febre Suína Africana/imunologia , Febre Suína Africana/prevenção & controle , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/classificação , Vírus da Febre Suína Africana/imunologia , Vírus da Febre Suína Africana/patogenicidade , Sus scrofa/virologia , Vacinas Atenuadas/imunologia , Proteínas Virais/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência , Replicação Viral , Genes Virais/genéticaRESUMO
While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel, enabling the pooled screening of entire viral genomes. Here, we applied this approach to Kaposi's sarcoma-associated herpesvirus (KSHV) by designing a sgRNA library containing all possible ~22,000 guides targeting the 154 kilobase viral genome, corresponding to one cut site approximately every 8 base pairs. We used the library to profile viral sequences involved in transcriptional activation of late genes, whose regulation involves several well characterized features including dependence on viral DNA replication and a known set of viral transcriptional activators. Upon phenotyping all possible Cas9-targeted viruses for transcription of KSHV late genes we recovered these established regulators and identified a new required factor (ORF46), highlighting the utility of the screening pipeline. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we identify the DNA binding but not catalytic domain of ORF46 to be required for viral DNA replication and thus late gene expression. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.
Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Genes Virais/genética , Herpesvirus Humano 8/genética , Sistemas CRISPR-Cas , Células HEK293 , HumanosRESUMO
Epstein-Barr virus (EBV) infection is associated with multiple malignancies, including pulmonary lymphoepithelioma-like carcinoma (pLELC), a particular subtype of primary lung cancer. However, the genomic characteristics of EBV related to pLELC remain unclear. Here, we obtained the whole-genome data set of EBV isolated from 78 pLELC patients and 37 healthy controls using EBV-captured sequencing. Compared with the reference genome (NC_007605), a total of 3,995 variations were detected across pLELC-derived EBV sequences, with the mutational hot spots located in latent genes. Combined with 180 published EBV sequences derived from healthy people in Southern China, we performed a genome-wide association study and identified 32 variations significantly related to pLELC (P < 2.56 × 10-05, Bonferroni correction), with the top signal of single nucleotide polymorphism (SNP) coordinate T7327C (OR = 1.22, P = 2.39 × 10-15) locating in the origin of plasmid replication (OriP). The results of population structure analysis of EBV isolates in East Asian showed the EBV strains derived from pLELC were more similar to those from nasopharyngeal carcinoma (NPC) than other EBV-associated diseases. In addition, typical latency type-II infection were recognized for EBV of pLELC at both transcription and methylation levels. Taken together, we defined the global view of EBV genomic profiles in pLELC patients for the first time, providing new insights to deepening our understanding of this rare EBV-associated primary lung carcinoma. IMPORTANCE Pulmonary lymphoepithelioma-like carcinoma (pLELC) is a rare, distinctive subtype of primary lung cancer closely associated with Epstein-Barr virus (EBV) infection. Here, we gave the first overview of pLELC-derived EBV at the level of genome, methylation and transcription. We obtained the EBV sequences data set from 78 primary pLELC patients, and revealed the sequences diversity across EBV genome and detected variability in known immune epitopes. Genome-wide association analysis combining 217 healthy controls identifies significant variations related to the risk of pLELC. Meanwhile, we characterized the integration landscapes of EBV at the genome-wide level. These results provided new insight for understanding EBV's role in pLELC tumorigenesis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/virologia , Infecções por Vírus Epstein-Barr/virologia , Genoma Viral/genética , Herpesvirus Humano 4/genética , Neoplasias Pulmonares/virologia , Povo Asiático , China , Metilação de DNA , Epitopos de Linfócito T/genética , Genes Virais/genética , Variação Genética , Estudo de Associação Genômica Ampla , Herpesvirus Humano 4/isolamento & purificação , Humanos , Integração Viral , Latência Viral/genéticaRESUMO
Compatibility among the influenza A virus (IAV) ribonucleoprotein (RNP) genes affects viral replication efficiency and can limit the emergence of novel reassortants, including those with potential pandemic risks. In this study, we determined the polymerase activities of 2,451 RNP reassortants among three seasonal and eight enzootic IAVs by using a minigenome assay. Results showed that the 2009 H1N1 RNP are more compatible with the tested enzootic RNP than seasonal H3N2 RNP and that triple reassortment increased such compatibility. The RNP reassortants among 2009 H1N1, canine H3N8, and avian H4N6 IAVs had the highest polymerase activities. Residues in the RNA binding motifs and the contact regions among RNP proteins affected polymerase activities. Our data indicates that compatibility among seasonal and enzootic RNPs are selective, and enzoosis of multiple strains in the animal-human interface can facilitate emergence of an RNP with increased replication efficiency in mammals, including humans.
Assuntos
Genes Virais/genética , Vírus da Influenza A/genética , Infecções por Orthomyxoviridae/genética , Vírus Reordenados/genética , Ribonucleoproteínas/genética , Animais , HumanosRESUMO
Maintenance of a balance between the levels of viral replication and selective pressure from the immune systems of insect vectors is one of the prerequisites for efficient transmission of insect-borne propagative phytoviruses. The mechanism regulating the adaptation of RNA viruses to insect vectors by genomic variation remains unknown. Our previous study demonstrated an extension of the 3'-untranslated terminal region (UTR) of two genomic segments of rice stripe virus (RSV). In the present study, a reverse genetic system for RSV in human cells and an insect vector, the small brown planthopper Laodelphax striatellus, was used to demonstrate that the 3'-terminal extensions suppressed viral replication in vector insects by inhibiting promoter activity due to structural interference with the panhandle structure formed by viral 3'- and 5'-UTRs. The extension sequence in the viral RNA1 segment was targeted by an endogenous insect microRNA, miR-263a, which decreased the inhibitory effect of the extension sequence on viral promoter activity. Surprisingly, the expression of miR-263a was negatively regulated by RSV infection. This elaborate coordination between terminal variation of the viral genome and endogenous insect microRNAs controls RSV replication in planthopper, thus reflecting a distinct strategy of adaptation of phytoviruses to insect vectors.
Assuntos
Genes de Insetos/genética , Genes Virais/genética , Insetos Vetores/genética , Insetos Vetores/virologia , Tenuivirus/genética , Animais , Humanos , MicroRNAs/genética , Replicação Viral/genéticaRESUMO
The genus Wolbachia is an archetype of maternally inherited intracellular bacteria that infect the germline of numerous invertebrate species worldwide. They can selfishly alter arthropod sex ratios and reproductive strategies to increase the proportion of the infected matriline in the population. The most common reproductive manipulation is cytoplasmic incompatibility, which results in embryonic lethality in crosses between infected males and uninfected females. Females infected with the same Wolbachia strain rescue this lethality. Despite more than 40 years of research and relevance to symbiont-induced speciation, as well as control of arbovirus vectors and agricultural pests, the bacterial genes underlying cytoplasmic incompatibility remain unknown. Here we use comparative and transgenic approaches to demonstrate that two differentially transcribed, co-diverging genes in the eukaryotic association module of prophage WO from Wolbachia strain wMel recapitulate and enhance cytoplasmic incompatibility. Dual expression in transgenic, uninfected males of Drosophila melanogaster crossed to uninfected females causes embryonic lethality. Each gene additively augments embryonic lethality in crosses between infected males and uninfected females. Lethality associates with embryonic defects that parallel those of wild-type cytoplasmic incompatibility and is notably rescued by wMel-infected embryos in all cases. The discovery of cytoplasmic incompatibility factor genes cifA and cifB pioneers genetic studies of prophage WO-induced reproductive manipulations and informs the continuing use of Wolbachia to control dengue and Zika virus transmission to humans.
Assuntos
Agentes de Controle Biológico , Citoplasma/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/microbiologia , Genes Virais/genética , Interações Hospedeiro-Patógeno , Prófagos/genética , Wolbachia/genética , Animais , Animais Geneticamente Modificados , Cruzamentos Genéticos , Citoplasma/patologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/fisiologia , Feminino , Masculino , Reprodução , Razão de Masculinidade , Simbiose , Wolbachia/classificação , Wolbachia/fisiologia , Wolbachia/virologiaRESUMO
The Betacoronaviruses comprise multiple subgenera whose members have been implicated in human disease. As with SARS, MERS and now SARS-CoV-2, the origin and emergence of new variants are often attributed to events of recombination that alter host tropism or disease severity. In most cases, recombination has been detected by searches for excessively similar genomic regions in divergent strains; however, such analyses are complicated by the high mutation rates of RNA viruses, which can produce sequence similarities in distant strains by convergent mutations. By applying a genome-wide approach that examines the source of individual polymorphisms and that can be tested against null models in which recombination is absent and homoplasies can arise only by convergent mutations, we examine the extent and limits of recombination in Betacoronaviruses. We find that recombination accounts for nearly 40% of the polymorphisms circulating in populations and that gene exchange occurs almost exclusively among strains belonging to the same subgenus. Although experimental studies have shown that recombinational exchanges occur at random along the coronaviral genome, in nature, they are vastly overrepresented in regions controlling viral interaction with host cells.
Assuntos
Betacoronavirus/classificação , Betacoronavirus/genética , Recombinação Genética/genética , Glicoproteína da Espícula de Coronavírus/genética , Troca Genética/genética , Genes Virais/genética , Genoma Viral/genética , Especificidade de Hospedeiro/genética , Modelos Genéticos , Polimorfismo Genético , SARS-CoV-2/classificação , SARS-CoV-2/genética , Tropismo Viral/genéticaRESUMO
Genome-wide epistasis analysis is a powerful tool to infer gene interactions, which can guide drug and vaccine development and lead to deeper understanding of microbial pathogenesis. We have considered all complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes deposited in the Global Initiative on Sharing All Influenza Data (GISAID) repository until four different cutoff dates, and used direct coupling analysis together with an assumption of quasi-linkage equilibrium to infer epistatic contributions to fitness from polymorphic loci. We find eight interactions, of which three are between pairs where one locus lies in gene ORF3a, both loci holding nonsynonymous mutations. We also find interactions between two loci in gene nsp13, both holding nonsynonymous mutations, and four interactions involving one locus holding a synonymous mutation. Altogether, we infer interactions between loci in viral genes ORF3a and nsp2, nsp12, and nsp6, between ORF8 and nsp4, and between loci in genes nsp2, nsp13, and nsp14. The paper opens the prospect to use prominent epistatically linked pairs as a starting point to search for combinatorial weaknesses of recombinant viral pathogens.
Assuntos
Epistasia Genética/genética , Genes Virais/genética , SARS-CoV-2/genética , COVID-19/patologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Exorribonucleases/genética , Genoma Viral/genética , Humanos , Metiltransferases/genética , RNA Helicases/genética , Seleção Genética/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Proteínas Viroporinas/genéticaRESUMO
Endogenous retroviruses and long terminal repeat (LTR) retrotransposons are mobile genetic elements that are closely related to retroviruses. Desilenced endogenous retroviruses are associated with human autoimmune disorders and neurodegenerative diseases. Caenorhabditis elegans and related Caenorhabditis spp. contain LTR retrotransposons and, as described here, numerous integrated viral genes including viral envelope genes that are part of LTR retrotransposons. We found that both LTR retrotransposons and endogenous viral elements are silenced by ADARs [adenosine deaminases acting on double-stranded RNA (dsRNA)] together with the endogenous RNA interference (RNAi) factor ERI-6/7, a homolog of MOV10 helicase, a retrotransposon and retrovirus restriction factor in human. siRNAs corresponding to integrated viral genes and LTR retrotransposons, but not to DNA transposons, are dependent on the ADARs and ERI-6/7. siRNAs corresponding to palindromic repeats are independent of the ADARs and ERI-6/7, and are in fact increased in adar- and eri-6/7-defective mutants because of an antiviral RNAi response to dsRNA. Silencing of LTR retrotransposons is dependent on downstream RNAi factors and P granule components but is independent of the viral sensor DRH-1/RIG-I and the nuclear Argonaute NRDE-3. The activation of retrotransposons in the ADAR- and ERI-6/7/MOV10-defective mutant is associated with the induction of the unfolded protein response (UPR), a common response to viral infection. The overlap between genes induced upon viral infection and infection with intracellular pathogens and genes coexpressed with retrotransposons suggests that there is a common response to different types of foreign elements that includes a response to proteotoxicity presumably caused by the burden of replicating pathogens and expressed retrotransposons.
Assuntos
Caenorhabditis elegans/genética , Retrovirus Endógenos/genética , Interações entre Hospedeiro e Microrganismos/genética , Interferência de RNA , Retroelementos/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Caenorhabditis elegans/virologia , Proteínas de Caenorhabditis elegans/metabolismo , DNA Helicases/metabolismo , DNA Viral/metabolismo , Estresse do Retículo Endoplasmático/genética , Regulação Viral da Expressão Gênica , Genes Virais/genética , Humanos , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Homologia de Sequência de Aminoácidos , Sequências Repetidas Terminais/genética , Resposta a Proteínas não Dobradas/genéticaRESUMO
Narnaviruses are RNA viruses detected in diverse fungi, plants, protists, arthropods, and nematodes. Though initially described as simple single-gene nonsegmented viruses encoding RNA-dependent RNA polymerase (RdRp), a subset of narnaviruses referred to as "ambigrammatic" harbor a unique genomic configuration consisting of overlapping open reading frames (ORFs) encoded on opposite strands. Phylogenetic analysis supports selection to maintain this unusual genome organization, but functional investigations are lacking. Here, we establish the mosquito-infecting Culex narnavirus 1 (CxNV1) as a model to investigate the functional role of overlapping ORFs in narnavirus replication. In CxNV1, a reverse ORF without homology to known proteins covers nearly the entire 3.2-kb segment encoding the RdRp. Additionally, two opposing and nearly completely overlapping novel ORFs are found on the second putative CxNV1 segment, the 0.8-kb "Robin" RNA. We developed a system to launch CxNV1 in a naive mosquito cell line and then showed that functional RdRp is required for persistence of both segments, and an intact reverse ORF is required on the RdRp segment for persistence. Mass spectrometry of persistently CxNV1-infected cells provided evidence for translation of this reverse ORF. Finally, ribosome profiling yielded a striking pattern of footprints for all four CxNV1 RNA strands that was distinct from actively translating ribosomes on host mRNA or coinfecting RNA viruses. Taken together, these data raise the possibility that the process of translation itself is important for persistence of ambigrammatic narnaviruses, potentially by protecting viral RNA with ribosomes, thus suggesting a heretofore undescribed viral tactic for replication and transmission. IMPORTANCE Fundamental to our understanding of RNA viruses is a description of which strand(s) of RNA are transmitted as the viral genome relative to which encode the viral proteins. Ambigrammatic narnaviruses break the mold. These viruses, found broadly in fungi, plants, and insects, have the unique feature of two overlapping genes encoded on opposite strands, comprising nearly the full length of the viral genome. Such extensive overlap is not seen in other RNA viruses and comes at the cost of reduced evolutionary flexibility in the sequence. The present study is motivated by investigating the benefits which balance that cost. We show for the first time a functional requirement for the ambigrammatic genome configuration in Culex narnavirus 1, which suggests a model for how translation of both strands might benefit this virus. Our work highlights a new blueprint for viral persistence, distinct from strategies defined by canonical definitions of the coding strand.
Assuntos
Culex/virologia , Genoma Viral/genética , Fases de Leitura Aberta/genética , Vírus de RNA/genética , Animais , Linhagem Celular , Genes Virais/genética , Elongação Traducional da Cadeia Peptídica/genética , Vírus de RNA/classificação , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Replicação Viral/genéticaRESUMO
African swine fever (ASF) is a severe hemorrhagic infectious disease in pigs caused by African swine fever virus (ASFV), leading to devastating economic losses in epidemic regions. Its control currently depends on thorough culling and clearance of the diseased and surrounding suspected pigs. An ASF vaccine has been extensively explored for years worldwide, especially in hog-intensive areas where it is highly desired, but it is still unavailable for numerous reasons. Here, we report another ASF vaccine candidate, named SY18ΔI226R, bearing a deletion of the I226R gene with a replacement of an enhanced green fluorescent protein (eGFP) expression cassette at the right end of the viral genome. This deletion results in the complete loss of virulence of SY18 as the gene-deleted strain does not cause any clinical symptoms in all pigs inoculated with a dosage of either 104.0 or 107.0 50% tissue culture infective doses (TCID50). Apparent viremia with a gradual decline was monitored, while virus shedding was detected only occasionally in oral or anal swabs. ASFV-specific antibody appeared at 9 days postinoculation. After intramuscular challenge with its parental strain ASFV SY18 at 21 days postinoculation, all the challenged pigs survived, without obvious febrile or abnormal clinical signs. No viral DNA could be detected upon the dissection of any tissue when viremia disappeared. These results indicated that SY18ΔI226R is safe in swine and elicits robust immunity to virulent ASFV infection. IMPORTANCE Outbreaks of African swine fever have resulted in devastating losses to the swine industry worldwide, but there is currently no commercial vaccine available. Although several vaccine candidates have been reported, none has been approved for use for several reasons, especially ones concerning biosafety. Here, we identified a new undescribed functional gene, I226R. When deleted from the ASFV genome, the virus completely loses its virulence in swine. Importantly, pigs infected with this gene-deleted virus were resistant to infection by intramuscular challenge with 102.5 or 104.0 TCID50 of its virulent parental virus. Furthermore, the nucleic acid of the gene-deleted virus and its virulent parental virus was rarely detected from oral or anal swabs. Viruses could not be detected in any tissues after necropsy when viremia became negative, indicating that robust immunity was achieved. Therefore, SY18ΔI226R is a novel, ideal, and efficacious vaccine candidate for genotype II ASF.