Replication of Tobamovirus RNA Ishibashi, Kazuhiro; Ishikawa, Masayuki
Annual review of phytopathology,
08/2016, Letnik:
54, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Tobacco mosaic virus
and other tobamoviruses have served as models for studying the mechanisms of viral RNA replication. In tobamoviruses, genomic RNA replication occurs via several steps: (
a
) ...synthesis of viral replication proteins by translation of the genomic RNA; (
b
) translation-coupled binding of the replication proteins to a 5′-terminal region of the genomic RNA; (
c
) recruitment of the genomic RNA by replication proteins onto membranes and formation of a complex with host proteins TOM1 and ARL8; (
d
) synthesis of complementary (negative-strand) RNA in the complex; and (
e
) synthesis of progeny genomic RNA. This article reviews current knowledge on tobamovirus RNA replication, particularly regarding how the genomic RNA is specifically selected as a replication template and how the replication proteins are activated. We also focus on the roles of the replication proteins in evading or suppressing host defense systems.
Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1-20 genomes). ...This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus. The model showed that stochastic processes that govern the replication or degradation of individual genomes result in the infection by a small number of genomes, while a large number of infectious genomes are introduced in the cell. It also predicted two interesting characteristics regarding cell infection patterns: stochastic variation among cells in the number of viral genomes that establish infection and stochastic inequality in the accumulation of their progenies in each cell. Both characteristics were validated experimentally by inoculating tobacco cells with a library of nucleotide sequence-tagged ToMV and analyzing the viral genomes that accumulated in each cell using a high-throughput sequencer. An additional simulation model revealed that these two characteristics enhance selection during tissue infection. The cell infection model also predicted a mechanism that enhances selection at the cellular level: a small difference in the replication abilities of coinfected variants results in a large difference in individual accumulation via the multiple-round formation of the replication complex (i.e., the replication machinery). Importantly, this predicted effect was observed in vivo. The cell infection model was robust to changes in the parameter values, suggesting that other viruses could adopt similar adaptation mechanisms. Taken together, these data reveal a comprehensive picture of viral infection processes including replication, cell-to-cell transmission, and evolution, which are based on the stochastic behavior of the viral genome molecules in each cell.
Plant cells have lytic vacuoles, which contain ribonucleases and proteinases. The vacuoles are fragile and easily collapsed upon homogenization of plant tissues or cells. Thus, with a few exceptions, ...plant cell extracts are contaminated by vacuole-derived lytic enzymes and unsuitable for biochemical analyses. Here, we describe a method for removing the vacuoles from intact tobacco BY-2 protoplasts and for cell-free translation and replication of genomic RNA of positive-strand RNA viruses using the extract of evacuolated protoplasts. We also describe a method for the identification and functional characterization of a plant resistance gene product using this system.
Mosaic diseases caused by tobamoviruses have posed significant threats to tomato production. In this review, we overview studies of tomato mosaic diseases published over the past century, which have ...led to several important discoveries in plant virology, such as the application of attenuated strains. A resistance breeding program established in the 1970s successfully controlled tomato mosaic virus for over 40 years; however, newly emerging tobamoviruses are posing serious challenges in current tomato production. We introduce recent biotechnological attempts to engineer tobamovirus-resistant tomato plants, which offer promising technologies for eradicating the current outbreak.
The tobamovirus, tomato brown rugose fruit virus (ToBRFV), is a significant concern in global tomato production due to the ineffectiveness of the widely used
Tm-2
2
resistance gene. Our previous ...study showed that the tomato variety GCR237, a
Tm-1
homozygote, resisted an Israeli isolate of ToBRFV (DSMZ PV-1241) for up to 35 days post inoculation (dpi), suggesting
Tm-1
-homozygous cultivars could control ToBRFV. In the present study, we inoculated GCR237 plants with ToBRFV and cultivated them for a longer period of time. The plants resisted systemic infection up to 50 dpi, but mosaic symptoms appeared on the upper leaves by 100 dpi. We retrieved the virus from symptomatic leaves and established four single local lesion isolates. These isolates had several amino acid (AA) substitutions in the helicase domain of 126-kDa/183-kDa replication proteins, where the
Tm-1
protein likely binds to inhibit viral RNA replication. Back-inoculating these isolates onto GCR237 plants confirmed they had acquired the ability to overcome GCR237’s resistance and induced mosaic symptoms as early as 14 dpi. About 90% of 229 ToBRFV isolates in the NCBI database had identical AA sequences in the corresponding region to DSMZ PV-1241, while ~ 10% inherently had AA substitutions that would confer complete breaking ability to the
Tm-1
resistance. These results suggest that while
Tm-1
can inhibit ToBRFV RNA replication, ToBRFV can easily overcome
Tm-1
homozygotes.
Replication proteins of tobacco mosaic virus (TMV), a positive-sense RNA virus, co-translationally bind to a 5'-proximal ~70-nucleotide (nt) region of the genomic RNA, referred to as the ...nuclease-resistant (NR) region for replication template selection. Therefore, disruption of the interaction between the viral replication proteins and viral genomic RNA is expected to inhibit the replication of TMV. In this study, we demonstrate that the addition of small RNA fragments (18-33 nts in length) derived from different regions within the NR region inhibit the binding of TMV replication proteins to viral RNA and TMV RNA replication in a cell-free system. Intriguingly, some of the small RNA fragments also inhibited the translation of mRNA in a sequence-nonspecific manner. These results highlight the pleiotropic roles of the 5'-proximal region of the TMV genome.
Eukaryotic positive-strand RNA viruses replicate their genomes in membranous compartments formed in a host cell, which sequesters the dsRNA replication intermediate from antiviral immune ...surveillance. Here, we find that soybean has developed a way to overcome this sequestration. We report the positional cloning of the broad-spectrum soybean mosaic virus resistance gene Rsv4, which encodes an RNase H family protein with dsRNA-degrading activity. An active-site mutant of Rsv4 is incapable of inhibiting virus multiplication and is associated with an active viral RNA polymerase complex in infected cells. These results suggest that Rsv4 enters the viral replication compartment and degrades viral dsRNA. Inspired by this model, we design three plant-gene-derived dsRNases that can inhibit the multiplication of the respective target viruses. These findings suggest a method for developing crops resistant to any target positive-strand RNA virus by fusion of endogenous host genes.
•5′- and 3′-UTRs of tobamovirus RNA play roles in translation and RNA replication.•The 3′-UTR contains pseudoknot structures and a tRNA-like structure.•TMV replication proteins cotranslationally bind ...to the 5′-UTR.•This binding leads to cis-preferential template selection for RNA replication.•The binding also inhibits further translation of TMV RNA.
The tobamovirus genome is a 5′-m7G-capped RNA that carries a tRNA-like structure at its 3′-terminus. The genomic RNA serves as the template for both translation and negative-strand RNA synthesis. The 5′- and 3′-untranslated regions (UTRs) of the genomic RNA contain elements that enhance translation, and the 3′-UTR also contains the elements necessary for the initiation of negative-strand RNA synthesis. Recent studies using a cell-free viral RNA translation–replication system revealed that a 70-nucleotide region containing a part of the 5′-UTR is bound cotranslationally by tobacco mosaic virus (TMV) replication proteins translated from the genomic RNA and that the binding leads the genomic RNA to RNA replication pathway. This mechanism explains the cis-preferential replication of TMV by the replication proteins. The binding also inhibits further translation to avoid a fatal ribosome–RNA polymerase collision, which might arise if translation and negative-strand synthesis occur simultaneously on a single genomic RNA molecule. Therefore, the 5′- and 3′-UTRs play multiple important roles in the life cycle of tobamovirus.
Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes. Before replication complex formation, virus-encoded ...replication proteins specifically recognize genomic RNA molecules and recruit them to sites of replication. Moreover, in many of these viruses, selection of replication templates by the replication proteins occurs preferentially in cis. This property is advantageous to the viruses in several aspects of viral replication and evolution, but the underlying molecular mechanisms have not been characterized. Here, we used an in vitro translation system to show that a 126-kDa replication protein of tobacco mosaic virus (TMV), a positive-strand RNA virus, binds a 5'-terminal ∼70-nucleotide region of TMV RNA cotranslationally, but not posttranslationally. TMV mutants that carried nucleotide changes in the 5'-terminal region and showed a defect in the binding were unable to synthesize negative-strand RNA, indicating that this binding is essential for template selection. A C-terminally truncated 126-kDa protein, but not the full-length 126-kDa protein, was able to posttranslationally bind TMV RNA in vitro, suggesting that binding of the 126-kDa protein to the 70-nucleotide region occurs during translation and before synthesis of the C-terminal inhibitory domain. We also show that binding of the 126-kDa protein prevents further translation of the bound TMV RNA. These data provide a mechanistic explanation of how the 126-kDa protein selects replication templates in cis and how fatal collision between translating ribosomes and negative-strand RNA-synthesizing polymerases on the genomic RNA is avoided.
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