Imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast.
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We show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (GFP) can be monitored by imaging microscopy in living yeast. This work is based on the constitutive expression of a GFP-containing fusion protein and the inducible expression of the tobacco etch virus (TEV) protease. In the fusion protein, the P4.3 GFP mutant is linked to the YS65T GFP mutant by a spacer bearing the TEV protease-specific cleavage site. (+info)
Infectious virus in transgenic plants inoculated with a nonviable, P1-proteinase defective mutant of a potyvirus.
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A mutant (P1-616) of the tobacco vein mottling potyvirus that contains a four-codon insertion in the P1 protein coding region of the viral RNA is unable to infect the normal host plant of the virus. Processing of the P1/HC-Pro cleavage site does not occur during in vitro translation of the mutant viral RNA. When plants transformed with the P1/HC-Pro/P3 coding region of tobacco vein mottling potyvirus RNA were inoculated with P1-616, some of them became infected, although there was a delay in the production of disease symptoms. Virus isolated from these plants was able to infect nontransgenic plants. Two variants of the recovered, infectious virus contained single-nucleotide alterations in the four-codon insertion in the P1-616 genome. In vitro translation of the variant genomic RNAs resulted in partial processing of the P1/HC-Pro cleavage site, although serological analysis of infected tissue showed complete processing in vivo. These results indicate that limited complementation of P1-616 occurs in the transgenic plants and that eventually there arises one or more variants of the mutant sequence that can effect P1/HC-Pro processing and therefore be replicated. (+info)
Potyvirus helper component-proteinase self-interaction in the yeast two-hybrid system and delineation of the interaction domain involved.
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Using the yeast two-hybrid system, a screen was performed for possible interactions between the proteins encoded by the 5' region of potyviral genomes [P1, helper component-proteinase (HC-Pro), and P3]. A positive self-interaction involving HC-Pro was detected with lettuce mosaic virus (LMV) and potato virus Y (PVY). The possibility of heterologous interaction between the HC-Pro of LMV and of PVY was also demonstrated. No interaction involving either the P1 or the P3 proteins was detected. A series of ordered deletions from either the N- or C-terminal end of the LMV HC-Pro was used to map the domain involved in interaction to the 72 N-terminal amino acids of the protein, a region known to be dispensable for virus viability but necessary for aphid transmission. A similar but less detailed analysis mapped the interacting domain to the N-terminal half of the PVY HC-Pro. (+info)
Self-association and mapping of interaction domains of helper component-proteinase of potato A potyvirus.
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Potyviral helper component-proteinase (HC-Pro) is a multifunctional protein involved in aphid transmission, long-distance movement, polyprotein processing, genome amplification and symptom expression. It has been proposed that the active form of HC-Pro is a dimer and that coat protein (CP)-HC-Pro interaction is required for aphid transmission. To test these proposed interactions between CP and HC-Pro of potato A potyvirus (PVA), the yeast two-hybrid system was used. HC-Pro was shown to interact with itself in vivo in yeast cells, as did CP. Taken together with previous observations, we conclude that the functional HC-Pro is a homodimer. Deletion analysis showed that a 24 aa domain in the N-terminal half and the C-terminal proteinase part of HC-Pro were required for the interaction between HC-Pro molecules. No interactions were found between HC-Pro and CP using the genes of aphid-transmissible as well as aphid non-transmissible strains of PVA. (+info)
Potyvirus helper component-proteinase and coat protein (CP) have coordinated functions in virus-host interactions and the same CP motif affects virus transmission and accumulation.
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Amino acid differences between helper component-proteinase (HC-Pro) and coat protein (CP) that are putatively associated with biological differences between isolates PVA-B11 and PVA-U of potato A potyvirus (PVA) were studied using an infectious cDNA clone of PVA-B11. Replacement of the entire CP gene of PVA-B11 with the CP gene of PVA-U reduced virus accumulation in tobacco 5-fold, to the level of PVA-U. In contrast, four simultaneous amino acid substitutions made in PVA-B11 HC-Pro (according to PVA-U HC-Pro) increased virus accumulation 2- to 4-fold. A single substitution (S7G) at the CP N terminus reduced virus accumulation 10-fold, but restored aphid-transmissibility of PVA-B11. Simultaneous mutation of HC-Pro and replacement of CP in B11 delayed systemic movement in tobacco and limited cell-to-cell movement in potato. These results imply coordinated functions of HC-Pro and CP in accumulation and movement of PVA, because the phenotypic effects caused by simultaneous mutation of the two genes were different from the expected 'sum' of phenotypic changes observed following mutation of only one gene at a time. (+info)
Functional analysis of the interaction between VPg-proteinase (NIa) and RNA polymerase (NIb) of tobacco etch potyvirus, using conditional and suppressor mutants.
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The tobacco etch potyvirus (TEV) RNA-dependent RNA polymerase (NIb) has been shown to interact with the proteinase domain of the VPg-proteinase (NIa). To investigate the significance of this interaction, a Saccharomyces cerevisiae two-hybrid assay was used to isolate conditional NIa mutant proteins with temperature-sensitive (ts) defects in interacting with NIb. Thirty-six unique tsNIa mutants with substitutions affecting the proteinase domain were recovered. Most of the mutants coded for proteins with little or no proteolytic activity at permissive and nonpermissive temperatures. However, three mutant proteins retained proteolytic activity at both temperatures and, in two cases (tsNIa-Q384P and tsNIa-N393D), the mutations responsible for the ts interaction phenotype could be mapped to single positions. One of the mutations (N393D) conferred a ts-genome-amplification phenotype when it was placed in a recombinant TEV strain. Suppressor NIb mutants that restored interaction with the tsNIa-N393D protein at the restrictive temperature were recovered by a two-hybrid selection system. Although most of the suppressor mutants failed to stimulate amplification of genomes encoding the tsNIa-N393D protein, two suppressors (NIb-I94T and NIb-C380R) stimulated amplification of virus containing the N393D substitution by approximately sevenfold. These results support the hypothesis that interaction between NIa and NIb is important during TEV genome replication. (+info)
Identification and characterization of the functional elements within the tobacco etch virus 5' leader required for cap-independent translation.
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Translation in plants is highly cap dependent, and the only plant mRNAs known to naturally lack a cap structure (m(7)GpppN) are viral in origin. The genomic RNA of tobacco etch virus (TEV), a potyvirus that belongs to the picornavirus superfamily, is a polyadenylated mRNA that is naturally uncapped and yet is a highly competitive mRNA during translation. The 143-nucleotide 5' leader is responsible for conferring cap-independent translation even on reporter mRNAs. We have carried out a deletion analysis of the TEV 5' leader to identify the elements responsible for its regulatory function and have identified two centrally located cap-independent regulatory elements (CIREs) that promote cap-independent translation. The introduction of a stable stem-loop structure upstream of each element demonstrated that CIRE-1 is less 5' end dependent in function than CIRE-2. In a dicistronic mRNA, the presence of the TEV 5' leader sequence in the intercistronic region increased expression of the second cistron, suggesting that the viral sequence can function in a 5'-distal position. Interestingly, the introduction of a stable stem-loop upstream of the TEV leader sequence or upstream of either CIRE in dicistronic constructs markedly increased their regulatory function. These data suggest that the TEV 5' leader contains two elements that together promote internal initiation but that the function of one element, in particular, is facilitated by proximity to the 5' end. (+info)
Mapping of a hypovirus p29 protease symptom determinant domain with sequence similarity to potyvirus HC-Pro protease.
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Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica results in a spectrum of phenotypic changes that can include alterations in colony morphology and significant reductions in pigmentation, asexual sporulation, and virulence (hypovirulence). Deletion of 88% [Phe(25) to Pro(243)] of the virus-encoded papain-like protease, p29, in the context of an infectious cDNA clone of the prototypic hypovirus CHV1-EP713 (recombinant virus Deltap29) partially relieved virus-mediated suppression of pigmentation and sporulation without altering the level of hypovirulence. We now report mapping of the p29 symptom determinant domain to a region extending from Phe(25) through Gln(73) by a gain-of-function analysis following progressive repair of the Deltap29 deletion mutant. This domain was previously shown to share sequence similarity [including conserved cysteine residues Cys(38), Cys(48), Cys(70), and Cys(72)] with the N-terminal portion of the potyvirus-encoded helper component-proteinase (HC-Pro), a multifunctional protein implicated in aphid-mediated transmission, genome amplification, polyprotein processing, long-distance movement, and suppression of posttranscriptional silencing. Substitution of a glycine residue for either Cys(38) or Cys(48) resulted in no qualitative or quantitative changes in virus-mediated symptoms. Unexpectedly, mutation of Cys(70) resulted in a very severe phenotype that included significantly reduced mycelial growth and profoundly altered colony morphology. In contrast, substitution for Cys(72) resulted in a less severe symptom phenotype approaching that observed for Deltap29. The finding that p29-mediated symptom expression is influenced by two cysteine residues that are conserved in the potyvirus-encoded HC-Pro raises the possibility that these related viral-papain-like proteases function in their respective fungal and plant hosts by impacting ancestrally related regulatory pathways. (+info)