Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus

Qian, Shasha; Chen, Xiaolan; Sun, Kai; Zhang, Yang; Li, Zhenghe

doi:10.1186/s12985-017-0776-7

Cited by 9 publications

(11 citation statements)

References 33 publications

(61 reference statements)

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“…The strategy was to clone the full-length viral genome encoding cDNA into binary vectors next to the 35S promoter and provide the nucleocapsid (NC) and replication factors on separate plasmids (7, 25, 26). Success in this system required the co-delivery of the viral replicase as well as several viral silencing suppressor proteins to reduce the effects of the cellular silencing machinery while galvanizing the initial replication steps (6, 7, 26). There is also a breakthrough in developing a reverse genetic system for tospoviruses by the Tao Xiaorong lab (personal communication).…”

Section: Discussionmentioning

confidence: 99%

“…The infectious cDNA technology was rapidly adopted for studying positive strand RNA viruses. Since exact 5’ ends are critical for launching the first round of translation and replication of transcripts to generate infectious virus genomes, plasmids and infectious cDNA inserts are often combined with bacteriophage T7, SP6, RNA pol I, or RNA pol II promoters fused to the exact 5’ end of the virus genomic cDNA (6–11). Exact 3’ ends are also critical and so transcriptional terminators or hepatitis delta virus ribozymes are often located at the 3’ ends to produce transcripts with exact 3’ ends (7, 8, 12–14).…”

Section: Introductionmentioning

confidence: 99%

“…Plasmids encoding the nucleocapsid core or subunits of the viral polymerase are co-delivered with the agRNA encoding cDNAs that successfully spur the replication process. The first infectious clone of a negative strand RNA virus that infects plants was Sonchus yellow net virus (SYNV) (6, 7, 25, 30). The SYNV full-length cDNA was introduced into a binary vector fused to a duplicated Cauliflower mosaic virus 35S promoter.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Rose rosette virus and development of reverse genetic system for studying virus accumulation and movement in whole plants

Verchot¹,

Herath²,

Urrutia

et al. 2019

Preprint

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6 that includes all seven segments of the negative-strand RNA genome. 7 8 Abstract 32The ability to mutate the genomic nucleic acid of viruses is the most straight-forward approach 33to understanding virus genetics. Reverse genetics of RNA viruses involves the introduction of 34 mutations at the cDNA level, and then introducing cDNAs into cells to produce infectious 35 progeny virus. Reverse genetic tools are used to investigate the products of viral genes, virus-36host interactions relating to pathogenicity and immunity, and requirements for vector 37 transmission. The technology has been slow to develop for viruses with negative strand RNA 38 genomes and has been especially difficult for plant viruses with multicomponent negative strand 39RNA genomes, many of which require an insect vector for transmission to be successful. Rose 40 rosette virus (RRV; Emaravirus) is a negative-sense RNA virus with a 7-segmented genome 41 that is enclosed by a double membrane (1-4). We devised a technology for delivery of plant sap 42 inoculum which can also deliver agrobacterium containing infectious clones to rose plants. We 43 report the first reverse genetic system for a member of the Emaravirus genus, Rose rosette 44 virus (RRV). We introduced fluorescent proteins at three locations in the seven segmented 45 genome and learned that such reporters can be stably maintained during systemic infection. 46This study demonstrates that RRV can infect Arabidopsis causing significant growth alterations 47 of the plant, while causing mild to more serious disease symptoms in Nicotiana benthamiana 48and two varieties of roses. This reverse genetic system creates new opportunities for studying 49 negative strand RNA viruses infecting plants. 50 Significant Statement 51Since an infectious clone for influenza virus was developed in 1998, little progress has been 52 made in infectious clone technology for viruses with negative strand genomes. We constructed 53 an infectious clone of the seven-segmented RRV genome that is contained in a binary vector 54 and delivered by Agrobacterium. RRV has emerged as a serious threat to cultivated roses, 55 causing millions of dollars in losses to commercial producers. This technology is a game 56 changer for investigations into Emaravirus genetics, studies of molecular virus-host interactions, 57 as well as for rose breeders who can use the infectious clone for rapid germplasm screening to 58 identify useful resistance for breeding programs. 59

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of Rose rosette virus and development of reverse genetic system for studying virus accumulation and movement in whole plants

Verchot¹,

Herath²,

Urrutia

et al. 2019

Preprint

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“…Agroinfiltration and agroinfection assays. SYNV minireplicon assays were carried out by infiltration of N. benthamiana leaf tissues with Agrobacterium tumefaciens strains to deliver the pSYNV MR GFP-RFP plasmid, the pGD-NPL plasmid, and the p19, ␥b, and P1/HC-Pro viral suppressors of RNA silencing (VSR) expression plasmid, as described previously (51,88). In addition, agrobacterial strains containing individual plasmids for expression of wild-type or mutated M proteins or of the sc4 protein were mixted with the above-mentioned mixtures at a ratio of 1:6 (vol/vol) with a total optical density (OD) A 600 of 0.7.…”

Section: Methodsmentioning

confidence: 99%

“…To generate RNA size markers for GFP and MR, we amplified the cDNAs using the primer pair T7GFPF (5=-TAATACGACTCACTATAGGGATGGTGAGCAAGGGCGAGGA-3=) and GFPR (5=-TTACTTGTACAGCTCGTCCATGC-3=) and the pair T7MRF (5=-TAATACGACTCACTATAGAGAG ACAGAAACTCAGAAAATACAATC-3=) and MRR (5=-AGAGACAAAAGCTCAGAACAATCC-3=), respectively. The forward primers contained the T7 promoter positioned at the 5=terminus (underlined), and the PCR products were transcribed in vitro by T7 RNA polymerase as described previously (88).…”

Section: Methodsmentioning

confidence: 99%

The Matrix Protein of a Plant Rhabdovirus Mediates Superinfection Exclusion by Inhibiting Viral Transcription

Zhou

Sun

Zhou

et al. 2019

J Virol

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Superinfection exclusion (SIE) or cross-protection phenomena have been documented for plant viruses for nearly a century and are widespread among taxonomically diverse viruses, but little information is available about SIE of plant negative-strand RNA viruses. Here, we demonstrate that SIE by sonchus yellow net nucleorhabdovirus virus (SYNV) is mediated by the viral matrix (M) protein, a multifunctional protein involved in transcription regulation, virion assembly, and virus budding. We show that fluorescent protein-tagged SYNV variants display mutual exclusion/cross-protection in Nicotiana benthamiana plants. Transient expression of the SYNV M protein, but not other viral proteins, interfered with SYNV local infections. In addition, SYNV M deletion mutants failed to exclude superinfection by wild-type SYNV. An SYNV minireplicon reporter gene expression assay showed that the M protein inhibited viral transcription. However, M protein mutants with weakened nuclear localization signals (NLS) and deficient nuclear interactions with the SYNV nucleocapsid protein were unable to suppress transcription. Moreover, SYNV with M NLS mutations exhibited compromised SIE against wild-type SYNV. From these data, we propose that M protein accumulating in nuclei with primary SYNV infections either coils or prevents uncoiling of nucleocapsids released by the superinfecting SYNV virions and suppresses transcription of superinfecting genomes, thereby preventing superinfection. Our model suggests that the rhabdovirus M protein regulates the transition from replication to virion assembly and renders the infected cells nonpermissive for secondary infections. IMPORTANCE Superinfection exclusion (SIE) is a widespread phenomenon in which an established virus infection prevents reinfection by closely related viruses. Understanding the mechanisms governing SIE will not only advance our basic knowledge of virus infection cycles but may also lead to improved design of antiviral measures. Despite the significance of SIE, our knowledge about viral SIE determinants and their modes of actions remain limited. In this study, we show that sonchus yellow net virus (SYNV) SIE is mediated by the viral matrix (M) protein. During primary infections, accumulation of M protein in infected nuclei results in coiling of genomic nucleocapsids and suppression of viral transcription. Consequently, nucleocapsids released by potential superinfectors are sequestered and are unable to initiate new infections. Our data suggest that SYNV SIE is caused by M protein-mediated transition from replication to virion assembly and that this process prevents secondary infections.

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