scholarly journals RNA1-Independent Replication and GFP Expression from Tomato marchitez virus Isolate M Cloned cDNA

2016 ◽  
Vol 106 (5) ◽  
pp. 500-509 ◽  
Author(s):  
I. Ferriol ◽  
M. Turina ◽  
E. J. Zamora-Macorra ◽  
B. W. Falk
Keyword(s):  
Fluorescent Protein ◽  
Rna Virus ◽  
Cell Movement ◽  
Full Length ◽  
Wild Type Virus ◽  
Cdna Clones ◽  
Genomic Rna ◽  
Positive Strand Rna Virus ◽  
Green Fluorescent ◽  
Positive Strand Rna

Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus–host–vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA.

scholarly journals A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses

2008 ◽  
Vol 83 (4) ◽  
pp. 1981-1991 ◽  
Author(s):  
Huiquan Liu ◽  
Yanping Fu ◽  
Daohong Jiang ◽  
Guoqing Li ◽  
Jun Xie ◽  
...  
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Hepatitis E Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Hepatitis E ◽  
Genomic Rna ◽  
Human Virus ◽  
Positive Strand ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT Previously, we reported that three double-stranded RNA (dsRNA) segments, designated L-, M-, and S-dsRNAs, were detected in Sclerotinia sclerotiorum strain Ep-1PN. Of these, the M-dsRNA segment was derived from the genomic RNA of a potexvirus-like positive-strand RNA virus, Sclerotinia sclerotiorum debilitation-associated RNA virus. Here, we present the complete nucleotide sequence of the L-dsRNA, which is 6,043 nucleotides in length, excluding the poly(A) tail. Sequence analysis revealed the presence of a single open reading frame (nucleotide positions 42 to 5936) that encodes a protein with significant similarity to the replicases of the “alphavirus-like” supergroup of positive-strand RNA viruses. A sequence comparison of the L-dsRNA-encoded putative replicase protein containing conserved methyltransferase, helicase, and RNA-dependent RNA polymerase motifs showed that it has significant sequence similarity to the replicase of Hepatitis E virus, a virus infecting humans. Furthermore, we present convincing evidence that the virus-like L-dsRNA could replicate independently with only a slight impact on growth and virulence of its host. Our results suggest that the L-dsRNA from strain Ep-1PN is derived from the genomic RNA of a positive-strand RNA virus, which we named Sclerotinia sclerotiorum RNA virus L (SsRV-L). As far as we know, this is the first report of a positive-strand RNA mycovirus that is related to a human virus. Phylogenetic and sequence analyses of the conserved motifs of the RNA replicase of SsRV-L showed that it clustered with the rubi-like viruses and that it is related to the plant clostero-, beny- and tobamoviruses and to the insect omegatetraviruses. Considering the fact that these related alphavirus-like positive-strand RNA viruses infect a wide variety of organisms, these findings suggest that the ancestral positive-strand RNA viruses might be of ancient origin and/or they might have radiated horizontally among vertebrates, insects, plants, and fungi.

scholarly journals The 3a cell-to-cell movement gene is dispensable for cell-to-cell transmission of brome mosaic virus RNA replicons in yeast but retained over 1045-fold amplification

2000 ◽  
Vol 81 (9) ◽  
pp. 2307-2311 ◽  
Author(s):  
Masayuki Ishikawa ◽  
Michael Janda ◽  
Paul Ahlquist
Keyword(s):  
Cell Division ◽  
Mosaic Virus ◽  
Rna Virus ◽  
Cell Movement ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Virus Rna ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna ◽  
Rna Replicon

In yeast expressing the RNA replication proteins encoded by brome mosaic virus (BMV), B3URA3, a BMV RNA3 derivative that harbours the 3a cell-to-cell movement protein gene and the yeast uracil biosynthesis gene URA3, was replicated and maintained in 85–95% of progeny at each cell division. Transmission of the B3URA3 RNA replicon from mother to daughter yeast did not require the 3a gene. Nevertheless, even after passaging for 165 cycles of RNA replication and yeast cell division, each of 40 independent Ura+ colonies tested retained B3URA3 RNAs whose electrophoretic mobilities and accumulation levels were indistinguishable from those of the original B3URA3. These and other results suggest that unselected genes in many positive-strand RNA virus replicons can be stably retained if the presence of the gene does not confer a selective disadvantage in RNA replication.

scholarly journals Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-Derived Membranes

2002 ◽  
Vol 76 (17) ◽  
pp. 8808-8819 ◽  
Author(s):  
C. Ritzenthaler ◽  
C. Laporte ◽  
F. Gaire ◽  
P. Dunoyer ◽  
C. Schmitt ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Virus Replication ◽  
Fluorescent Protein ◽  
De Novo ◽  
Rna Virus ◽  
Lipid Synthesis ◽  
Cell Movement ◽  
Brefeldin A ◽  
Cell Periphery ◽  
Green Fluorescent

ABSTRACT Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear “viral compartment.” We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.

Cellular Protein HuR Regulates the Switching of Genomic RNA Templates for Differential Functions during the Coxsackievirus B3 Life Cycle

2021 ◽  
Vol 95 (21) ◽  
Author(s):  
Biju George ◽  
Pratik Dave ◽  
Priya Rani ◽  
Padmanava Behera ◽  
Saumitra Das
Keyword(s):  
Life Cycle ◽  
Rna Virus ◽  
Coxsackievirus B3 ◽  
Cellular Protein ◽  
Genomic Rna ◽  
Host Proteins ◽  
Positive Strand ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna ◽  
Genomic Template

A positive-strand RNA virus must balance the availability of its genomic template for different viral processes at different stages of its life cycle. A few host proteins are shown to be important to help the virus in switching the usage of a template between these processes.

scholarly journals A Core35S Promoter of Cauliflower Mosaic Virus Drives More Efficient Replication of Turnip Crinkle Virus

Plants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1700
Author(s):  
Md Emran Ali ◽  
Sumyya Waliullah
Keyword(s):  
Virus Replication ◽  
Fluorescent Protein ◽  
Rna Virus ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Cdna Clones ◽  
Post Inoculation ◽  
Turnip Crinkle Virus ◽  
Green Fluorescent ◽  
High Level

The 35S promoter with a duplicated enhancer (frequently referred to as 2X35S) is a strong dicotyledonous plant-specific promoter commonly used in generating transgenic plants to enable high-level expression of genes of interest. It is also used to drive the initiation of RNA virus replication from viral cDNA, with the consensus understanding that high levels of viral RNA production powered by 2X35S permit a more efficient initiation of virus replication. Here, we showed that the exact opposite is true. We found that, compared to the Core35S promoter, the 2X35S promoter-driven initiation of turnip crinkle virus (TCV) infection was delayed by at least 24 h. We first compared three versions of 35S promoter, namely 2X35S, 1X35S, and Core35S, for their ability to power the expression of a non-replicating green fluorescent protein (GFP) gene, and confirmed that 2X35S and Core35S correlated with the highest and lowest GFP expression, respectively. However, when inserted upstream of TCV cDNA, 2X35S-driven replication was not detected until 72 h post-inoculation (72 hpi) in inoculated leaves. By contrast, Core35S-driven replication was detected earlier at 48 hpi. A similar delay was also observed in systemically infected leaves (six versus four days post-inoculation). Combining our results, we hypothesized that the stronger 2X35S promoter might enable a higher accumulation of a TCV protein that became a repressor of TCV replication at higher cellular concentration. Extending from these results, we propose that the Core35S (or mini35S) promoter is likely a better choice for generating infectious cDNA clones of TCV.

scholarly journals RNA-Protein Interaction Analysis of SARS-CoV-2 5′ and 3′ Untranslated Regions Reveals a Role of Lysosome-Associated Membrane Protein-2a during Viral Infection

mSystems ◽  
2021 ◽  
Author(s):  
Rohit Verma ◽  
Sandhini Saha ◽  
Shiv Kumar ◽  
Shailendra Mani ◽  
Tushar Kanti Maiti ◽  
...  
Keyword(s):  
Interaction Analysis ◽  
Rna Virus ◽  
Untranslated Regions ◽  
Host Proteins ◽  
Positive Strand ◽  
Replication Complex ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna ◽  
Viral Genomic Rna

Replication of a positive-strand RNA virus involves an RNA-protein complex consisting of viral genomic RNA, host RNA(s), virus-encoded proteins, and host proteins. Dissecting out individual components of the replication complex will help decode the mechanism of viral replication. 5′ and 3′ UTRs in positive-strand RNA viruses play essential regulatory roles in virus replication.

scholarly journals SARS-CoV-2 Isolation and Propagation from Turkish COVID-19 patients

2020 ◽  
Author(s):  
Cihan Tastan ◽  
Bulut Yurtsever ◽  
Gozde Sir ◽  
Derya Dilek Kancagi ◽  
Sevda Demir ◽  
...  
Keyword(s):  
Rna Virus ◽  
The Novel ◽  
Isolation And Characterization ◽  
Characterization Studies ◽  
Positive Strand Rna Virus ◽  
The Republic ◽  
Novel Coronavirus ◽  
Positive Strand Rna ◽  
Vaccine Testing ◽  
Republic Of Turkey

AbstractThe novel coronavirus pneumonia, which was named later as Coronavirus Disease 2019 (COVID-19), is caused by the Severe Acute Respiratory Syndrome Coronavirus 2, namely SARS-CoV-2. It is a positive-strand RNA virus that is the seventh coronavirus known to infect humans. The COVID-19 outbreak presents enormous challenges for global health behind the pandemic outbreak. The first diagnosed patient in Turkey has been reported by the Republic of Turkey Ministry of Health on March 11, 2020. Today, over ninety thousand cases in Turkey, and two million cases around the world have been declared. Due to the urgent need for vaccine and anti-viral drug, isolation of the virus is crucial. Here, we report one of the first isolation and characterization studies of SARS-CoV-2 from nasopharyngeal and oropharyngeal specimens of diagnosed patients in Turkey. This study provides an isolation and replication methodology, and cell culture tropism of the virus that will be available to the research communities.Article SummaryScientists have isolated virus from Turkish COVID-19 patients. The isolation, propagation, and plaque and immune response assays of the virus described here will serve in following drug discovery and vaccine testing.

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