scholarly journals Rescue of Tomato spotted wilt tospovirus entirely from cDNA clones, establishment of the first reverse genetics system for a segmented (-)RNA plant virus

2019 ◽  
Author(s):  
Mingfeng Feng ◽  
Ruixiang Cheng ◽  
Minglong Chen ◽  
Rong Guo ◽  
Luyao Li ◽  
...  
Keyword(s):  
Life Cycle ◽  
Reverse Genetics ◽  
Molecular Genetic ◽  
Cdna Clones ◽  
Genome Replication ◽  
Negative Strand ◽  
Replication System ◽  
Ribonucleoprotein Complexes ◽  
Tomato Spotted Wilt ◽  
Rna Genome

AbstractThe group of negative strand RNA viruses (NSVs) includes not only dangerous pathogens of medical importance but also serious plant pathogens of agronomical importance. Tomato spotted wilt tospovirus (TSWV) is one of those plant NSVs that cause severe diseases on agronomic crops and pose major threats to global food security. Its negative-strand segmented RNA genome has, however, always posed a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from cDNA clones, the first reverse genetics (RG) system for a segmented plant NSV. First, a replication and transcription competent mini-genome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by a 5’ Hammerhead and 3’ Ribozyme sequence of Hepatitis Delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA dependent RNA polymerase (RdRp) gene. Next, a movement competent mini-genome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. After further optimization, infectious TSWV and derivatives carrying eGFP reporters were successfully rescuedin plantavia simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA and L(+)-agRNA. Viral rescue occurred in the additional presence of various viral suppressors of RNAi, but TSWV NSs interfered with the rescue of genomic RNA. The establishment of a RG system for TSWV now allows detailed molecular genetic analysis of all aspects of tospovirus life cycle and their pathogenicity.SignificanceFor many different animal-infecting segmented negative-strand viruses (NSVs), a reverse genetics system has been established that allows the generation of mutant viruses to study disease pathology and the role ofcis- andtrans-acting elements in the virus life cycle. In contrast to the relative ease to establish RG systems for animal-infecting NSVs, establishment of such system for the plant-infecting NSVs with a segmented RNA genome so far has not been successful. Here we report the first reverse genetics system for a segmented plant NSV, the Tomato spotted wilt tospovirus, a virus with a tripartite RNA genome. The establishment of this RG system now provides us with a new and powerful platform to study their disease pathology during a natural infection.

scholarly journals Rescue of tomato spotted wilt virus entirely from complementary DNA clones

2019 ◽  
Vol 117 (2) ◽  
pp. 1181-1190 ◽  
Author(s):  
Mingfeng Feng ◽  
Ruixiang Cheng ◽  
Minglong Chen ◽  
Rong Guo ◽  
Luyao Li ◽  
...  
Keyword(s):  
Tomato Spotted Wilt Virus ◽  
Plant Pathogens ◽  
Genetic Manipulation ◽  
Molecular Genetic ◽  
Cdna Clones ◽  
Complementary Dna ◽  
Replication System ◽  
Ribonucleoprotein Complexes ◽  
Tomato Spotted Wilt ◽  
Wilt Virus

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S(−)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by the 5′ hammerhead and 3′ ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M(−)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(−)-gRNA, and L(+)-agRNA in which the glycoprotein gene sequence of M(−)-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and γb, but TSWV NSs interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity.

scholarly journals Generation of Measles Virus with a Segmented RNA Genome

2006 ◽  
Vol 80 (9) ◽  
pp. 4242-4248 ◽  
Author(s):  
Makoto Takeda ◽  
Yuichiro Nakatsu ◽  
Shinji Ohno ◽  
Fumio Seki ◽  
Maino Tahara ◽  
...  
Keyword(s):  
Measles Virus ◽  
Reverse Genetics ◽  
Cultured Cells ◽  
Rna Viruses ◽  
Genome Replication ◽  
The Family ◽  
New Strategy ◽  
Foreign Proteins ◽  
Transcriptional Units ◽  
Rna Genome

ABSTRACT Viruses classified in the order Mononegavirales have a single nonsegmented RNA molecule as the genome and employ similar strategies for genome replication and gene expression. Infectious particles of Measles virus (MeV), a member of the family Paramyxoviridae in the order Mononegavirales, with two or three RNA genome segments (2 seg- or 3 seg-MeV) were generated using a highly efficient reverse genetics system. All RNA segments of the viruses were designed to have authentic 3′ and 5′ self-complementary termini, similar to those of negative-stranded RNA viruses that intrinsically have multiple RNA genome segments. The 2 seg- and 3 seg-MeV were viable and replicated well in cultured cells. 3 seg-MeV could accommodate up to six additional transcriptional units, five of which were shown to be capable of expressing foreign proteins efficiently. These data indicate that the MeV genome can be segmented, providing an experimental insight into the divergence of the negative-stranded RNA viruses with nonsegmented or segmented RNA genomes. They also illustrate a new strategy to develop mononegavirus-derived vectors harboring multiple additional transcriptional units.

scholarly journals Attenuation of Bunyavirus Replication by Rearrangement of Viral Coding and Noncoding Sequences

2005 ◽  
Vol 79 (11) ◽  
pp. 6940-6946 ◽  
Author(s):  
Anice C. Lowen ◽  
Amanda Boyd ◽  
John K. Fazakerley ◽  
Richard M. Elliott
Keyword(s):  
Mammalian Cells ◽  
Reverse Genetics ◽  
Viral Rna ◽  
Wild Type Virus ◽  
Coding Region ◽  
Reading Frame ◽  
Ribonucleoprotein Complexes ◽  
The Family ◽  
Prototype Virus ◽  
Negative Sense

ABSTRACT Bunyamwera virus (BUN) is the prototype virus of the family Bunyaviridae. BUN has a tripartite negative-sense RNA genome comprising small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments, while the internal regions are unique. The UTRs direct replication and transcription of viral RNA and are sufficient to allow encapsidation of viral RNA into ribonucleoprotein complexes. To investigate the segment-specific functions of the UTRs, we have used reverse genetics to recover a recombinant virus (called BUN MLM) in which the L segment open reading frame (ORF) is flanked by the M segment UTRs. Compared to wild-type virus, BUN MLM virus shows growth attenuation in cultured mammalian cells and a slower disease progression in mice, produces small plaques, expresses reduced levels of L mRNA and L (RNA polymerase) protein, synthesizes less L genomic and antigenomic RNA, and has an increased particle-to-PFU ratio. Our data suggest that the packaging of BUN RNAs is not segment specific. In addition, the phenotype of BUN MLM virus supports the finding that BUN UTRs differ in their regulation of RNA synthesis but suggests that the interplay between each segment UTR and its cognate ORF may contribute to that regulation. Since BUN MLM virus is attenuated due to an essentially irreversible mutation, the rearrangement of UTRs is a feasible strategy for vaccine design for the more pathogenic members of the Bunyaviridae.

scholarly journals The Combined Use of Alphavirus Replicons and Pseudoinfectious Particles for the Discovery of Antivirals Derived from Natural Products

2014 ◽  
Vol 20 (5) ◽  
pp. 673-680 ◽  
Author(s):  
Phillip C. Delekta ◽  
Avi Raveh ◽  
Martha J. Larsen ◽  
Pamela J. Schultz ◽  
Giselle Tamayo-Castillo ◽  
...  
Keyword(s):  
Life Cycle ◽  
Antiviral Activity ◽  
Viral Entry ◽  
Particle System ◽  
Antiviral Agents ◽  
Viral Life Cycle ◽  
Genome Replication ◽  
Bacterial Strains ◽  
Antiviral Compounds ◽  
Western Equine Encephalitis

Alphaviruses are a prominent class of reemergent pathogens due to their globally expanding ranges, potential for lethality, and possible use as bioweapons. The absence of effective treatments for alphaviruses highlights the need for innovative strategies to identify antiviral agents. Primary screens that use noninfectious self-replicating RNAs, termed replicons, have been used to identify potential antiviral compounds for alphaviruses. Only inhibitors of viral genome replication, however, will be identified using replicons, which excludes many other druggable steps in the viral life cycle. To address this limitation, we developed a western equine encephalitis virus pseudoinfectious particle system that reproduces several crucial viral life cycle steps in addition to genome replication. We used this system to screen a library containing ~26,000 extracts derived from marine microbes, and we identified multiple bacterial strains that produce compounds with potential antiviral activity. We subsequently used pseudoinfectious particle and replicon assays in parallel to counterscreen candidate extracts, and followed antiviral activity during biochemical fractionation and purification to differentiate between inhibitors of viral entry and genome replication. This novel process led to the isolation of a known alphavirus entry inhibitor, bafilomycin, thereby validating the approach for the screening and identification of potential antiviral compounds.

Engineered Resistance to Tomato Spotted Wilt Virus, a Negative-Strand RNA Virus

1993 ◽  
pp. 386-386
Author(s):  
Jan J. L. Gielen ◽  
Peter de Haan ◽  
Mart Q. J. M. van Grinsven ◽  
Rob Goldbach ◽  
André W. Schram
Keyword(s):  
Tomato Spotted Wilt Virus ◽  
Rna Virus ◽  
Negative Strand ◽  
Tomato Spotted Wilt ◽  
Engineered Resistance ◽  
Wilt Virus

scholarly journals Going beyond Integration: The Emerging Role of HIV-1 Integrase in Virion Morphogenesis

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1005 ◽  
Author(s):  
Jennifer L. Elliott ◽  
Sebla B. Kutluay
Keyword(s):  
Life Cycle ◽  
Viral Genome ◽  
Critical Role ◽  
Viral Rna ◽  
Target Cells ◽  
Host Chromosome ◽  
Virion Morphogenesis ◽  
Rna Genome ◽  
Hiv 1

The HIV-1 integrase enzyme (IN) plays a critical role in the viral life cycle by integrating the reverse-transcribed viral DNA into the host chromosome. This function of IN has been well studied, and the knowledge gained has informed the design of small molecule inhibitors that now form key components of antiretroviral therapy regimens. Recent discoveries unveiled that IN has an under-studied yet equally vital second function in human immunodeficiency virus type 1 (HIV-1) replication. This involves IN binding to the viral RNA genome in virions, which is necessary for proper virion maturation and morphogenesis. Inhibition of IN binding to the viral RNA genome results in mislocalization of the viral genome inside the virus particle, and its premature exposure and degradation in target cells. The roles of IN in integration and virion morphogenesis share a number of common elements, including interaction with viral nucleic acids and assembly of higher-order IN multimers. Herein we describe these two functions of IN within the context of the HIV-1 life cycle, how IN binding to the viral genome is coordinated by the major structural protein, Gag, and discuss the value of targeting the second role of IN in virion morphogenesis.

scholarly journals Genus-specific recruitment of filovirus ribonucleoprotein complexes into budding particles

2011 ◽  
Vol 92 (12) ◽  
pp. 2900-2905 ◽  
Author(s):  
Larissa Spiegelberg ◽  
Victoria Wahl-Jensen ◽  
Larissa Kolesnikova ◽  
Heinz Feldmann ◽  
Stephan Becker ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Spherical Particles ◽  
Target Cells ◽  
Genome Replication ◽  
Replication Competent Virus ◽  
Ribonucleoprotein Complexes ◽  
Virus Morphogenesis ◽  
Rnp Complex ◽  
Model Virus ◽  
Transcription And Replication

The filoviral matrix protein VP40 orchestrates virus morphogenesis and budding. To do this it interacts with both the glycoprotein (GP1,2) and the ribonucleoprotein (RNP) complex components; however, these interactions are still not well understood. Here we show that for efficient VP40-driven formation of transcription and replication-competent virus-like particles (trVLPs), which contain both an RNP complex and GP1,2, the RNP components and VP40, but not GP1,2 and VP40, must be from the same genus. trVLP preparations contained both spherical and filamentous particles, but only the latter were able to infect target cells and to lead to genome replication and transcription. Interestingly, the genus specificity of the VP40–RNP interactions was specific to the formation of filamentous trVLPs, but not to spherical particles. These results not only further our understanding of VP40 interactions, but also suggest that special care is required when using trVLP or VLP systems to model virus morphogenesis.

scholarly journals Negative-Strand Tospoviruses and Tenuiviruses Carry a Gene for a Suppressor of Gene Silencing at Analogous Genomic Positions

2003 ◽  
Vol 77 (2) ◽  
pp. 1329-1336 ◽  
Author(s):  
Etienne Bucher ◽  
Titia Sijen ◽  
Peter de Haan ◽  
Rob Goldbach ◽  
Marcel Prins
Keyword(s):  
Rna Silencing ◽  
Tomato Spotted Wilt Virus ◽  
Fluorescent Protein ◽  
Negative Strand ◽  
Tomato Spotted Wilt ◽  
Ns3 Protein ◽  
Genes Encoding ◽  
Green Fluorescent ◽  
Wilt Virus ◽  
Suppressor Of Rna Silencing

ABSTRACT Posttranscriptional silencing of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana plants was suppressed when these plants were infected with Tomato spotted wilt virus (TSWV), a plant-infecting member of the Bunyaviridae. Infection with TSWV resulted in complete reactivation of GFP expression, similar to the case for Potato virus Y, but distinct from that for Cucumber mosaic virus, two viruses known to carry genes encoding silencing suppressor proteins. Agrobacterium-based leaf injections with individual TSWV genes identified the NSS gene to be responsible for the RNA silencing-suppressing activity displayed by this virus. The absence of short interfering RNAs in NSS-expressing leaf sectors suggests that the tospoviral NSS protein interferes with the intrinsic RNA silencing present in plants. Suppression of RNA silencing was also observed when the NS3 protein of the Rice hoja blanca tenuivirus, a nonenveloped negative-strand virus, was expressed. These results indicate that plant-infecting negative-strand RNA viruses carry a gene for a suppressor of RNA silencing.

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