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M C Schaad

Publications and source records attributed to M C Schaad.

15 recordsLinked to original sources
PubMed2000

Strain-specific interaction of the tobacco etch virus NIa protein with the translation initiation factor eIF4E in the yeast two-hybrid system.

The NIa protein of potyviruses provides VPg and proteolytic functions during virus replication. It has also been shown to confer host genotype-specific movement functions in plants. Specifically, NIa from tobacco etch virus (TEV)-Oxnard, but not from most other strains, confers the ability to move long distances in Nicotiana tabacum cultivar "V-20." This led to the hypothesis that all or part of NIa may interact with one or more cellular factors. To identify cellular proteins that interact with NIa in a host- or strain-specific manner, a yeast two-hybrid search of a tomato cDNA library was done. Ten proteins that interacted with NIa were recovered, with translation initiation factor eIF4E being by far the most common protein identified. Interaction of eIF4E with NIa was shown to be TEV strain-specific. eIF4E from both tomato and tobacco interacted well with NIa from the HAT strain, but not from the Oxnard strain. However, using chimeric NIa proteins, the determinant for systemic infection of V20 plants was found to be genetically distinct from the determinant controlling eIF4E interaction. In TEV-eIF4E coexpression experiments, evidence suggesting that eIF4E provides a positive effect on genome amplification was obtained.

Chimera↗
PubMed1999

Functional analysis of the interaction between VPg-proteinase (NIa) and RNA polymerase (NIb) of tobacco etch potyvirus, using conditional and suppressor mutants.

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.

Endopeptidases↗
PubMed1998

Genetic evidence for an essential role for potyvirus CI protein in cell-to-cell movement.

The potyvirus cylindrical inclusion (CI) protein, an RNA helicase required for genome replication, was analyzed genetically using alanine-scanning mutagenesis. Thirty-one mutations were introduced into the CI protein coding region of modified tobacco etch virus (TEV) genomes expressing either beta-glucuronidase or green fluorescent protein reporters. Twelve of the mutants were replication-defective in protoplast inoculation assays. Among the 19 replication-competent mutants, several possessed cell-to-cell or long-distance movement defects in tobacco plants. Two mutants, AS1 and AS8, were restricted to single cells in inoculated leaves despite genome amplification levels that were equivalent to that of parental virus. Other mutants, such as AS9 and AS14, were able to move cell to cell slowly but were debilitated in long-distance movement. These data provide genetic evidence for a direct role of CI protein in potyvirus intercellular movement, and for distinct roles of the CI protein in genome replication and movement. In combination with high-resolution ultrastructural analyzes and previous genetic data, these results support a model in which CI protein interacts directly with plasmodesmata and capsid protein-containing ribonucleoprotein complexes to facilitate potyvirus cell-to-cell movement.

Alanine↗
PubMed1997

Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein.

The mechanisms that direct positive-stranded RNA virus replication complexes to plant and animal cellular membranes are poorly understood. We describe a specific interaction between a replication protein of an RNA plant virus and membranes in vitro and in live cells. The tobacco etch virus (TEV) 6 kDa protein associated with membranes as an integral protein via a central 19 amino acid hydrophobic domain. In the presence or absence of other viral proteins, fluorescent fusion proteins containing the 6 kDa protein associated with large vesicular compartments derived from the endoplasmic reticulum (ER). Infection by TEV was associated with a collapse of the ER network into a series of discrete aggregated structures. Viral RNA replication complexes from infected cells were also associated with ER-like membranes. Targeting of TEV RNA replication complexes to membranous sites of replication is proposed to involve post-translational interactions between the 6 kDa protein and the ER.

Amino Acid Sequence↗
PubMed1997

VPg of tobacco etch potyvirus is a host genotype-specific determinant for long-distance movement.

The V20 cultivar of Nicotiana tabacum was shown previously to exhibit a strain-specific restriction of long-distance movement of tobacco etch potyvirus (TEV). In V20, both TEV-HAT and TEV-Oxnard strains are capable of genome amplification and cell-to-cell movement, but only TEV-Oxnard is capable of systemic infection by vasculature-dependent long-distance movement. To investigate the basis for host-specific movement of TEV, chimeric virus genomes were assembled from TEV-HAT and TEV-Oxnard. Viruses containing the TEV-Oxnard coding regions for HC-Pro and/or capsid protein (CP), two proteins that are known to be essential for TEV long-distance movement, failed to infect V20 systemically. In contrast, chimeric viruses encoding the TEV-Oxnard VPg domain of NIa were able to infect V20 systemically. The critical region controlling the infection phenotype in V20 was mapped to a 67-nucleotide segment containing 10-nucleotide differences, but only five amino acid differences, between TEV-HAT and TEV-Oxnard. In V20 coinfection experiments, a restricted strain had no effect on systemic infection by a long-distance movement-competent chimeric strain, suggesting that the restricted strain was not inducing a generalized systemic resistance response. These data suggest that the VPg domain, which is covalently attached to the 5' end of genomic RNA, interacts either directly or indirectly with host components to facilitate long-distance movement.

Amino Acid Sequence↗
PubMed1996

Analysis of the VPg-proteinase (NIa) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing, and genome amplification.

A mutational analysis was conducted to investigate the functions of the tobacco etch potyvirus VPg-proteinase (NIa) protein in vivo. The NIa N-terminal domain contains the VPg attachment site, whereas the C-terminal domain contains a picornavirus 3C-like proteinase. Cleavage at an internal site separating the two domains occurs in a subset of NIa molecules. The majority of NIa molecules in TEV-infected cells accumulate within the nucleus. By using a reporter fusion strategy, the NIa nuclear localization signal was mapped to a sequence within amino acid residues 40 to 49 in the VPg domain. Mutations resulting in debilitation of NIa nuclear translocation also debilitated genome amplification, suggesting that the NLS overlaps a region critical for RNA replication. The internal cleavage site was shown to be a poor substrate for NIa proteolysis because of a suboptimal sequence context around the scissile bond. Mutants that encoded NIa variants with accelerated internal proteolysis exhibited genome amplification defects, supporting the hypothesis that slow internal processing provides a regulatory function. Mutations affecting the VPg attachment site and proteinase active-site residues resulted in amplification-defective viruses. A transgenic complementation assay was used to test whether NIa supplied in trans could rescue amplification-defective viral genomes encoding altered NIa proteins. Neither cells expressing NIa alone nor cells expressing a series of NIa-containing polyproteins supported increased levels of amplification of the mutants. The lack of complementation of NIa-defective mutants is in contrast to previous results obtained with RNA polymerase (NIb)-defective mutants, which were relatively efficiently rescued in the transgenic complementation assay. It is suggested that, unlike NIb polymerase, NIa provides replicative functions that are cis preferential.

Biological Transport↗
PubMed1996

Suppression of long-distance movement of tobacco etch virus in a nonsusceptible host.

To investigate host functions involved in the tobacco etch potyvirus (TEV) infection process, a tobacco line (V20) with a strain-specific defect in supporting systemic infection was analyzed. Using a modified TEV encoding a reporter protein, beta-glucuronidase (GUS), genome amplification, cell-to-cell movement, and long-distance movement were measured in V20 and a susceptible line, Havana425. Comparable levels of TEV-GUS genome amplification were measured in inoculated protoplasts from both tobacco lines. The rates of cell-to-cell movement of virus in inoculated leaves were nearly identical in V20 and Havana425 between 48 and 72 h postinoculation. In contrast, long-distance movement from leaf to leaf was markedly restricted in V20 relative to Havana425. In situ histochemical analysis of inoculated leaves revealed that infection foci expanded radially over time, providing the potential for contact of virus with veins. Immunocytochemical analysis of V20 tissue from infection foci indicated that TEV-GUS entered the phloem parenchyma or companion cells adjacent to the sieve elements, suggesting that the block in long-distance movement was associated with entry into, or exit from, sieve elements. The genetic basis for the V20 restriction was characterized in a segregation analysis of a cross between V20 and Havana425. The heterozygous F1 progeny displayed the susceptible phenotype, indicating that the V20 restriction was a recessive trait. Segregation in the F2 progeny indicated that the restriction was likely due to the interaction of recessive genes at two nonlinked loci. These data support the hypothesis that long-distance movement requires a set of host functions that are distinct from those involved in cell-to-cell movement.

Genes, Plant↗
PubMed1995

Long-distance movement factor: a transport function of the potyvirus helper component proteinase.

Transport of viruses from cell to cell in plants typically involves one or more viral proteins that supply dedicated movement functions. Transport from leaf to leaf through phloem, or long-distance transport, is a poorly understood process with requirements differing from those of cell-to-cell movement. Through genetic analysis of tobacco etch virus (TEV; potyvirus group), a novel long-distance movement factor was identified that facilitates vascular-associated movement in tobacco. A mutation in the central region of the helper component proteinase (HC-Pro), a TEV-encoded protein with previously described activities in aphid-mediated transmission and polyprotein processing, inactivated long-distance movement. This mutant virus exhibited only minor defects in genome amplification and cell-to-cell movement functions. In situ histochemical analysis revealed that the mutant was capable of infecting mesophyll, bundle sheath, and phloem cells within inoculated leaves, suggesting that the long-distance movement block was associated with entry into or exit from sieve elements. The long-distance movement defect was specifically complemented by HC-Pro supplied in trans by a transgenic host. The data indicate that HC-Pro functions in one or more steps unique to long-distance transport.

Biological Transport↗
PubMed1994

Genetics of mouse hepatitis virus transcription: evidence that subgenomic negative strands are functional templates.

Mouse hepatitis virus (MHV) A59 temperature-sensitive (ts) mutants belonging to complementation group C were characterized and mapped by standard genetic recombination techniques. Temperature shift experiments early in infection suggested that the group C allele can be divided into two phenotypically distinct subgroups, designated C1 and C2. Since previous data indicated that the group C1 mutants probably contained an early defect which affects negative-strand synthesis, RNA synthesis was further examined by analyzing replicative-form (RF) RNA. Full-length as well as subgenomic-length RF RNAs were radiolabeled from 3 to 12 h postinfection (p.i.) and labeled late in infection after shift to the nonpermissive temperature (39.5 degrees C). The relative percent molar ratios of each mRNA and corresponding RF RNA were roughly equivalent throughout infection. Temperature shift experiments at 5.5 or 6.0 h p.i. resulted in an 83 to 92% reduction in the amount of total RF RNA at 39.5 degrees C. Radiolabeling time course experiments after temperature shift to 39.5 degrees C also demonstrated incorporation (6 to 9 h p.i.) into both subgenomic-length and full-length RF RNAs, suggesting that previously transcribed negative strands were functional templates throughout infection. To determine if the reduction in RF RNA was due to a decrease in positive- or negative-strand RNA synthesis, rates of mRNA synthesis were calculated from both full-length and subgenomic-length templates. The rate of mRNA synthesis after the shift was increased at 39.5 degrees C compared with that at 32 degrees C regardless of the template used; however, transcription rates calculated from subgenomic-length templates were similar to those of other viral and eukaryotic polymerases. These findings support the notion that the group C1 allele regulates negative-strand RNA synthesis and strongly suggest that the subgenomic negative-strand RNAs are probably the predominant functional templates for the synthesis of positive-strand RNAs late in infection.

Animals↗
PubMed1993

Evidence for new transcriptional units encoded at the 3' end of the mouse hepatitis virus genome.

Prior studies have demonstrated that the mouse hepatitis virus (MHV) genome is divided into at least seven coding regions each transcribed into a distinct mRNA. The majority of these mRNAs are synthesized from a highly conserved intergenic start site (UCU/CAAAC), contain a 65-72 nt leader RNA at their 5' end and form a 3' co-terminal nested set. In this study, we have used radiolabeling experiments to demonstrate the presence of a small approximately 900 nt mRNA and its corresponding RF RNA in MHV-infected cells. Surprisingly, PCR amplification and sequence analysis revealed the presence of not one, but two small leader-containing RNAs that initiate from highly conserved intergenic start sites (UCCAAAC and UCUAAAU) which are located within the 3'-most nucleocapsid gene sequence. These studies provide evidence suggesting that one or two additional small mRNAs are encoded from the 3' end of the MHV genome.

Amino Acid Sequence↗
PubMed1993

Studies into the mechanism for MHV transcription.

Previous studies have demonstrated that the MHV genome is divided into seven transcriptional units which are transcribed from highly conserved intergenic start sites (UCU/CAAAC) into mRNA containing a common leader RNA at the 5' end and a coterminal 3' end. In this manuscript, we provide evidence that an additional transcriptional unit is encoded at the 3' end of the MHV genome and is transcribed from a perfect intergenic region into a leader-containing approximately 800 nt mRNA. This mRNA could potentially encode a small 17-18 kDa protein which is identical to the C-terminal third of the nucleocapsid gene.

Base Sequence↗
PubMed1990

Genetics of mouse hepatitis virus transcription: identification of cistrons which may function in positive and negative strand RNA synthesis.

A panel of 26 temperature-sensitive mutants of MHV-A59 were selected by mutagenesis with either 5-fluorouracil or 5-azacytidine. Complementation analysis revealed the presence of one RNA+ and five RNA- complementation groups. None of the RNA- complementation groups transcribed detectable levels of positive- or negative-stranded RNA at the restrictive temperature. Temperature shift experiments after the onset of mRNA synthesis revealed at least two classes of RNA- mutants. RNA- complementation groups A, B, D, and E were blocked in the ability to release infectious virus and transcribe mRNA and genome, while group C mutants continued to release infectious virus and transcribe both mRNA and genome. Temperature shift experiments at different times postinfection suggest that the group C mutants encode a function required early in viral transcription which affects the overall rate of positive strand synthesis. Analysis of steady state levels of negative strand RNA after the shift indicate that the group C mutants were probably blocked in the ability to synthesize additional minus strand RNA under conditions in which the group E mutants continued low levels of minus strand synthesis. These data suggest that at least four cistrons may be required for positive strand synthesis while the group C cistron functions during minus strand synthesis.

Animals↗
PubMed1990

Establishing a genetic recombination map for murine coronavirus strain A59 complementation groups.

MHV-A59 temperature-sensitive mutants, representing one RNA+ and five RNA- complementation groups, were isolated and characterized by genetic recombination techniques. Maximum recombination frequencies occurred under multiplicities of infection greater than 10 each in which 99.99% of the cells were co-infected. Recombination frequencies between different ts mutants increased steadily during infection and peaked late in the virus growth cycle. These data suggest that recombination is a late event in the virus replication cycle. Recombination frequencies were also found to range from 63 to 20,000 times higher than the sum of the spontaneous reversion frequencies of each ts mutant used in the cross. Utilizing standard genetic recombination techniques, the five RNA- complementation groups of MHV-A59 were arranged into an additive, linear, genetic map located at the 5' end of the genome in the 23-kb polymerase region. These data indicate that at least five distinct functions are encoded in the MHV polymerase region which function in virus transcription. Moreover, using well-characterized ts mutants the recombination frequency for the entire 32-kb MHV genome was found to approach 25% or more. This is the highest recombination frequency described for a nonsegmented, linear, plus-polarity RNA virus.

Animals↗