G1 Phase Cell Cycle Arrest Induced by SARS-CoV 3a Protein via the Cyclin D3/pRb Pathway

Abstract

SARS-CoV 3a is a structural protein, mainly localizing to Golgi apparatus and co-localizing with SARS-CoV M in co-transfected cells. Here we observed that transient expression of 3a inhibited cell growth and prevented 5-bromodeoxyuridine incorporation, suggesting that 3a deregulated cell cycle progression. Cell cycle analysis demonstrated that 3a expression was associated with blockage of cell cycle progression at G1 phase in HEK 293, COS-7, and Vero cells 24-60 h after transfection. Mutation analysis of 3a revealed that C-terminal region (176 aa approximately 274 aa), including a potential calcium ATPase motif, was essential for induction of cell cycle arrest. Topological analysis showed that 3a predominantly located in Golgi apparatus, with its N-terminus residing in the lumen (Nlum) and C-terminus in the cytosol (Ccyt). Analyzing the cellular proteins involving in regulation of cell cycle progression, we demonstrated that 3a expression was correlated with a significant reduction of cyclin D3 level and phosphorylation of retinoblastoma (Rb) protein at Ser-795 and Ser-809/811, not with the expression of cyclin D1, D2, cdk4, and cdk6 in 293 cells. Increases in p53 phosphorylation on Ser-15 were observed in both SARS-CoV M and 3a transfected cells, suggesting that it might not correlate with the 3a-induced G0/G1 phase arrest. The reduction of cyclin D3 level and phosphorylation of Rb were further confirmed in SARS-CoV infected Vero cells. These results indicate that SARS-CoV 3a protein, through limiting the expression of cyclin D3, may inhibit Rb phosphorylation, which in turn leads to a block in the G1 phase of the cell cycle and an inhibition of cell proliferation.

Accessory proteins of SARS-CoV and other coronaviruses

“…Huang et al demonstrated by sucrose-gradient centrifugation and densitometric scans the presence of a 37-kDa form (protein 3a-1) and a 31-kDa form (protein 3a-2). The 37-kDa form was detected in fractions with similar densities (1.18-1.20 g/ml) to the SARS-CoV particles, as opposed to fractions containing the 31-kDa form (1.13-1.15 g/ml) Yuan et al, 2007, suggesting that the 31-kDa form may be present as extracellular membrane structures while the 37-kDa form may be assembled into the mature virons. The presence of these two forms is due to the protein 3a being able to be modified post-translationally.…”

“…The C-terminal domain is hydrophilic in nature and contains both the YxxU (where x represents any aa and U is an amino acid with a hydrophobic, bulky side-chain) and the diacidic motifs (ExD, Asp-x-Glu, where x represents any aa). This domain can play a role in G1 cell-cycle arrest by depletion of cyclin D3 (Yuan et al, 2007). Interestingly, p3a seems to share a similar topology with the M protein (Marra et al, 2003).…”

Alteronol inhibits proliferation in HeLa cells through inducing a G1-phase arrest

“…Western blot assays were carried out as described previously [12,13] . HeLa Cells were incubated with increasing concentrations of alteronol (1.5, 3.0, 4.5 µg/ml) for 24 h. Western blot analysis using total protein extracts from cultured cells was performed as previously described [12,13] .…”

“…Western blot assays were carried out as described previously [12,13] . HeLa Cells were incubated with increasing concentrations of alteronol (1.5, 3.0, 4.5 µg/ml) for 24 h. Western blot analysis using total protein extracts from cultured cells was performed as previously described [12,13] . Proteins were size fractionated by 10% gel electrophoresis and the nitrocellulose membranes were probed overnight at 4°C with a 1 : 200 dilution of mouse anti‐human cyclinD1 antibody (Santa Cruz Biotechnology).…”

Hybridising Genetic Algorithm-Neural Network (GANN) in marker genes detection

“…Cyclins function as regulators of CDK kinases and different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal co-ordination of each mitotic event. The protein encoded by this gene has been shown to interact with and be involved in the phosphorylation of tumour suppression protein Rb [17]. Cyclin D3 is higher associated with Tlymphoblastic leukaemia/lymphomas (T-LBLL), a neoplastic counterpart of T cells at the early developmental stages of differentiation [13].…”