Wheat streak mosaic virus Coat Protein Deletion Mutants Elicit More Severe Symptoms Than Wild-Type Virus in Multiple Cereal Hosts

Previously, we reported that coat protein (CP) of Wheat streak mosaic virus (WSMV) (genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. In this study, we demonstrated that WSMV mutants with deletion of CP amino acids 58 to 84 but not of 36 to 57 induced severe chlorotic streaks and spots, followed by acute chlorosis in wheat, maize, barley, and rye compared with mild to moderate chlorotic streaks and mosaic symptoms by wild-type virus. Deletion of CP amino acids 58 to 84 from the WSMV genome accelerated cell-to-cell movement, with increased accumulation of genomic RNAs and CP, compared with the wild-type virus. Microscopic examination of wheat tissues infected by green fluorescent protein–tagged mutants revealed that infection by mutants lacking CP amino acids 58 to 84 caused degradation of chloroplasts, resulting in acute macroscopic chlorosis. The profile of CP-specific proteins was altered in wheat infected by mutants causing acute chlorosis, compared with mutants eliciting wild-type symptoms. All deletion mutants accumulated CP-specific major protein similarly to that in wild-type virus; however, mutants that elicit acute chlorosis failed to accumulate a 31-kDa minor protein compared with wild-type virus or mutants lacking amino acids 36 to 57. Taken together, these data suggest that deletion of CP amino acids 58 to 84 from the WSMV genome enhanced accumulation of CP and genomic RNA, altered CP-specific protein profiles, and caused severe symptom phenotypes in multiple cereal hosts.

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Plants Transformed with a Mutant Alfalfa Mosaic Virus Coat Protein Gene Are Resistant to the Mutant but Not to Wild-Type Virus

Virology ◽

10.1006/viro.1994.1484 ◽

1994 ◽

Vol 203 (2) ◽

pp. 269-276 ◽

Cited By ~ 11

Author(s):

Peter E.M. Taschner ◽

Guido Van Marle ◽

Frans Th. Brederode ◽

Nilgun E. Tumer ◽

John F. Bol

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Coat Protein Gene ◽

Protein Gene ◽

Alfalfa Mosaic Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virus Coat Protein ◽

Virus Coat

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Noncapped Genomic RNA Are Critical for Alphaviral Infection and Pathogenicity

Proceedings ◽

10.3390/proceedings2020050044 ◽

2020 ◽

Vol 50 (1) ◽

pp. 44

Author(s):

Autumn T. LaPointe ◽

Kevin J Sokoloski

Keyword(s):

Growth Kinetics ◽

Particle Production ◽

Immune Cell ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Genomic Rnas ◽

Viral Rnas ◽

The Impact

Alphaviruses are positive-sense RNA arthropod-borne viruses that represent a significant threat to public health. During alphaviral replication, significant quantities of viral genomic RNAs that lack a canonical 5’ cap structure are produced and packaged into viral particles, despite the fact that the noncapped genomes cannot be translated and are essentially noninfectious. Previously, we have reported that the capping efficiency of nsP1, the alphaviral capping enzyme, of Sindbis virus (SINV) could be modulated via point mutation. It was found that increasing RNA capping efficiency led to decreased viral growth kinetics via decreased particle production, despite increased innate immune evasion, whereas decreasing capping efficiency led to wild-type growth kinetics and particle production. This led to the conclusion that the noncapped viral RNAs meaningfully contribute to the biology of alphaviral infections at the molecular level. To determine the importance of the noncapped viral RNAs in vivo, we characterized the impact of altered capping efficiency in a murine model of infection utilizing a neurovirulent strain of SINV. Mice infected with the nsP1 mutant with decreased capping exhibited wild-type rates of mortality, weight loss, and neurological symptoms. Conversely, the mice infected with the increased capping nsP1 mutant showed significantly reduced mortality and morbidity compared to mice infected with the wild-type virus. Interestingly, viral titers in the ankle, serum, and brain were equivalent between the wild-type virus and the two mutant viruses. Importantly, examination of the brain tissue revealed that mice infected with the increased capping mutant had significantly reduced immune cell infiltration and expression of proinflammatory cytokines compared to the decreased capping mutant and wild-type virus. Collectively, these data indicate that the noncapped viral RNAs have important roles during the early and late stages of alphaviral infection and suggest a novel mechanism by which noncapped viral RNA aids in viral pathogenesis.

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Restoration of Wild-Type Virus by Double Recombination of Tombusvirus Mutants with a Host Transgene

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.2.153 ◽

1999 ◽

Vol 12 (2) ◽

pp. 153-162 ◽

Cited By ~ 50

Author(s):

Marise Borja ◽

Teresa Rubio ◽

Herman B. Scholthof ◽

Andrew O. Jackson

Keyword(s):

Coat Protein ◽

Transgenic Plants ◽

Coat Protein Gene ◽

Protein Gene ◽

Wild Type Virus ◽

Type Virus ◽

Virus Infections ◽

Wild Type ◽

Transgenic Lines ◽

Double Recombination

Nicotiana benthamiana plants transformed with the coat protein gene of tomato bushy stunt virus (TBSV) failed to elicit effective virus resistance when inoculated with wild-type virus. Subsequently, R1 and R2 progeny from 13 transgenic lines were inoculated with a TBSV mutant containing a defective coat protein gene. Mild symptoms typical of those elicited in nontransformed plants infected with the TBSV mutant initially appeared. However, within 2 to 4 weeks, up to 20% of the transgenic plants sporadically began to develop the lethal syndrome characteristic of wild-type virus infections. RNA hybridization and immunoblot analyses of these plants and nontransformed N. benthamiana inoculated with virus from the transgenic lines indicated that wild-type virus had been regenerated by a double recombination event between the defective virus and the coat protein transgene. Similar results were obtained with a TBSV deletion mutant containing a nucleotide sequence marker, and with a chimeric cucumber necrosis virus (CNV) containing the defective TBSV coat protein gene. In both cases, purified virions contained wild-type TBSV RNA or CNV chimeric RNA derived by recombination with the transgenic coat protein mRNA. These results thus demonstrate that recombinant tombusviruses can arise frequently from viral genes expressed in transgenic plants.

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Production of Noncapped Genomic RNAs is Critical to Sindbis Virus Disease and Pathogenicity

10.1101/2020.06.11.145847 ◽

2020 ◽

Author(s):

Autumn T. LaPointe ◽

V. Douglas Landers ◽

Claire E. Westcott ◽

Kevin J. Sokoloski

Keyword(s):

Viral Replication ◽

Sindbis Virus ◽

Viral Rna ◽

Wild Type Virus ◽

Type Virus ◽

Genomic Rna ◽

Wild Type ◽

Genomic Rnas ◽

The Brain

ABSTRACTAlphaviruses are positive-sense RNA viruses that utilize a 5’ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5’ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to wild type infection. The mice infected with the D355A nsP1 mutant showed significantly reduced mortality and morbidity compared to mice infected with wild type virus. Interestingly, both capping mutants had roughly equivalent viral titer in the brain compared to wild type virus, illustrating that the changes in mortality were not due to deficits in viral replication or dissemination. Examination of the brain tissue revealed that mice infected with the D355A capping mutant had significantly reduced cell death and immune cell infiltration compared to the N376A mutant and wild type virus. Finally, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNA aid in viral pathogenesis.AUTHOR SUMMARYMosquito transmitted alphaviruses have been the cause of widespread outbreaks of disease which can range from mild illness to lethal encephalitis or severe polyarthritis. In order to successfully replicate, the alphavirus RNA genome needs a 5’ cap structure so that the genome can be translated and produce the viral replication machinery. Despite this, a large number of viral genomes produced during infection do not have a 5’ cap structure, and their role during infection is unknown. Using mouse models of infection and point mutations in the nsP1 protein of Sindbis virus which alter the amount of noncapped genomic RNA (ncgRNA) produced, we found the decreasing the production of ncgRNA greatly reduced morbidity and mortality as well as proinflammatory cytokine expression, resulting in less tissue-damaging inflammation in the brain. These studies suggest that the ncgRNAs contribute to pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

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Expression of herpes simplex virus type 1 major DNA-binding protein, ICP8, in transformed cell lines: complementation of deletion mutants and inhibition of wild-type virus.

Journal of Virology ◽

10.1128/jvi.61.4.1136-1146.1987 ◽

1987 ◽

Vol 61 (4) ◽

pp. 1136-1146 ◽

Cited By ~ 11

Author(s):

P K Orberg ◽

P A Schaffer

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Dna Binding Protein ◽

Wild Type Virus ◽

Deletion Mutants ◽

Type Virus ◽

Wild Type ◽

Simplex Virus ◽

Transformed Cell Lines

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The AAUAAA Motif of Bamboo Mosaic Virus RNA Is Involved in Minus-Strand RNA Synthesis and Plus-Strand RNA Polyadenylation

Journal of Virology ◽

10.1128/jvi.79.23.14555-14561.2005 ◽

2005 ◽

Vol 79 (23) ◽

pp. 14555-14561 ◽

Cited By ~ 23

Author(s):

I-Hsuan Chen ◽

Wen-Jen Chou ◽

Pei-Yu Lee ◽

Yau-Heiu Hsu ◽

Ching-Hsiu Tsai

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Rna Synthesis ◽

Wild Type Virus ◽

Protein Accumulation ◽

Minus Strand ◽

Genomic Rna ◽

Genomic Rnas ◽

Virus Rna ◽

Rna Accumulation

ABSTRACT Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome with a 5′-cap structure and a 3′ poly(A) tail. Deleting the internal loop that contains the putative polyadenylation signal (AAUAAA) in the 3′ untranslated region (UTR) of BaMV genomic RNA appeared to diminish coat protein accumulation to 2% (C. P. Cheng and C. H. Tsai, J. Mol. Biol. 288:555-565, 1999). To investigate the function of the AAUAAA motif, mutations were introduced into an infectious BaMV cDNA at each residue except the first nucleotide. After transfection of Nicotiana benthamiana protoplasts with RNA transcript, the accumulations of viral coat protein and RNAs were determined. Based on the results, three different categories could be deduced for the mutants. Category 1 includes two mutants expressing levels of the viral products similar to those of the wild-type virus. Six mutations in category 2 led to decreased to similar levels of both minus-strand and genomic RNAs. Category 3 includes the remaining seven mutations that also bring about decreases in both minus- and plus-strand RNA levels, with more significant effects on genomic RNA accumulation. Mutant transcripts from each category were used to infect N. benthamiana plants, from which viral particles were isolated. The genomic RNAs of mutants in category 3 were found to have shorter poly(A) tails. Taken together, the results suggest that the AAUAAA motif in the 3′ UTR of BaMV genomic RNA is involved not only in the formation of the poly(A) tail of the plus-strand RNA, but also in minus-strand RNA synthesis.

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A Conserved Capsid Protein Surface Domain of Cucumber Mosaic Virus Is Essential for Efficient Aphid Vector Transmission

Journal of Virology ◽

10.1128/jvi.76.19.9756-9762.2002 ◽

2002 ◽

Vol 76 (19) ◽

pp. 9756-9762 ◽

Cited By ~ 68

Author(s):

Sijun Liu ◽

Xiaohua He ◽

Gyungsoon Park ◽

Caroline Josefsson ◽

Keith L. Perry

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Capsid Protein ◽

Wild Type Virus ◽

Cdna Clones ◽

Wild Type ◽

Vector Transmission ◽

Aphid Vector

ABSTRACT A prominent feature on the surfaces of virions of Cucumber mosaic virus (CMV) is a negatively charged loop structure (the βH-βI loop). Six of 8 amino acids in this capsid protein loop are highly conserved among strains of CMV and other cucumoviruses. Five of these amino acids were individually changed to alanine or lysine (an amino acid of opposite charge) to create nine mutants (the D191A, D191K, D192A, D192K, L194A, E195A, E195K, D197A, and D197K mutants). Transcripts of cDNA clones were infectious when they were mechanically inoculated onto tobacco, giving rise to symptoms of a mottle-mosaic typical of the wild-type virus (the D191A, D191K, D192A, E195A, E195K, and D197A mutants), a systemic necrosis (the D192K mutant), or an atypical chlorosis with necrotic flecking (the L194A mutant). The mutants formed virions and accumulated to wild-type levels, but eight of the nine mutants were defective in aphid vector transmission. The aspartate-to-lysine mutation at position 197 interfered with infection; the only recovered progeny (the D197K∗ mutant) harbored a second-site mutation (denoted by the asterisk) of alanine to glutamate at position 193, a proximal site in the βH-βI loop. Since the disruption of charged amino acid residues in the βH-βI loop reduces or eliminates vector transmissibility without grossly affecting infectivity or virion formation, we hypothesize that this sequence or structure has been conserved to facilitate aphid vector transmission.

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