Abstract
The parapoxvirus, orf virus (OV) encodes a RING-H2 protein that is a homologue of subunit 11 (APC11) of the Anaphase Promoting Complex or Cyclosome (APC/C). APC/C is a ubiquitin ligase and through this activity it functions as a master regulator of the cell cycle responsible for controlling mitotic exit and G1 phase progression. The OV homologue of APC11, referred to as PACR (Poxvirus APC/C Regulator), shares with APC11 the ability to bind APC2 and competes with APC11 for incorporation into APC/C. However, unlike its cellular counterpart, PACR does not support ubiquitination. This led us to postulate that OV may be capable of modulating the cell cycle. In the present study, we show that PACR causes G2/M arrest, consistent with the inhibition of APC/C. Interestingly, OV infection causes G1 phase arrest and prevents cellular DNA replication. These effects were not dependent on the presence of PACR, although, the inhibitory effects occurred more quickly in its presence. The rapid arrest is believed to be contributed to by the accumulation of the cyclin-dependent kinase (CDK) inhibitor, p27, which is a known inhibitor of S phase entry. Surprisingly on top of this, there are also indications of OV having a cell cycle promoting affect similar to mitogenic stimulation.
OV replication was shown to be more sensitive to the inhibitory effects of the nucleoside analogue, cidofovir (CDV) when PACR is absent. This suggests a role for PACR in generating a nucleotide pool able to support efficient OV replication. Curiously, all of the small number of poxviruses that encode PACR homologues do not encode a version of the viral thymidine kinase, which is otherwise common among poxviruses, and all of the PACR-encoding viruses are primarily epitheliotropic. The current study provides a glimpse into OV's modulation of the cell cycle and to the possible roles PACR plays in ensuring successful OV replication in the skin.
The present study also shows that the M026L protein, a PACR homologue encoded by Molluscum contagiosum virus, is functionally similar to PACR in its binding with APC/C and inability to support ubiquitination. Structural modelling of PACR and M026L shows substitution of amino acids in loop 2 and the α2-helix region, which in APC11 form a hydrophobic cleft needed for the recruitment of E2. It is hypothesized these differences explain the absence of ubiquitination function in M026L and PACR. A similar substitution pattern is conserved in all PACR homologues suggesting conservation of function as well. Based on these data it is proposed that the PACR-encoding epitheliotropic poxviruses diverged from a common ancestor after the acquisition of the PACR progenitor from a host species.