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1.
NAR Genom Bioinform ; 6(3): lqae104, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39157584

RESUMO

The traditional view of plus (+)-strand RNA virus transcriptomes is that infected cells contain a limited variety of viral RNAs, such as full-length (+)-strand genomic RNA(s), (-)-strand replication intermediate(s), 3' co-terminal subgenomic RNA(s), and viral recombinant defective (D)-RNAs. To ascertain the full complement of viral RNAs associated with the simplest plant viruses, long-read direct RNA nanopore sequencing was used to perform transcriptomic analyses of two related umbra-like viruses: citrus yellow vein-associated virus (CY1) from citrus and CY2 from hemp. Analysis of different timepoints/tissues in CY1- and CY2-infected Nicotiana benthamiana plants and CY2-infected hemp revealed: (i) three 5' co-terminal RNAs of 281 nt, 442 nt and 671 nt, each generated by a different mechanism; (ii) D-RNA populations containing the 671 fragment at their 5'ends; (iii) many full-length genomic RNAs and D-RNAs with identical 3'end 61 nt truncations; (iv) virtually all (-)-strand reads missing 3 nt at their 3' termini; (v) (±) foldback RNAs comprising about one-third of all (-)-strand reads and (vi) a higher proportion of full-length gRNAs in roots than in leaves, suggesting that roots may be functioning as a gRNA reservoir. These findings suggest that viral transcriptomes are much more complex than previously thought.

2.
PLoS Biol ; 22(4): e3002600, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38662792

RESUMO

The signature feature of all plant viruses is the encoding of movement proteins (MPs) that supports the movement of the viral genome into adjacent cells and through the vascular system. The recent discovery of umbravirus-like viruses (ULVs), some of which only encode replication-associated proteins, suggested that they, as with umbraviruses that lack encoded capsid proteins (CPs) and silencing suppressors, would require association with a helper virus to complete an infection cycle. We examined the infection properties of 2 ULVs: citrus yellow vein associated virus 1 (CY1), which only encodes replication proteins, and closely related CY2 from hemp, which encodes an additional protein (ORF5CY2) that was assumed to be an MP. We report that both CY1 and CY2 can independently infect the model plant Nicotiana benthamiana in a phloem-limited fashion when delivered by agroinfiltration. Unlike encoded MPs, ORF5CY2 was dispensable for infection of CY2, but was associated with faster symptom development. Examination of ORF5CY2 revealed features more similar to luteoviruses/poleroviruses/sobemovirus CPs than to 30K class MPs, which all share a similar single jelly-roll domain. In addition, only CY2-infected plants contained virus-like particles (VLPs) associated with CY2 RNA and ORF5CY2. CY1 RNA and a defective (D)-RNA that arises during infection interacted with host protein phloem protein 2 (PP2) in vitro and in vivo, and formed a high molecular weight complex with sap proteins in vitro that was partially resistant to RNase treatment. When CY1 was used as a virus-induced gene silencing (VIGS) vector to target PP2 transcripts, CY1 accumulation was reduced in systemic leaves, supporting the usage of PP2 for systemic movement. ULVs are therefore the first plant viruses encoding replication and CPs but no MPs, and whose systemic movement relies on a host MP. This explains the lack of discernable helper viruses in many ULV-infected plants and evokes comparisons with the initial viruses transferred into plants that must have similarly required host proteins for movement.


Assuntos
Nicotiana , Doenças das Plantas , Proteínas do Movimento Viral em Plantas , Nicotiana/virologia , Nicotiana/genética , Nicotiana/metabolismo , Doenças das Plantas/virologia , Proteínas do Movimento Viral em Plantas/metabolismo , Proteínas do Movimento Viral em Plantas/genética , Vírus de RNA/genética , Vírus de RNA/fisiologia , Vírus de RNA/metabolismo , Vírus de Plantas/fisiologia , Vírus de Plantas/genética , Vírus de Plantas/metabolismo , Vírus de Plantas/patogenicidade , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , RNA Viral/genética , RNA Viral/metabolismo , Genoma Viral , Floema/virologia , Floema/metabolismo
3.
J Virol ; 97(4): e0024523, 2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37017521

RESUMO

Viruses constantly evolve and adapt to the antiviral defenses of their hosts. The biology of viral circumvention of these selective pressures can often be attributed to the acquisition of novel antagonistic gene products or by rapid genome change that prevents host recognition. To study viral evasion of RNA interference (RNAi)-based defenses, we established a robust antiviral system in mammalian cells using recombinant Sendai virus designed to be targeted by endogenous host microRNAs (miRNAs) with perfect complementarity. Using this system, we previously demonstrated the intrinsic ability of positive-strand RNA viruses to escape this selective pressure via homologous recombination, which was not observed in negative-strand RNA viruses. Here, we show that given extensive time, escape of miRNA-targeted Sendai virus was enabled by host adenosine deaminase acting on RNA 1 (ADAR1). Independent of the viral transcript targeted, ADAR1 editing resulted in disruption of the miRNA-silencing motif, suggesting an intolerance for extensive RNA-RNA interactions necessary for antiviral RNAi. This was further supported in Nicotiana benthamiana, where exogenous expression of ADAR1 interfered with endogenous RNAi. Together, these results suggest that ADAR1 diminishes the effectiveness of RNAi and may explain why it is absent in species that utilize this antiviral defense system. IMPORTANCE All life at the cellular level has the capacity to induce an antiviral response. Here, we examine the result of imposing the antiviral response of one branch of life onto another and find evidence for conflict. To determine the consequences of eliciting an RNAi-like defense in mammals, we applied this pressure to a recombinant Sendai virus in cell culture. We find that ADAR1, a host gene involved in regulation of the mammalian response to virus, prevented RNAi-mediated silencing and subsequently allowed for viral replication. In addition, the expression of ADAR1 in Nicotiana benthamiana, which lacks ADARs and has an endogenous RNAi system, suppresses gene silencing. These data indicate that ADAR1 is disruptive to RNAi biology and provide insight into the evolutionary relationship between ADARs and antiviral defenses in eukaryotic life.


Assuntos
Adenosina Desaminase , Interações entre Hospedeiro e Microrganismos , MicroRNAs , Interferência de RNA , Infecções por Respirovirus , Animais , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Antivirais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Replicação Viral/genética , Vírus Sendai/classificação , Inativação Gênica , Humanos , Mutação , Fases de Leitura Aberta , Evolução Biológica , Interações entre Hospedeiro e Microrganismos/genética , Infecções por Respirovirus/metabolismo , Infecções por Respirovirus/virologia
4.
Viruses ; 15(3)2023 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-36992347

RESUMO

The cap-independent translation of plus-strand RNA plant viruses frequently depends on 3' structures to attract translation initiation factors that bind ribosomal subunits or bind directly to ribosomes. Umbraviruses are excellent models for studying 3' cap-independent translation enhancers (3'CITEs), as umbraviruses can have different 3'CITEs in the central region of their lengthy 3'UTRs, and most also have a particular 3'CITE (the T-shaped structure or 3'TSS) near their 3' ends. We discovered a novel hairpin just upstream of the centrally located (known or putative) 3'CITEs in all 14 umbraviruses. These CITE-associated structures (CASs) have conserved sequences in their apical loops and at the stem base and adjacent positions. In 11 umbraviruses, CASs are preceded by two small hairpins joined by a putative kissing loop interaction (KL). Converting the conserved 6-nt apical loop to a GNRA tetraloop in opium poppy mosaic virus (OPMV) and pea enation mosaic virus 2 (PEMV2) enhanced translation of genomic (g)RNA, but not subgenomic (sg)RNA reporter constructs, and significantly repressed virus accumulation in Nicotiana benthamiana. Other alterations throughout OPMV CAS also repressed virus accumulation and only enhanced sgRNA reporter translation, while mutations in the lower stem repressed gRNA reporter translation. Similar mutations in the PEMV2 CAS also repressed accumulation but did not significantly affect gRNA or sgRNA reporter translation, with the exception of deletion of the entire hairpin, which only reduced translation of the gRNA reporter. OPMV CAS mutations had little effect on the downstream BTE 3'CITE or upstream KL element, while PEMV2 CAS mutations significantly altered KL structures. These results introduce an additional element associated with different 3'CITEs that differentially affect the structure and translation of different umbraviruses.


Assuntos
Tombusviridae , Regiões 3' não Traduzidas , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Ribossomos/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Tombusviridae/genética , Tombusviridae/metabolismo , RNA Subgenômico/genética
5.
Viruses ; 14(12)2022 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-36560619

RESUMO

The 3' untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3'UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3' cap-independent translation enhancers (3'CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3'TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3' terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed "Trio", that are exclusively found in a subset of umbraviruses and are located just upstream of the 3'TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3' terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3'CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement.


Assuntos
Carmovirus , Tombusviridae , Tombusviridae/genética , Tombusviridae/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Replicação Viral , Regiões 3' não Traduzidas , Biossíntese de Proteínas
6.
mBio ; 11(2)2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32156817

RESUMO

The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3' untranslated regions (UTRs). The umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3' UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant's vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana, p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3' UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor.IMPORTANCE Nonsense-mediated decay (NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3' untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.


Assuntos
Interações entre Hospedeiro e Microrganismos/genética , Degradação do RNAm Mediada por Códon sem Sentido , Pisum sativum/virologia , Tombusviridae/genética , Proteínas Virais/genética , Regiões 3' não Traduzidas/genética , Vírus de RNA/genética , RNA Viral/genética , RNA-Seq , Nicotiana/virologia , Tombusviridae/metabolismo , Proteínas Virais/metabolismo
7.
PLoS Pathog ; 14(11): e1007459, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30452463

RESUMO

Nonsense-mediated decay (NMD) is a host RNA control pathway that removes aberrant transcripts with long 3' untranslated regions (UTRs) due to premature termination codons (PTCs) that arise through mutation or defective splicing. To maximize coding potential, RNA viruses often contain internally located stop codons that should also be prime targets for NMD. Using an agroinfiltration-based NMD assay in Nicotiana benthamiana, we identified two segments conferring NMD-resistance in the carmovirus Turnip crinkle virus (TCV) genome. The ribosome readthrough structure just downstream of the TCV p28 termination codon stabilized an NMD-sensitive reporter as did a frameshifting element from umbravirus Pea enation mosaic virus. In addition, a 51-nt unstructured region (USR) at the beginning of the TCV 3' UTR increased NMD-resistance 3-fold when inserted into an unrelated NMD-sensitive 3' UTR. Several additional carmovirus 3' UTRs also conferred varying levels of NMD resistance depending on the construct despite no sequence similarity in the analogous region. Instead, these regions displayed a marked lack of RNA structure immediately following the NMD-targeted stop codon. NMD-resistance was only slightly reduced by conversion of 19 pyrimidines in the USR to purines, but resistance was abolished when a 2-nt mutation was introduced downstream of the USR that substantially increased the secondary structure in the USR through formation of a stable hairpin. The same 2-nt mutation also enhanced the NMD susceptibility of a subgenomic RNA expressed independently of the genomic RNA. The conserved lack of RNA structure among most carmoviruses at the 5' end of their 3' UTR could serve to enhance subgenomic RNA stability, which would increase expression of the encoded capsid protein that also functions as the RNA silencing suppressor. These results demonstrate that the TCV genome has features that are inherently NMD-resistant and these strategies could be widespread among RNA viruses and NMD-resistant host mRNAs with long 3' UTRs.


Assuntos
Carmovirus/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Regiões 3' não Traduzidas/genética , Carmovirus/patogenicidade , Códon sem Sentido/genética , Códon de Terminação/genética , Biossíntese de Proteínas , Interferência de RNA/fisiologia , Estabilidade de RNA/genética , Vírus de RNA/genética , RNA Viral/genética , Ribossomos , Nicotiana/genética
8.
J Virol ; 89(22): 11603-18, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26355083

RESUMO

UNLABELLED: Turnip crinkle virus (TCV) contains a structured 3' region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3' untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3'UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or in dcl-2/dcl4 or ago1/ago2 backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCV-rev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs. IMPORTANCE: Genomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3' UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3' UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.


Assuntos
Regiões 3' não Traduzidas/genética , Carmovirus/genética , Interações Hospedeiro-Patógeno/genética , Dobramento de RNA/genética , Interferência de RNA , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas Argonautas/genética , Proteínas do Capsídeo/genética , Mutação/genética , Fases de Leitura Aberta/genética , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , RNA Viral/genética , Nicotiana/virologia
9.
J Virol ; 85(10): 4638-53, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21389125

RESUMO

Plus-strand RNA viruses without 5' caps require noncanonical mechanisms for ribosome recruitment. A translational enhancer in the 3' untranslated region (UTR) of Turnip crinkle virus (TCV) contains an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. We now report that the 63-nucleotide (nt) 5' UTR of TCV contains a 19-nt pyrimidine-rich element near the initiation codon that supports translation of an internal open reading frame (ORF) independent of upstream 5' UTR sequences. Addition of 80S ribosomes to the 5' UTR reduced the flexibility of the polypyrimidine residues and generated a toeprint consistent with binding to this region. Binding of salt-washed 40S ribosomal subunits was reduced 6-fold when the pyrimidine-rich sequence was mutated. 40S subunit binding generated the same toeprint as 80S ribosomes but also additional ones near the 5' end. Generation of out-of-frame AUGs upstream of the polypyrimidine region reduced translation, which suggests that 5'-terminal entry of 40S subunits is followed by scanning and that the polypyrimidine region is needed for an alternative function that requires ribosome binding. No evidence for RNA-RNA interactions between 5' and 3' sequences was found, suggesting that TCV utilizes an alternative means for circularizing its genome. Combining 5' and 3' UTR fragments in vitro had no discernible effect on the structures of the RNAs. In contrast, when 80S ribosomes were added to both fragments, structural changes were found in the 5' UTR polypyrimidine tract that were not evident when ribosomes interacted with the individual fragments. This suggests that ribosomes can promote an interaction between the 5' and 3' UTRs of TCV.


Assuntos
Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Carmovirus/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Ribossomos/metabolismo , Conformação de Ácido Nucleico , Proteínas Virais/biossíntese
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