Different Effect of Proteasome Inhibition on Vesicular Stomatitis Virus and Poliovirus Replication

Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2α, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection

Different proteasome inhibitors affect VSV replication.

<p>(A) Proteasome inhibitor 1 and Bortezomib decreased VSV replication. Titration of VSV from the medium of overnight infected HeLa cells. VSV infection (MOI = 1) for one hour was substituted by the regular medium with indicated concentration of proteasome inhibitors. VSV was titrated by plaque assay after overnight growth. (B) Analysis of P-protein synthesis in the cells treated with proteasome inhibitor 1. HeLa cells were infected with VSV (MOI = 5) for 4 h and treated with proteasome inhibitor 1 (PI) or MG132 (MG) at a time of VSV infection. The total protein extracts (5 µg) from these cells were analyzed by Western blotting with anti-P-protein Abs. The concentrations of proteasome inhibitors varied from 5 to 20 µM. Keratin 18 (K18) was a protein loading control. (C) Bortezomib suppressed VSV replication. HeLa cells were infected with VSV, treated with Bortezomib (100 nM) and MG132 (5 µM), and analyzed as described in panel B. K18 was a protein loading control.</p

Functional Specialization of Human Salivary Glands and Origins of Proteins Intrinsic to Human Saliva.

Salivary proteins are essential for maintaining health in the oral cavity and proximal digestive tract, and they serve as potential diagnostic markers for monitoring human health and disease. However, their precise organ origins remain unclear. Through transcriptomic analysis of major adult and fetal salivary glands and integration with the saliva proteome, the blood plasma proteome, and transcriptomes of 28+ organs, we link human saliva proteins to their source, identify salivary-gland-specific genes, and uncover fetal- and adult-specific gene repertoires. Our results also provide insights into the degree of gene retention during gland maturation and suggest that functional diversity among adult gland types is driven by specific dosage combinations of hundreds of transcriptional regulators rather than by a few gland-specific factors. Finally, we demonstrate the heterogeneity of the human acinar cell lineage. Our results pave the way for future investigations into glandular biology and pathology, as well as saliva's use as a diagnostic fluid

Functional Specialization of Human Salivary Glands and Origins of Proteins Intrinsic to Human Saliva.

Salivary proteins are essential for maintaining health in the oral cavity and proximal digestive tract, and they serve as potential diagnostic markers for monitoring human health and disease. However, their precise organ origins remain unclear. Through transcriptomic analysis of major adult and fetal salivary glands and integration with the saliva proteome, the blood plasma proteome, and transcriptomes of 28+ organs, we link human saliva proteins to their source, identify salivary-gland-specific genes, and uncover fetal- and adult-specific gene repertoires. Our results also provide insights into the degree of gene retention during gland maturation and suggest that functional diversity among adult gland types is driven by specific dosage combinations of hundreds of transcriptional regulators rather than by a few gland-specific factors. Finally, we demonstrate the heterogeneity of the human acinar cell lineage. Our results pave the way for future investigations into glandular biology and pathology, as well as saliva's use as a diagnostic fluid

Different activation of eIF2α phosphorylation by VSV and poliovirus infections.

<p>HeLa cells were infected with VSV for 4 h, infected with poliovirus for 4 h, or treated with 1 µM of thapsigargin for 1 hour. Cytoplasmic protein extracts from these and control cells were analyzed with Abs against eIF2α and phosphorylated form of eIF2α (panel A). Same membrane was analyzed with Abs against VSV P- protein and poliovirus capsid proteins (panel B).</p

Proteolytic cleavage of p65-RelA and eIF4G occurred later during poliovirus infection of the cells with inhibited proteasome activity.

<p>HeLa cells and MG 132 2 h pretreated HeLa cells were infected with poliovirus (MOI = 5) for 1 h. After change of medium, total protein extracts were collected every hour and tested with anti-p65-RelA C-terminus specific Abs (A) or with anti eIF4G N-terminus specific Abs (B). 10 µg of protein were tested in Western blotting experiments.</p

Poliovirus protein synthesis was delayed in MG132-pretreated cells.

<p>Control HeLa cells and HeLa cells pre-treated with MG132 for 2 h were infected with poliovirus (MOI = 5) for 2, 3 and 4 h. All cells were incubated in methionine/cysteine free medium supplemented with S³⁵-methionine/cysteine for last 30 min before harvesting. To study general translation, 10 µg of cytoplasmic protein extracts were separated by electrophoresis and analyzed by autoradiography (A). To study poliovirus capsid protein accumulation, capsid proteins were precipitated by specific Abs from 100 µg of cytoplasmic protein extracts and analyzed by electrophoresis and autoradiography (B).</p

The effect of MG132 and virus infection on cellular protein synthesis.

<p>(A) Protein extracts were purified from control HeLa cells, cells infected with VSV for 4 h, cells treated with 5 µM of MG132 for 4 h, and cells infected with VSV and treated with MG132 for 4 h. All cells were incubated with S³⁵ methionine/cysteine for last 30 min before the protein extracts purification. Cytoplasmic protein extracts were analyzed by electrophoresis and autoradiography. (B) Cytoplasmic protein extracts from control, VSV infected, and MG treated cells were precipitated with anti-actin Abs, and the complexes were purified on protein A agarose. S³⁵ labeled actin was analyzed by electrophoresis and autoradiography. (C) Cytoplasmic S³⁵-labeled protein extracts from MG-treated and poliovirus-infected cells were precipitated with anti-actin Abs and analyzed as described in panel B. All protein bands' intensity was detected by ImageJ software to calculate percentage of protein synthesis inhibition.</p