Characterization of tomato golden mosaic virus (TGMV) infection in planta and its effects on plant cell cycle, nuclear process, and DNA replication dynamics

Tomato golden mosaic virus (TGMV) is a single-stranded DNA geminivirus that infects dicotyledonous plants. Because TGMV does not encode DNA polymerases, viral replication is completely dependent on host enzymes. Many geminiviruses are restricted to developing vascular tissues, which contain meristematic cells competent for DNA replication and cell division. Infection of Nicotiana benthamiana with TGMV, however, revealed nuclear localization of TGMV DNA and the TGMV replication proteins AL1 and AL3 in a variety of differentiated cell types. This presented a paradox: How can TGMV replicate in cells that have exited the cell cycle and no longer contain DNA replication machinery? One possibility was that like many mammalian DNA tumor viruses, TGMV might coordinate reentry of quiescent cells into the cell cycle and thus create a nuclear environment conducive to DNA synthesis. I examined several parameters of TGMV infection to address this hypothesis. Morphological evidence such as nuclear hypertrophy, movement of nuclei toward the cell center, and condensation of chromatin characteristic of prophase, supported the possibility that TGMV induces cell cycle functions. TGMV was also found to induce the accumulation of the host-encoded DNA synthesis factor proliferating cell nuclear antigen (PCNA) in the same TGMV-infected cells that contained TGMV AL1. TGMV AL1 was also found to interact with the host cell cycle regulatory protein pRb. One outstanding question that arose was is the chromosomal DNA also replicated during infection? To address this question, I examined nascent DNA synthesis using 5-bromo-2-deoxyuridine (BrdU) incorporation and found that both viral and chromosomal DNA were replicated. The patterns of BrdU incorporation demonstrated that host and viral DNA replication could also be separated spatially and temporally within a single cell. Many nuclei showed BrdU incorporation in discrete foci that were reminiscent of host replication factories, and diffuse chromatin characteristic of S phase. Together, the evidence suggests that TGMV induces differentiated cells to reenter the cell cycle, establishes conditions favorable for DNA replication, and causes extensive reorganization of plant nuclear processes. This work sets the stage for the further characterization of TGMV infection dynamics, TGMV interaction with the plant cell cycle, and viral and plant DNA replication.