Functional analysis of poxvirus anaphase-promoting complex regulator (PACR), an orf virus cell cycle regulator.
Orsbourn, Conor Bryce
Viruses, after long periods of time infecting and co-evolving with their hosts have developed complex and ingenious ways of manipulating host cell environments to better suit their needs. One such example of this is modulation of the cell cycle. One important regulator of the cell cycle is the anaphase promoting complex/cyclosome (APC), a ubiquitin ligase. It is important in the exit from mitosis, in maintaining the cell in G1 phase and inhibiting the transition into S phase until conditions are appropriate. Orf virus expresses a protein called the poxvirus anaphase promoting complex/cyclosome regulator (PACR), which interacts with, and influences the action of the APC. It is currently suggested that PACR’s inhibition of the APC leads the cell to enter a pseudo-S phase like state which increases the levels of cellular replicative products for orf virus to use for viral replication. The fact that a PACR deleted mutant orf virus (OV PACR KO) shows reduced growth characteristics supports this model, but there is no definitive evidence linking PACR’s effects on APC and its role in viral replication. Restoration of wild type (WT) levels of growth to the mutant virus when APC is inactivated by RNA interference is one means of establishing such a link, and formed the basis of this study.A first step was to identify suitable cells. The limited availability of ovine reagents meant that the standard cells used in our studies of orf virus, primary lamb testes (LT cells), were not suitable. The growth of WT and OV PACR KO orf virus in LT cells were therefore compared to growth in two human cell lines, HeLa and human dermal fibroblasts (HDFs). These studies revealed that the impaired growth of OV PACR KO relative to WT seen in LT cells was replicated in HDFs. Furthermore, a derivative of OV PACR KO in which a functional PACR gene had been restored (OV PACR Restore) showed the same growth characteristics as WT orf virus. Intriguingly, all three viruses grew equally well in HeLa cells, perhaps reflecting the transformed nature of these cervical carcinoma-derived cells. HDF cells were therefore selected to examine the effect of the inactivation of APC following siRNA knockdown (KD) of APC11 subunit 11 (APC11). Western blot analysis confirmed effective knock down of APC11. APC11 KD cells were infected 72 hours after transfection with WT, OV PACR KO and OV PACR Restore virus and viral yields compared to those obtained in normal HDF cells. When the growth curves were compared we saw that the WT and Restore viruses had similar growth characteristics in both sets of cells and growth of the OV PACR KO virus was not enhanced in the APC11 KD HDF cells.FACS analysis of propidium iodide stained APC11 KD showed no obvious disruption of the cell cycle, including no accumulation of cells in G2/M phase as would be expected upon the knock down of APC11. In another functional check on the knock down, western blot analysis of thymidine kinase (TK) levels was undertaken. APC directs TK’s proteosomal degradation during G1 phase and TK levels rise as cells go through the G1/S transition. Our analysis showed a transient increase in TK levels in APC11 KD cells. These results showed that the APC11 KD was having the desired effect on APC controlled substrates. However, elevated TK levels occurring at 48 post transfection would have been unlikely to benefit viral growth in the transfection-infection schedule employed in this study.An assay of viral β-glucuronidase reporter gene activity was developed as a way of comparing virus levels without having to complete lengthy plaque titrations. The assay showed similar trends to those seen in the plaque titrations and could be used as a fast method for confirming differences in viral load in future studies.In summary it has been shown that HDF cells are appropriate cells for these studies. Efficient knock down of APC11 can be achieved in these cells and resulted in elevated levels of cellular TK. Further experiments using a modified schedule will be required to complete the examination of the mechanism by which PACR assists orf virus replication.
Advisor: Mercer, Andrew
Degree Name: Master of Science
Degree Discipline: Microbiology and Immunology
Publisher: University of Otago
Keywords: PACR; orf
Research Type: Thesis