Novel Regulatory Mechanisms by Which Large T Antigen Coordinates the Merkel Cell Polyomavirus Life Cycle

Due to its association with Merkel cell carcinoma (MCC), a substantial effort has been made to better understand how Merkel cell polyomavirus (MCPyV) proteins drive oncogenesis; however, our understanding of the early steps of MCPyV infection remains poor. The polyomavirus Large Tumor antigen (LT) is a highly multi-functional protein with a wide range of activities, including: stimulation of cellular proliferation through its interaction with retinoblastoma protein and DnaJ heatshock protein family members; arrest of the cell cycle through a poorly understood activity localized to the C-terminal region; and regulation of the initiation of viral DNA replication. LT proteins also play important roles in regulating viral transcription. How these various functions are regulated to ensure an orderly progression of events conducive for the viral life cycle has not been well established. In this study, I show how phosphorylation of MCPyV LT plays an important role in regulating its many functions. I identify threonines 297 and 299 as key phospho-sites which regulate LT\u27s ability to initiate replication. T297 phosphorylation inhibits LT binding to the viral origin of replication and acts as an off switch, while phosphorylation of T299 is required to stimulate LT-mediated replication of viral genomes. This study was the first to identify phosphorylation sites of LT and link them to important protein functions. Cross-reactivity to a phospho-specific antibody revealed yet another phosphorylation site on MCPyV LT as S816. We discovered that this phosphorylation event is mediated by ATM kinase, and may play a role in the MCPyV LT C-terminal domain\u27s ability to arrest the cell cycle. This study helps to further elucidate MCPyV\u27s association with the host DNA Damage Response (DDR) and provides some rational for the recruitment of these factors to viral replication centers. Finally, studies of the viral non-coding control region (NCCR) reveal a surprising interaction between LT and sT on the late promoter. MCPyV LT is able to robustly stimulate the late promoter only in the context of an intact Ori and sT co-expression. Using phosphomutant LTs and mutant Ori sequences, I highlight the importance of LT binding to the Ori and stimulation of replication as key factors in LT-mediated activation of the late promoter in the context of sT co-expression. LT alone actually represses the late promoter and requires sT coexpression to efficiently stimulate the late promoter after replication. This study therefore reveals an important dependence on sT expression for the regulation of transcription that has not yet been reported with other polyomaviruses. In sum, this study demonstrates multiple mechanisms of regulation including protein phosphorylation, protein-DNA interactions, and co-expression of key viral proteins as regulators of LT function. These studies may help elucidate critical factors required for establishing a robust cellular infection system which is greatly needed to further our understanding of the basic virology of this important human tumor virus

Two key events associated with a transposable element burst occurred during rice domestication

Transposable elements shape genome evolution through periodic bursts of amplification. In this study we exploited knowledge of the components of the mPing/Ping/Pong TE family in four rice strains undergoing mPing bursts to track their copy numbers and distribution in a large collection of genomes from the wild progenitor Oryza rufipogon and domesticated Oryza sativa (rice). We characterized two events that occurred to the autonomous Ping element and appear to be critical for mPing hyperactivity. First, a point mutation near the end of the element created a Ping variant ( Ping16A ) with reduced transposition. The proportion of strains with Ping16A has increased during domestication while the original Ping (Ping16G) has been dramatically reduced. Second, transposition of Ping16A into a Stowaway element generated a locus ( Ping16A_Stow ) whose presence correlates with strains that have high mPing copies. Finally, demonstration that Pong elements have been stably silenced in all strains analyzed indicates that sustained activity of the mPing/Ping family during domestication produced the components necessary for the mPing burst, not the loss of epigenetic regulation

Calibration of the Actical Accelerometer in Adults

The purpose of this investigation was to develop cut-points for the Actical accelerometer in adults that correspond to light, moderate, and vigorous physical activity using a percentage of maximal oxygen uptake (VO2max). Twenty five young adults completed a progressive submaximal exercise test on a treadmill wearing an Actical accelerometer while oxygen uptake was measured. The VO2max based cut-points for light-to-moderate was 4952 counts per minute (cpm), for moderate-to-vigorous intensity was 9714 cpm. VO2 based cut-points were significantly greater than MET based cut-points. The results of this investigation suggest that MET definitions of moderate and vigorous intensity are too light for young adults and may lead to misclassification of physical activity levels. The results also suggest that individual calibration of the Actical accelerometer may be needed due to the high variability of VO2max based cut-points