Steven Nagar

Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation.

Geminiviruses have small, single-stranded DNA genomes that replicate through double-stranded intermediates in the nuclei of infected plant cells. Viral double-stranded DNA also assembles into minichromosomes and is transcribed in infected cells. Geminiviruses encode only a few proteins for their replication and transcription and rely on host enzymes for these processes. However, most plant cells, which have exited the cell cycle and undergone differentiation, do not contain the replicative enzymes necessary for viral DNA synthesis. To overcome this barrier, geminiviruses induce the accumulation of DNA replication machinery in mature plant cells, most likely by modifying cell cycle and transcriptional controls. In animals, several DNA viruses depend on host replication and transcription machinery and can alter their hosts to create an environment that facilitates efficient viral replication. Analysis of these viruses and their proteins has contributed significantly to our understanding of DNA replication, transcription, and cell cycle regulation in mammalian cells. Geminiviruses have the same potential for plant systems. Plants offer many advantages for these types of studies, including ease of transformation, well-defined cell populations and developmental programs, and greater tolerance of cell cycle perturbation and polyploidy. Our knowledge of the molecular and cellular events that mediate geminivirus infection has increased significantly during recent years. The goal of this review is to summarize recent research addressing geminivirus DNA replication and its integration with transcriptional and cell cycle regulatory processes.

A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells.

Geminiviruses are plant DNA viruses that replicate through DNA intermediates in plant nuclei. The viral components required for replication are known, but no host factors have yet been identified. We used immunolocalization to show that the replication proteins of the geminivirus tomato golden mosaic virus (TGMV) are located in nuclei of terminally differentiated cells that have left the cell cycle. In addition, TGMV infection resulted in a significant accumulation of the host DNA synthesis protein proliferating cell nuclear antigen (PCNA). PCNA, an accessory factor for DNA polymerase delta, was not present at detectable levels in healthy differentiated cells. The TGMV replication protein AL1 was sufficient to induce accumulation of PCNA in terminally differentiated cells of transgenic plants. Analysis of the mechanism(s) whereby AL1 induces the accumulation of host replication machinery in quiescent plant cells will provide a unique opportunity to study plant DNA synthesis.

Host DNA Replication Is Induced by Geminivirus Infection of Differentiated Plant Cells

The geminivirus Tomato golden mosaic virus (TGMV) replicates in differentiated plant cells using host DNA synthesis machinery. We used 5-bromo-2-deoxyuridine (BrdU) incorporation to examine DNA synthesis directly in infected Nicotiana benthamiana plants to determine if viral reprogramming of host replication controls had an impact on host DNA replication. Immunoblot analysis revealed that up to 17-fold more BrdU was incorporated into chromosomal DNA of TGMV-infected versus mock-infected, similarly treated healthy leaves. Colocalization studies of viral DNA and BrdU demonstrated that BrdU incorporation was specific to infected cells and was associated with both host and viral DNA. TGMV and host DNA synthesis were inhibited differentially by aphidicolin but were equally sensitive to hydroxyurea. Short BrdU labeling times resulted in some infected cells showing punctate foci associated with host DNA. Longer periods showed BrdU label uniformly throughout host DNA, some of which showed condensed chromatin, only in infected nuclei. By contrast, BrdU associated with viral DNA was centralized and showed uniform, compartmentalized labeling. Our results demonstrate that chromosomal DNA is replicated in TGMV-infected cells.

Iron metabolism in the eye: A review

This review article covers all aspects of iron metabolism, which include studies of iron levels within the eye and the processes used to maintain normal levels of iron in ocular tissues. In addition, the involvement of iron in ocular pathology is explored. In each section there is a short introduction to a specific metabolic process responsible for iron homeostasis, which for the most part has been studied in non-ocular tissues. This is followed by a summary of our current knowledge of the process in ocular tissues.

Altered Ferritin Subunit Composition: Change in Iron Metabolism in Lens Epithelial Cells and Downstream Effects on Glutathione Levels and VEGF Secretion

PURPOSE The iron storage protein ferritin is necessary for the safe storage of iron and for protection against the production of iron-catalyzed oxidative damage. Ferritin is composed of 24 subunits of two types: heavy (H) and light (L). The ratio of these subunits is tissue specific, and alteration of this ratio can have profound effects on iron storage and availability. In the present study, siRNA for each of the chains was used to alter the ferritin H:L chain ratio and to determine the effect of these changes on ferritin synthesis, iron metabolism, and downstream effects on iron-responsive pathways in canine lens epithelial cells. METHODS Primary cultures of canine lens epithelial cells were used. The cells were transfected with custom-made siRNA for canine ferritin H- and L-chains. De novo ferritin synthesis was determined by labeling newly synthesized ferritin chains with 35S-methionine, immunoprecipitation, and separation by SDS-PAGE. Iron uptake into cells and incorporation into ferritin was measured by incubating the cells with 59Fe-labeled transferrin. Western blot analysis was used to determine the presence of transferrin receptor, and ELISA was used to determine total ferritin concentration. Ferritin localization in the cells was determined by immunofluorescence labeling. VEGF, glutathione secretion levels, and cystine uptake were measured. RESULTS FHsiRNA decreased ferritin H-chain synthesis, but doubled ferritin L-chain synthesis. FLsiRNA decreased both ferritin H- and L-chain synthesis. The degradation of ferritin H-chain was blocked by both siRNAs, whereas only FHsiRNA blocked the degradation of ferritin L-chain, which caused significant accumulation of ferritin L-chain in the cells. This excess ferritin L-chain was found in inclusion bodies, some of which were co-localized with lysosomes. Iron storage in ferritin was greatly reduced by FHsiRNA, resulting in increased iron availability, as noted by a decrease in transferrin receptor levels and iron uptake from transferrin. Increased iron availability also increased cystine uptake and glutathione concentration and decreased nuclear translocation of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor (VEGF) accumulation in the cell-conditioned medium. CONCLUSIONS Most of the effects of altering the ferritin H:L ratio with the specific siRNAs were due to changes in the availability of iron in a labile pool. They caused significant changes in iron uptake and storage, the rate of ferritin synthesis and degradation, the secretion of VEGF, and the levels of glutathione in cultured lens epithelial cells. These profound effects clearly demonstrate that maintenance of a specific H:L ratio is part of a basic cellular homeostatic mechanism.

Hypoxia controls iron metabolism and glutamate secretion in retinal pigmented epithelial cells.

BACKGROUND Blood-barrier systems are essential in controlling iron levels in organs such as the brain and eye, both of which experience hypoxia in pathological conditions. While hypoxias effects on numerous iron regulatory and storage proteins have been studied, little is known about how hypoxia affects iron metabolism. Iron also controls glutamate production and secretion; therefore the effects of hypoxia on iron metabolism and glutamate secretion were studied in polarized retinal pigmented epithelial (RPE) cells. METHODS Primary canine RPE were cultured in Millicells to create polarized cell cultures. Iron uptake and efflux were measured in hypoxic and normoxic conditions. RPE were loaded with ⁵⁹Fe-transferrin. Glutamate concentrations in the cell conditioned media were also measured. RESULTS Hypoxia induced a large increase in iron efflux from RPE in the basolateral direction. Glutamate secretion occurred mainly in the basolateral direction which is away from the retina and out of the eye in vivo. Glutamate secretion was doubled under hypoxic conditions. CONCLUSIONS Hypoxia is known to induce oxidative damage. The current results show that iron, a key catalyst of free radical generation, is removed from RPE under hypoxic conditions which may help protect RPE from oxidative stress. Results obtained here indicate the importance of using polarized tight junctional cells as more physiologically relevant models for blood-barrier-like systems. GENERAL SIGNIFICANCE While the effects of hypoxia on iron efflux and glutamate secretion may be protective for RPE cells and retina, increased glutamate secretion in the brain could cause some of the damaging neurological effects seen in stroke.

CHANGES IN FERRITIN H- AND L-CHAINS IN CANINE LENSES WITH AGE-RELATED NUCLEAR CATARACT

PURPOSE To determine potential differences in the characteristics of the iron storage protein ferritin and its heavy (H) and light (L) subunits in fiber cells from cataractous and noncataractous lenses of older dogs. METHODS Lens fiber cell homogenates were analyzed by SDS-PAGE, and ferritin chains were immunodetected with ferritin chain-specific antibodies. Ferritin concentration was measured by ELISA. Immunohistochemistry was used to localize ferritin chains in lens sections. RESULTS The concentration of assembled ferritin was comparable in noncataractous and cataractous lenses of similarly aged dogs. The ferritin L-chain detected in both lens types was modified and was approximately 11 kDa larger (30 kDa) than standard L-chain (19 kDa) purified from canine liver. The H-chain identified in cataractous fiber cells (29 kDa) differed from the 21-kDa standard canine H-chain and from the 12-kDa modified H-chain present in fiber cells of noncataractous lenses. Histologic analysis revealed that the H-chain was distributed differently throughout cataractous lenses compared with noncataractous lenses. There was also a difference in subunit makeup of assembled ferritin between the two lens types. Ferritin from cataractous lenses contained more H-chain and bound 11-fold more iron than ferritin from noncataractous lenses. CONCLUSIONS There are significant differences in the characteristics of ferritin H-chain and its distribution in canine cataractous lenses compared with noncataractous lenses. The higher content of H-chain in assembled ferritin allows this molecule to sequester more iron. In addition, the accumulation of H-chain in deeper fiber layers of the lens may be part of a defense mechanism by which the cataractous lens limits iron-catalyzed oxidative damage.

Source-Dependent Intracellular Distribution of Iron in Lens Epithelial Cells Cultured Under Normoxic and Hypoxic Conditions

PURPOSE Intracellular iron trafficking and the characteristics of iron distribution from different sources are poorly understood. We previously determined that the lens removes excess iron from fluids of inflamed eyes. In the current study, we examined uptake and intracellular distribution of ⁵⁹Fe from iron transport protein transferrin or ferric chloride (nontransferrin-bound iron [NTBI]) in cultured canine lens epithelial cells (LECs). Because lens tissue physiologically functions under low oxygen tension, we also tested effects of hypoxia on iron trafficking. Excess iron, not bound to proteins, can be damaging to cells due to its ability to catalyze formation of reactive oxygen species. METHODS LECs were labeled with ⁵⁹Fe-Tf or ⁵⁹FeCl₃ under normoxic or hypoxic conditions. Cell lysates were fractioned into mitochondria-rich, nuclei-rich, and cytosolic fractions. Iron uptake and its subcellular distribution were measured by gamma counting. RESULTS ⁵⁹Fe accumulation into LECs labeled with ⁵⁹Fe-Tf was 55-fold lower as compared with that of ⁵⁹FeCl₃. Hypoxia (24 hours) decreased uptake of iron from transferrin but not from FeCl₃. More iron from ⁵⁹FeCl₃ was directed to the mitochondria-rich fraction (32.6%-47.7%) compared with ⁵⁹Fe from transferrin (10.6%-12.6%). The opposite was found for the cytosolic fraction (8.7%-18.3% and 54.2%-46.6 %, respectively). Hypoxia significantly decreased iron accumulation in the mitochondria-rich fraction of LECs labeled with ⁵⁹Fe-Tf . CONCLUSIONS There are source-dependent differences in iron uptake and trafficking. Uptake and distribution of NTBI are not as strictly regulated as that of iron from transferrin. Excessive exposure to NTBI, which could occur in pathological conditions, may oxidatively damage organelles, particularly mitochondria.