3′-Deoxyadenosine, also known as cordycepin, is a known polyadenylation inhibitor with a large spectrum of biological activities, including anti-proliferative, pro-apoptotic and anti-inflammatory effects. In this study we confirm that cordycepin reduces the length of poly(A) tails, with some mRNAs being much more sensitive than others. The low doses of cordycepin that cause poly(A) changes also reduce the proliferation of NIH3T3 fibroblasts. At higher doses of the drug we observed inhibition of cell attachment and a reduction of focal adhesions. Furthermore, we observed a strong inhibition of total protein synthesis that correlates with an inhibition of mammalian target of rapamycin (mTOR) signaling, as observed by reductions in Akt kinase and 4E-binding protein (4EBP) phosphorylation. In 4EBP knock-out cells, the effect of cordycepin on translation is strongly reduced, confirming the role of this modification. In addition, the AMP-activated kinase (AMPK) was shown to be activated. Inhibition of AMPK prevented translation repression by cordycepin and abolished 4EBP1 dephosphorylation, indicating that the effect of cordycepin on mTOR signaling and protein synthesis is mediated by AMPK activation. We conclude that many of the reported biological effects of cordycepin are likely to be due to its effects on mTOR and AMPK signaling.
The African swine fever virus (ASFV) DP71L protein is present in all isolates as either a short form of 70 to 72 amino acids or a long form of about 184 amino acids, and both of these share sequence similarity to the C-terminal domain of the herpes simplex virus ICP34.5 protein and cellular protein GADD34. In the present study we expressed DP71L in different mammalian cells and demonstrated that DP71L causes dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2␣) in resting cells and during chemical-induced endoplasmic reticulum stress and acts to enhance expression of cotransfected reporter genes. We showed that DP71L binds to all the three isoforms (␣, , and ␥) of the protein phosphatase 1 catalytic subunit (PP1c) and acts by recruiting PP1c to eIF2␣. We also showed that DP71L inhibits the induction of ATF4 and its downstream target, CHOP. We investigated the eIF2␣ phosphorylation status and induction of CHOP in porcine macrophages infected by two ASFV field isolates, Malawi Lil20/1 and Benin 97/1, and two DP71L deletion mutants, Malawi⌬NL and E70⌬NL. Our results showed that deletion of the DP71L gene did not cause an increase in the level of eIF2␣ phosphorylation or induction of CHOP, indicating that DP71L is not the only factor required by the virus to control the phosphorylation level of eIF2␣ during infection. We therefore hypothesize that ASFV has other mechanisms to prevent the eIF2␣ phosphorylation and the subsequent protein synthesis inhibition.
The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate the translation initiation factor 2α (eIF2α) and avoid shut-off of global protein synthesis and downstream activation of the pro-apoptotic factor CHOP. Residues V16 and F18A were critical for binding of DP71L to PP1. Mutation of this PP1 binding motif or deletion of residues between 52 and 66 reduced the ability of DP71L to cause dephosphorylation of eIF2α and inhibit CHOP induction. The residues LSAVL, between 57 and 61, were also required. PP1 was co-precipitated with wild type DP71L and the mutant lacking residues 52- 66 or the LSAVL motif, but not with the PP1 binding motif mutant. The residues in the LSAVL motif play a critical role in DP71L function but do not interfere with binding to PP1. Instead we propose these residues are important for DP71L binding to eIF2α.
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