Inhibition Of Cellular Proteins Research Articles

Characterization of the African Swine Fever Virus Decapping Enzyme during Infection.

ABSTRACTAfrican swine fever virus (ASFV) infection is characterized by a progressive decrease in cellular protein synthesis with a concomitant increase in viral protein synthesis, though the mechanism by which the virus achieves this is still unknown. Decrease of cellular mRNA is observed during ASFV infection, suggesting that inhibition of cellular proteins is due to an active mRNA degradation process. ASFV carries a gene (Ba71V D250R/Malawi g5R) that encodes a decapping protein (ASFV-DP) that has a Nudix hydrolase motif and decapping activity in vitro. Here, we show that ASFV-DP was expressed from early times and accumulated throughout the infection with a subcellular localization typical of the endoplasmic reticulum, colocalizing with the cap structure and interacting with the ribosomal protein L23a. ASFV-DP was capable of interaction with poly(A) RNA in cultured cells, primarily mediated by the N-terminal region of the protein. ASFV-DP also interacted with viral and cellular RNAs in the context of infection, and its overexpression in infected cells resulted in decreased levels of both types of transcripts. This study points to ASFV-DP as a viral decapping enzyme involved in both the degradation of cellular mRNA and the regulation of viral transcripts.IMPORTANCE Virulent ASFV strains cause a highly infectious and lethal disease in domestic pigs for which there is no vaccine. Since 2007, an outbreak in the Caucasus region has spread to Russia, jeopardizing the European pig population and making it essential to deepen knowledge about the virus. Here, we demonstrate that ASFV-DP is a novel RNA-binding protein implicated in the regulation of mRNA metabolism during infection, making it a good target for vaccine development.

Mechanism of Resistance of Hepatitis C Virus Replicons to Structurally Distinct Cyclophilin Inhibitors

The current standard of care for hepatitis C virus (HCV) infection, pegylated alpha interferon in combination with ribavirin, has a limited response rate and adverse side effects. Drugs targeting viral proteins are in clinical development, but they suffer from the development of high viral resistance. The inhibition of cellular proteins that are essential for viral amplification is thought to have a higher barrier to the emergence of resistance. Three cyclophilin inhibitors, the cyclosporine analogs DEBIO-025, SCY635, and NIM811, have shown promising results for the treatment of HCV infection in early clinical trials. In this study, we investigated the frequency and mechanism of resistance to cyclosporine (CsA), NIM811, and a structurally unrelated cyclophilin inhibitor, SFA-1, in replicon-containing Huh7 cells. Cross-resistance between all clones was observed. NIM811-resistant clones were selected only after obtaining initial resistance to either CsA or SFA-1. The time required to select resistance against cyclophilin inhibitors was significantly longer than that required for resistance selection against viral protein inhibitors, and the achievable resistance level was substantially lower. Resistance to cyclophilin inhibitors was mediated by amino acid substitutions in NS3, NS5A, and NS5B, with NS5A mutations conferring the majority of resistance. Mutation D320E in NS5A mediated most of the resistance conferred by NS5A. Taken together, the results indicate that there is a very low frequency and level of resistance to cyclophilin-binding drugs mediated by amino acid substitutions in three viral proteins. The interaction of cyclophilin with NS5A seems to be the most critical, since the NS5A mutations have the largest impact on resistance.

The effects of estrogen and progesterone in vivo on protein synthesis and secretion by cultured epithelial cells from sheep endometrium.

Ovariectomized ewes were treated with either nothing or implants of estrogen (E), progesterone (P), or E + P. Epithelial and stromal cells from caruncular and intercaruncular regions of sheep endometrium were dispersed by collagenase digestion and enriched by Ficoll gradient separation. Verification of cell types was by electron microscopy, keratin staining (epithelial cells), cell size, and appearance in culture. Epithelial cells were cultured under optimized conditions with [35S]methionine (S-met) and uptake of label by cells and its incorporation into cellular and secreted protein determined. Protein in the medium and lysed cells was analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells from E-treated animals had higher S-met uptake and incorporation into proteins (cellular and secreted) than cells from ewes treated with nothing and P-treated animals. E effects were not significantly reduced in the presence of P. When secreted protein was expressed as a percent of total incorporated S-met, P treatment either alone or with E increased the proportion of labeled protein secreted by cells. There were no significant differences between caruncular and intercaruncular. Two-dimensional polyacrylamide-gel electrophoresis of secreted proteins showed one major glycoprotein (mol wt, 46,000, isoelectric point, 5.8-6.5) and four minor proteins induced by E + P greater than E, and five minor proteins inhibited by the steroids. Both induction and inhibition of cellular proteins were also apparent, though of lesser magnitude. Overall, whereas E treatment in vivo influenced the rate of incorporation of S-met into proteins by epithelial cells in vitro, P treatment increased the proportion of newly synthesized protein which was secreted. Steroids caused significant alterations in the individual proteins secreted by ovine endometrium.

Therapeutic activity of pretazettine on Rauscher leukemia: combination of antiviral activity and cellular protein inhibition.

Pretazettine hydrochloride (PTZ) has been found to inhibit protein synthesis, without being inhibitory to DNA and RNA, in Rauscher leukemic blood cells in mice for at least 6 h after its administration. With comparison to Virazole and cycloheximide, the specific anti-Rauscher virus activity of PTZ has been demonstrated only in acutely-infected NIH/3T3 cells but not in chronically-infected cells. It is not certain that the inhibitory action of PTZ on reverse transcriptase is contributory to its therapeutic activity in leukemic mice.