Long-distance movement and replication maintenance functions correlate with silencing suppression activity of potyviral HC-Pro.

The tobacco etch potyviral protein, HC-Pro, is a multifunctional proteinase required for long-distance movement in plants and maintenance of genome replication at the single-cell level. It also functions in a counterdefensive capacity as a suppressor of posttranscriptional gene silencing (PTGS). To determine whether the requirements for HC-Pro during long distance movement and replication maintenance are due to the silencing suppressor function of the protein, a series of HC-Pro alanine scanning and other site-directed mutants were analyzed. Using a transient silencing suppression assay in Agrobacterium-injected leaf tissue, several suppression-defective mutants were identified. Each of six HC-Pro mutations, which were shown previously to confer long-distance movement and replication maintenance defects, conferred PTGS suppression defects. Interestingly, the genes encoding these defective HC-Pro derivatives were themselves susceptible targets of PTGS, resulting in low levels of mRNA and protein accumulation. Mutations that inactivated the proteinase domain active site had no effect on PTGS suppression function. The results are consistent with the hypothesis that the role of HC-Pro in long-distance movement and genome replication depends on PTGS suppression function and that this function is independent of HC-Pro proteolytic activity.

Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway.

Certain plant viruses encode suppressors of posttranscriptional gene silencing (PTGS), an adaptive antiviral defense response that limits virus replication and spread. The tobacco etch potyvirus protein, helper component-proteinase (HC-Pro), suppresses PTGS of silenced transgenes. The effect of HC-Pro on different steps of the silencing pathway was analyzed by using both transient Agrobacterium tumefaciens-based delivery and transgenic systems. HC-Pro inactivated PTGS in plants containing a preexisting silenced beta-glucuronidase (GUS) transgene. PTGS in this system was associated with both small RNA molecules (21-26 nt) corresponding to the 3' proximal region of the transcribed GUS sequence and cytosine methylation of specific sites near the 3' end of the GUS transgene. Introduction of HC-Pro into these plants resulted in loss of PTGS, loss of small RNAs, and partial loss of methylation. These results suggest that HC-Pro targets a PTGS maintenance (as opposed to an initiation or signaling) component at a point that affects accumulation of small RNAs and methylation of genomic DNA.

A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing.

Posttranscriptional gene silencing (PTGS) in plants inactivates some aberrant or highly expressed RNAs in a sequence-specific manner in the cytoplasm. A silencing mechanism similar to PTGS appears to function as an adaptive antiviral response. We demonstrate that the P1/HC-Pro polyprotein encoded by tobacco etch virus functions as a suppressor of PTGS. A locus comprised of a highly expressed beta-glucuronidase (GUS) transgene was shown to exhibit PTGS. Genetic crosses and segregation analyses revealed that a P1/ HC-Pro transgene suppressed PTGS of the GUS sequence. Nuclear transcription assays indicated that the silencing suppression activity of P1/HC-Pro was at the posttranscriptional level. These data reveal that plant viruses can condition enhanced susceptibility within a host through interdiction of a potent defense response.

Complementation of null CF mice with a human CFTR YAC transgene.

We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene. Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice. One line has approximately two copies of the intact YAC. Mice carrying this transgene and expressing no mouse cftr appear normal and breed well, in marked contrast to the null mice, where 50% die by approximately 5 days after birth. The chloride secretory responses in these mice are as large or larger than in wild-type tissues. Expression of the transgene is highly cell type specific and matches that of the endogenous mouse gene in the crypt epithelia throughout the gut and in salivary gland tissue. However, there is no transgene expression in some tissues, such as the Brunner's glands, where it would be expected. Where there are differences between the mouse and human pattern of expression, the transgene follows the mouse pattern. We have thus defined a cloned fragment of DNA which directs physiological levels of expression in many of the specific cells where CFTR is normally expressed.

Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase.

The tobacco etch potyvirus (TEV) helper component-proteinase (HC-Pro, 460 amino acid residues) is a multifunctional protein involved in aphid-mediated transmission, genome amplification, polyprotein processing, and long-distance movement. To investigate the interrelationships between three of these functions, 25 alanine-scanning mutations affecting clusters of charged residues were introduced into the HC-Pro coding sequence. The resulting mutants were analyzed with respect to HC-Pro proteolytic activity in vitro, genome amplification in protoplasts, and long-distance movement in tobacco plants. Three classes of mutants were identified. Class I mutants (total of 17) were capable of genome amplification, long-distance movement, and HC-Pro proteolysis with efficiencies similar to parental virus. The class III mutant (total of 1) encoded a proteolytically debilitated HC-Pro and was replication-defective. Class II mutants (total of 7) encoded proteolytically active HC-Pro, but each exhibited a suppressed amplification phenotype that was characterized by a progressive shutoff during the course of infection in protoplasts. The class II mutants also exhibited defects in long-distance movement, accumulating to relative levels of 0 to 7.5% in noninoculated tissue. Wild-type HC-Pro supplied in trans was able to partially rescue the class II mutant amplification defects in protoplasts and long-distance movement defects in plants, although the extent of complementation of movement function varied for each mutant. Six of the seven class II mutations affected the central region of HC-Pro between residues 126 and 300, whereas only one affected the C-terminal proteolytic domain. These results indicate that the central region of HC-Pro is necessary for efficient genome amplification and long-distance movement, and that the one or more HC-Pro functions involved in these processes is at least partially trans-active. Additionally, the long-distance movement properties of a previously characterized HC-Pro-defective mutant (TEV-GUS/CCCE) were characterized further using grafted nontransgenic and HC-Pro-expressing transgenic plants. The results indicated that HC-Pro is required in both inoculated and noninoculated tissues to complement the TEV-GUS/CCCE movement defects.

Requirement for HC-Pro processing during genome amplification of tobacco etch potyvirus.

The helper component-proteinase (HC-Pro) of tobacco etch potyvirus (TEV) is a multifunctional protein with several known activities. The N-terminal region is required for aphid transmission and efficient genome amplification, the central region is required for long-distance movement in plants, and the C-terminal domain is a cysteine-type proteinase that autocatalytically cleaves between itself and the P3 protein. To investigate the requirement for HC-Pro-mediated proteolysis during viral replication, a variety of mutations resulting in amino acid substitutions and insertions in the proteolytic domain and cleavage site motif were introduced into the viral genome. Mutations affecting the active site residues, His722 and Cys649, or the cleavage site P1' residue, Gly764, inhibited proteolytic activity of HC-Pro. Mutant genomes containing these modifications were amplification-defective in protoplasts and plants. Mutants with substitutions affecting several conserved, but non-active site, residues (Ser610, Cys694, and Asp715) within the proteinase domain encoded active HC-Pro proteinases and were similar to parental virus in protoplasts and plants. To determine if the replication defect of the proteinase-debilitated mutants was due to inactivation of HC-Pro proteolytic activity or simply to the inability of HC-Pro and P3 protein to separate, a sequence coding for a heterologous cleavage site recognized by the TEV NIa proteinase was inserted between the HC-Pro and P3 coding regions of an active site mutant. This cleavage site was functional in vitro using purified NIa proteinase. However, this modification was insufficient to restore amplification activity to the mutant. In addition, the active site mutant was not complemented by wild-type HC-Pro supplied in trans by transgenic plants. These results suggest that an active HC-Pro proteinase is required in cis for TEV genome amplification.

Translational enhancement of H-ferritin mRNA by interleukin-1 beta acts through 5' leader sequences distinct from the iron responsive element.

Interleukin-1 beta (Il-1 beta), a key cytokine in the acute phase response, elevates hepatic expression of both the heavy (H) and light (L) ferritin subunits without influencing the steady-state levels of either ferritin transcript. Transfection experiments with human hepatoma cells reveal that sequences within the 5' untranslated region (5'UTR) of H-ferritin mRNA confer translational regulation to chimaeric chloramphenicol acetyl transferase (CAT) mRNAs in response to Il-1 beta in the absence of marked changes in CAT mRNA levels. Il-1 beta dependent translational enhancement is mediated by a distinct G + C rich RNA sequence within 70 nucleotides (nt) of the start codon. The upstream Iron Responsive Element RNA stemloop does not confer increased expression to CAT mRNA in Il-1 beta stimulated hepatoma transfectants. A 38 nucleotide consensus sequence within the 5'UTRs of the mRNAs encoding the hepatic acute phase proteins alpha 1-antitrypsin (alpha 1AT), alpha 1-acid glycoprotein (AGP) and haptoglobin (Dente et al., 1985) is similar to sequences in the G + C rich H-ferritin mRNA translational regulatory element. Deletion of three nucleotides from this region of the 61 nt G + C rich element in the H-ferritin mRNA 5' leader eliminates Il-1 beta translational enhancement of the CAT reporter transcripts.