Analysis of geminivirus AL2 and L2 proteins reveals a novel AL2 silencing suppressor activity.

UNLABELLED: Both posttranscriptional and transcriptional gene silencing (PTGS and TGS, respectively) participate in defense against the DNA-containing geminiviruses. As a countermeasure, members of the genus Begomovirus (e.g., Cabbage leaf curl virus) encode an AL2 protein that is both a transcriptional activator and a silencing suppressor. The related L2 protein of Beet curly top virus (genus Curtovirus) lacks transcription activation activity. Previous studies showed that both AL2 and L2 suppress silencing by a mechanism that correlates with adenosine kinase (ADK) inhibition, while AL2 in addition activates transcription of cellular genes that negatively regulate silencing pathways. The goal of this study was to clarify the general means by which these viral proteins inhibit various aspects of silencing. We confirmed that AL2 inhibits systemic silencing spread by a mechanism that requires transcription activation activity. Surprisingly, we also found that reversal of PTGS and TGS by ADK inactivation depended on whether experiments were conducted in vegetative or reproductive Nicotiana benthamiana plants (i.e., before or after the vegetative-to-reproductive transition). While AL2 was able to reverse silencing in both vegetative and reproductive plants, L2 and ADK inhibition were effective only in vegetative plants. This suggests that silencing maintenance mechanisms can change during development or in response to stress. Remarkably, we also observed that AL2 lacking its transcription activation domain could reverse TGS in reproductive plants, revealing a third, previously unsuspected AL2 suppression mechanism that depends on neither ADK inactivation nor transcription activation. IMPORTANCE: RNA silencing in plants is a multivalent antiviral defense, and viruses respond by elaborating multiple and sometimes multifunctional proteins that inhibit various aspects of silencing. The studies described here add an additional layer of complexity to this interplay. By examining geminivirus AL2 and L2 suppressor activities, we show that L2 is unable to suppress silencing in Nicotiana benthamiana plants that have undergone the vegetative-to-reproductive transition. As L2 was previously shown to be effective in mature Arabidopsis plants, these results illustrate that silencing mechanisms can change during development or in response to stress in ways that may be species specific. The AL2 and L2 proteins are known to share a suppression mechanism that correlates with the ability of both proteins to inhibit ADK, while AL2 in addition can inhibit silencing by transcriptionally activating cellular genes. Here, we also provide evidence for a third AL2 suppression mechanism that depends on neither transcription activation nor ADK inactivation. In addition to revealing the remarkable versatility of AL2, this work highlights the utility of viral suppressors as probes for the analysis of silencing pathways.

Co-dependence of HTLV-1 p12 and p8 functions in virus persistence.

HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4+ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8.

A complex containing SNF1-related kinase (SnRK1) and adenosine kinase in Arabidopsis.

SNF1-related kinase (SnRK1) in plants belongs to a conserved family that includes sucrose non-fermenting 1 kinase (SNF1) in yeast and AMP-activated protein kinase (AMPK) in animals. These kinases play important roles in the regulation of cellular energy homeostasis and in response to stresses that deplete ATP, they inhibit energy consuming anabolic pathways and promote catabolism. Energy stress is sensed by increased AMP:ATP ratios and in plants, 5'-AMP inhibits inactivation of phosphorylated SnRK1 by phosphatase. In previous studies, we showed that geminivirus pathogenicity proteins interact with both SnRK1 and adenosine kinase (ADK), which phosphorylates adenosine to generate 5'-AMP. This suggested a relationship between SnRK1 and ADK, which we investigate in the studies described here. We demonstrate that SnRK1 and ADK physically associate in the cytoplasm, and that SnRK1 stimulates ADK in vitro by an unknown, non-enzymatic mechanism. Further, altering SnRK1 or ADK activity in transgenic plants altered the activity of the other kinase, providing evidence for in vivo linkage but also revealing that in vivo regulation of these activities is complex. This study establishes the existence of SnRK1-ADK complexes that may play important roles in energy homeostasis and cellular responses to biotic and abiotic stress.

Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation.

Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S-adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibition. However, at one exceptional locus, ADK inhibition was insufficient and TGS reversal required the transcriptional activation domain of AL2. Using restriction-sensitive PCR and bisulfite sequencing, we showed that AL2-mediated TGS suppression is accompanied by reduced cytosine methylation. Finally, using a methylation-sensitive single-nucleotide extension assay, we showed that transgenic expression of AL2 or L2 causes global reduction in cytosine methylation. Our results provide further evidence that viral chromatin methylation is an important host defense and allow us to propose that as a countermeasure, geminivirus proteins reverse TGS by nonspecifically inhibiting cellular transmethylation reactions. To our knowledge, this is the first report that viral proteins can inhibit TGS.

Viral genome methylation as an epigenetic defense against geminiviruses.

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.

Functional modulation of the geminivirus AL2 transcription factor and silencing suppressor by self-interaction.

The DNA genomes of geminiviruses have a limited coding capacity that is compensated for by the production of small multifunctional proteins. The AL2 protein encoded by members of the genus Begomovirus (e.g., Tomato golden mosaic virus) is a transcriptional activator, a silencing suppressor, and a suppressor of a basal defense. The related L2 protein of Beet curly top virus (genus Curtovirus) shares the pathogenicity functions of AL2 but lacks transcriptional activation activity. It is known that AL2 and L2 can suppress local silencing by interacting with adenosine kinase (ADK) and can suppress basal defense by interacting with SNF1 kinase. However, how the activities of these viral proteins are regulated remains an unanswered question. Here, we provide some answers by demonstrating that AL2, but not L2, interacts with itself. The zinc finger-like motif (CCHC) is required but is not sufficient for AL2 self-interaction. Alanine substitutions for the invariant cysteine residues that comprise the motif abolish self-interaction or cause aberrant subnuclear localization but do not abolish interaction with ADK and SNF1. Using bimolecular fluorescence complementation, we show that AL2:AL2 complexes accumulate primarily in the nucleus, whereas AL2:ADK and L2:ADK complexes accumulate mainly in the cytoplasm. Further, the cysteine residue mutations impair the ability of AL2 to activate the coat protein promoter but do not affect local silencing suppression. Thus, AL2 self-interaction correlates with nuclear localization and efficient activation of transcription, whereas AL2 and L2 monomers can suppress local silencing by interacting with ADK in the cytoplasm.

Adenosine kinase inhibition and suppression of RNA silencing by geminivirus AL2 and L2 proteins.

Most plant viruses are initiators and targets of RNA silencing and encode proteins that suppress this adaptive host defense. The DNA-containing geminiviruses are no exception, and the AL2 protein (also known as AC2, C2, and transcriptional activator protein) encoded by members of the genus Begomovirus has been shown to act as a silencing suppressor. Here, a three-component, Agrobacterium-mediated transient assay is used to further examine the silencing suppression activity of AL2 from Tomato golden mosaic virus (TGMV, a begomovirus) and to determine if the related L2 protein of Beet curly top virus (BCTV, genus Curtovirus) also has suppression activity. We show that TGMV AL2, AL2(1-100) (lacking the transcriptional activation domain), and BCTV L2 can all suppress RNA silencing directed against a green fluorescent protein (GFP) reporter gene when silencing is induced by a construct expressing an inverted repeat GFP RNA (dsGFP). We previously found that these viral proteins interact with and inactivate adenosine kinase (ADK), a cellular enzyme important for adenosine salvage and methyl cycle maintenance. Using the GFP-dsGFP system, we demonstrate here that codelivery of a construct expressing an inverted repeat ADK RNA (dsADK), or addition of an ADK inhibitor (the adenosine analogue A-134974), suppresses GFP-directed silencing in a manner similar to the geminivirus proteins. In addition, AL2/L2 suppression phenotypes and nucleic acid binding properties are shown to be different from those of the RNA virus suppressors HC-Pro and p19. These findings provide strong evidence that ADK activity is required to support RNA silencing, and indicate that the geminivirus proteins suppress silencing by a novel mechanism that involves ADK inhibition. Further, since AL2(1-100) is as effective a suppressor as the full-length AL2 protein, activation and silencing suppression appear to be independent activities.

Amplified crevicular leukocyte activity in aggressive periodontal disease.

Exaggerated neutrophil responses are a critical component in the pathogenesis of periodontal disease. We investigated whether leukocyte activity in aggressive periodontitis (AP) is increased compared with that in chronic periodontitis (CP) by gingival crevicular fluid (GCF) analysis of myeloperoxidase (MPO), beta-N-acetyl-hexosaminidase (beta-NAH), cathepsin D (CD), and elastase-alpha-1-proteinase inhibitor complex (alpha-1-EPI) before and 6 months after therapy. Initial AP neutrophil responses were significantly amplified compared with those in CP (MPO, 3.2-fold; beta-NAH, 37.5-fold; CD, 2.2-fold; alpha-1-EPI, 1.4-fold; p < 0.05). Surgical therapy resulted in a significant reduction of GCF markers compared with non-surgical treatment. However, the changes in clinical parameters were not different between AP and CP (P > 0.05). Analysis of the results suggests that the local inflammatory response in AP is characterized by increased release of inflammatory mediators of neutrophil origin into the GCF. Analysis of the data further suggests that surgical therapy is a more predictable method for removal of the pro-inflammatory etiology.

PMN responses following use of 2 biodegradable GTR membranes.

OBJECTIVES: In the present prospective trial, the PMN response following resorbable GTR barrier placement was evaluated in mandibular class II furcation lesions. MATERIALS AND METHODS: In 10 patients with treated chronic periodontitis, we randomly selected the 1st molars in the mandible with buccal degree II furcation involvement for either polylactic-citric-acid-ester (PLA) or glycolide-lactic-copolymer (PGL) GTR membrane therapy. We examined contralateral healthy molar sites as untreated controls. We then evaluated the PMN-derived inflammatory tissue response at baseline, weekly up to 6 weeks post-therapy and at 12 and 24 weeks using GCF myeloperoxidase (MPO), beta-glucuronidase (betaG) and beta-N-acetyl-hexosaminidase (betaNAH). RESULTS: The enzyme levels increased from baseline to the 6-week examination. After the 6-week reappointment, enzyme levels dropped reaching the baseline scores at both the 12- and 24-week visit. At PGL sites, the enzyme levels decreased earlier. Compared with healthy control sites, the MPO, betaNAH and betaG tests revealed different maximum levels at week 2 and 3 (PGL) and week 4, 5 and 6 (PLA). For both of the barriers the clinical parameters revealed a sustained improvement following therapy. CONCLUSION: The release of PMN enzymes following placement of bioabsorbable membranes reflects the early soft tissue healing process. Our results suggest that the PMN response is barrier-dependent with the maximum response occuring at different times. However, the host response did not measureably affect the course of clinical healing.

Surgical therapy of peri-implant disease: a 3-year follow-up study of cases treated with 3 different techniques of bone regeneration.

BACKGROUND: Advanced peri-implant intrabony defects require comprehensive surgical treatment regimens different from periodontal therapy strategies. The purpose of this longitudinal trial was to evaluate the peri-implant outcomes following guided bone regeneration with 3 treatment protocols. METHODS: In 25 patients, 41 peri-implant defects with supporting bone loss >50% of the implant length were treated with flap surgery plus autogenous bone grafts alone (FG) (controls, n = 12) plus non-resorbable (FGM) (test 1, n = 20) or bioabsorbable barriers (FGRM) (test 2, n = 9) and supportive antimicrobial therapy. Following submerged healing, the membranes were removed (FGM), and the peri-implant probing depths (PD), probing bone levels (BL), mobility scores (PT), and intrabony defect height (DH) were radiographically evaluated at baseline, 6 months, and 1 and 3 years post-therapy. RESULTS: Non-surgical/anti-infective therapy resulted in a limited improvement of PD scores after 6 months. At the 3-year visit, surgical treatment revealed significant changes from baseline for the controls and both of the test groups for PD: 5.1 +/- 2.7 mm (FG), 5.4 +/- 3.0 mm (FGM), and 2.6 +/- 1.6 mm (FGRM), and for BL: 3.2 +/- 2.4 mm (FG), 3.4 +/- 2.4 mm (FGM), and 2.3 +/- 1.6 mm (FGRM), Mann-Whitney test, P < or = 0.05. The changes for DH and PT were significant only for FG- and FGM-treated subjects. The overall improvement for FGRM-treated patients during the 3-year observation was less marked. However, the differences between the 3 surgical treatment protocols did not affect the treatment outcomes after 3 years. CONCLUSIONS: Autogenous bone grafting is an appropriate treatment regimen to augment open crater-formed peri-implant defects. Although certain clinical situations require an additional fixation of barrier membranes, their routine application should be approached with caution.

Actinobacillus actinomycetemcomitans in destructive periodontal disease. Three-year follow-up results.

BACKGROUND: Convincing data exist that A. actinomycetemcomitans is an etiologic agent of periodontal disease. The purpose of this longitudinal study was to evaluate A. actinomycetemcomitans as a diagnostic indicator for periodontal disease in treated and periodontally maintained patients. METHODS: Following comprehensive mechanical/surgical and supportive amoxicillin plus metronidazole therapy in 13 subjects with A. actinomycetemcomitans-associated destructive periodontal disease, we monitored subgingival A. actinomycetemcomitans at 4 individual sites in each patient up to 3 years post-therapy. The periodontal status was determined, and A. actinomycetemcomitans levels were quantitatively enumerated on TSBV agar in CFU/ml. Six patients with a persistence of subgingival A. actinomycetemcomitans at each reexamination within 3 years post-therapy were selected to be at risk for minor periodontal treatment outcomes and further recurrence of periodontal disease (test group). Seven subjects with a complete suppression of A. actinomycetemcomitans at each post-therapy visit served as controls. RESULTS: The periodontal parameters decreased from overall values of 6.39 mm (probing depth, PD) and 7.64 mm (clinical attachment level, CAL) at the outset to 3.81 mm (PD) and 5.62 mm (CAL) 2 years post-therapy (Friedman, P< or =0.05). At the 3-year reexamination, the PD/CAL scores increased to 4.03/5.78 mm. Among the 6 individuals (46%) with persistence of subgingival A. actinomycetemcomitans at the final 3-year visit (test group), periodontal status yielded increased levels of 4.45 mm (PD) and 6.60 mm (CAL). The control subjects (n = 7) revealed lower values of 3.67 mm (PD) and 5.09 mm (CAL). However, on a patient level, during the 3-year observational trial, the periodontal status of the 13 individuals was not statistically affected by subgingival infection with A. actinomycetemcomitans. CONCLUSIONS: Although in advanced periodontal disease, comprehensive mechanical and antimicrobial treatment is an appropriate regimen for sustained improvement of periodontal health, long-term control of subgingival infection with A. actinomycetemcomitans could not be achieved. In the maintenance care of destructive periodontitis, the persistence of A. actinomycetemcomitans is not a diagnostic parameter for periodontal disease.

[Effective treatment of a large muscle hernia by local botulinum toxin administration]. / Wirksame Entlastung einer grossen Muskelhernie durch lokale Botulinumtoxin-Anwendung.

A 53-year-old patient developed an impairing muscle hernia when a fascia lata graft was harvested as a substitute for a cruciate ligament of the knee and closure of the defect was not possible. The fascial defect enlarged with time, extending along the whole upper leg. The large muscle protrusion and incarceration in the distal fascial slit was extremely painful during walking and getting up from a chair. Since autologous grafts were disregarded because of the high tissue pressure and alloplastic substitutes seemed problematic, the large hernia was successfully reduced by local muscle denervation with injections of botulinum-A toxin into the protruding vastus lateralis muscle. This procedure achieved relief of pain and enabled the patient to walk without complaints. Side effects were not observed.

Peri-implant conditions in periodontally compromised patients following maxillary sinus augmentation. A long-term post-therapy trial.

Augmentation of the maxillary sinus in the atrophied edentulous posterior maxilla is an integral part of implant prosthodontics. This study examined the clinical outcome in 50 periodontally compromised successfully treated subjects with severe maxillary atrophy following oral implantation with Brånemark, IMZ or Frialit-2 endosseous implants between 1991 and 1994. Simultaneous sinus augmentation was achieved using autogenous bone grafts harvested from the anterior mandible. Oral implants in 37 periodontally healthy patients directly placed in the stable local maxillary bone served as controls. The oral rehabilitation included implant supported restorations or removable superstructures over a period between 3 and 5 years. The peri-implant status of implant abutments inserted in the periodontal compromised augmented maxilla resulted in values comparable to the local maxillary bone except for the GCF rates with enhanced levels of 63.9 +/- 49.9 (controls 37.9 +/- 40.7). The average peri-implant Periotest values in the augmented maxillary sinus (test group) were -3.1 PT and +0.2 PT in the controls. The Periotest scores in the sinus area ranked between -7.0 and +5.0 with mean PT values of -1.5 for IMZ, -3.2 for Brånemark and -4.0 for Frialit-2 abutments. The functional integration of oral implants following sinus augmentation with autologous bone grafts and conventionally placed endosseous implants in the local bone was similar. The additional implant stabilization within the mandibular cortical bone grafts resulted in very low Periotest scores. In periodontally compromised subjects treated for chronic adult periodontitis with minimal maxillary bone height less than 5 mm the endosseous implantation with simultaneous sinus augmentation is recommended as an appropriate technique for long-term oral implant rehabilitation.

[Effect of fluoride iontophoresis in dentin hypersensitivity of periodontally treated patients]. / Wirkung der Fluorid-Ionotophorese bei Dentinhypersensibilität parodontal behandelter Patienten.

Independent of chosen stimulus, age or differences in selected groups of teeth, a reduction in pain intensity of about 50% can be attained following initial treatment with fluoride iontophoresis. The second treatment after 4 weeks leads to a nearly complete reduction in dentin-hypersensitivity. Subsequent to a third application of fluoride iontophoresis only minimal hypersensitivity is detectable. Precipitation of calcium-fluoride and blockage of dentinal tubules should be discussed. A decrease in hypersensitivity is likewise observed in the control group. The results of the final clinical examination show that slightly greater sensitivity remains. Electroanalgetic effects are probably responsible for the reduction of dentinal pain. Treatment of hypersensitivity is dependent upon such factors as age and oral hygiene. Supervised therapy for refractive dentinal pain appears to have greater success when fluoride iontophoresis is used.

Immunoglobulin allotypes and familial cutaneous malignant melanoma/dysplastic nevi. A family study.

Gm and Km allotypes were examined in 355 members of 14 large pedigrees segregating for autosomal dominant cutaneous malignant melanoma/dysplastic nevi. No evidence for linkage was found between the disease locus and the immunoglobulin loci. Furthermore, there was no association of G1m(2) or Gm(-1) with cutaneous malignant melanoma in the 14 independent family probands.

[Femoropatellar pain syndrome. Conservative treatment and results 7-10 years following Maquet operation]. / Das femoropatelläre Schmerzsyndrom. Konservative Behandlung und Resultate 7-10 Jahre nach Maquet-Operation.

Owing to the largely unsatisfactory results achieved to date following surgery for chondromalacia patellae, and owing to our greater knowledge of the functional relationships within the knee joint, the range of aetiological factors in femoropatellar pain needs to be expanded. Functional variants in the muscular and ligamentous system must be considered as well as anatomical variants. On the basis of symptomatology and the results of clinical examination, two extreme forms can be distinguished, depending on ligamentous laxity and muscular stabilisation capacity. Physiotherapy must be determined by these. There remain few cases in which surgery is indicated.