The herpes simplex virus 1 Us11 protein inhibits autophagy through its interaction with the protein kinase PKR.

Autophagy is now known to be an essential component of host innate and adaptive immunity. Several herpesviruses have developed various strategies to evade this antiviral host defense. Herpes simplex virus 1 (HSV-1) blocks autophagy in fibroblasts and in neurons, and the ICP34.5 protein is important for the resistance of HSV-1 to autophagy because of its interaction with the autophagy machinery protein Beclin 1. ICP34.5 also counteracts the shutoff of protein synthesis mediated by the double-stranded RNA (dsRNA)-dependent protein kinase PKR by inhibiting phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) in the PKR/eIF2α signaling pathway. Us11 is a late gene product of HSV-1, which is also able to preclude the host shutoff by direct inhibition of PKR. In the present study, we unveil a previously uncharacterized function of Us11 by demonstrating its antiautophagic activity. We show that the expression of Us11 is able to block autophagy and autophagosome formation in both HeLa cells and fibroblasts. Furthermore, immediate-early expression of Us11 by an ICP34.5 deletion mutant virus is sufficient to render the cells resistant to PKR-induced and virus-induced autophagy. PKR expression and the PKR binding domain of Us11 are required for the antiautophagic activity of Us11. However, unlike ICP34.5, Us11 did not interact with Beclin 1. We suggest that the inhibition of autophagy observed in cells infected with HSV-1 results from the activity of not only ICP34.5 on Beclin 1 but also Us11 by direct interaction with PKR.

The human cytomegalovirus protein TRS1 inhibits autophagy via its interaction with Beclin 1.

Human cytomegalovirus modulates macroautophagy in two opposite directions. First, HCMV stimulates autophagy during the early stages of infection, as evident by an increase in the number of autophagosomes and a rise in the autophagic flux. This stimulation occurs independently of de novo viral protein synthesis since UV-inactivated HCMV recapitulates the stimulatory effect on macroautophagy. At later time points of infection, HCMV blocks autophagy (M. Chaumorcel, S. Souquere, G. Pierron, P. Codogno, and A. Esclatine, Autophagy 4:1-8, 2008) by a mechanism that requires de novo viral protein expression. Exploration of the mechanisms used by HCMV to block autophagy unveiled a robust increase of the cellular form of Bcl-2 expression. Although this protein has an anti-autophagy effect via its interaction with Beclin 1, it is not responsible for the inhibition induced by HCMV, probably because of its phosphorylation by c-Jun N-terminal kinase. Here we showed that the HCMV TRS1 protein blocks autophagosome biogenesis and that a TRS1 deletion mutant is defective in autophagy inhibition. TRS1 has previously been shown to neutralize the PKR antiviral effector molecule. Although phosphorylation of eIF2α by PKR has been described as a stimulatory signal to induce autophagy, the PKR-binding domain of TRS1 is dispensable to its inhibitory effect. Our results show that TRS1 interacts with Beclin 1 to inhibit autophagy. We mapped the interaction with Beclin 1 to the N-terminal region of TRS1, and we demonstrated that the Beclin 1-binding domain of TRS1 is essential to inhibit autophagy.

A protein kinase A-dependent mechanism by which rotavirus affects the distribution and mRNA level of the functional tight junction-associated protein, occludin, in human differentiated intestinal Caco-2 cells.

We found that at the tight junctions (TJs) of Caco-2 cell monolayers, rhesus monkey rotavirus (RRV) infection induced the disappearance of occludin. Confocal laser scanning microscopy showed the disappearance of occludin from the cell-cell boundaries without modifying the expression of the other TJ-associated proteins, ZO-1 and ZO-3. Western immunoblot analysis of RRV-infected cells showed a significant fall in the levels of the nonphosphorylated form of occludin in both Triton X-100-insoluble and Triton X-100-soluble fractions, without any change in the levels of the phosphorylated form of occludin. Quantitative reverse transcription-PCRs revealed that the level of transcription of the gene that encodes occludin was significantly reduced in RRV-infected cells. Treatment of RRV-infected cells with Rp-cyclic AMP and protein kinase A inhibitors H89 and KT5720 during the time course of the infection restored the distribution of occludin and a normal level of transcription of the gene that encodes occludin.

An NSP4-dependant mechanism by which rotavirus impairs lactase enzymatic activity in brush border of human enterocyte-like Caco-2 cells.

Lactase-phlorizin hydrolase (LPH, EC 3.2.1.23-62) is a brush border membrane (BBM)-associated enzyme in intestinal cells that hydrolyse lactose, the most important sugar in milk. Impairing in lactase activity during rotavirus infection has been described in diseased infants but the mechanism by which the functional lesion occurs remains unknown. We undertook a study to elucidate whether rotavirus impairs the lactase enzymatic activity in BBM of human enterocyte cells. In this study we use cultured human intestinal fully differentiated enterocyte-like Caco-2 cells to demonstrate how the lactase enzymatic activity at BBM is significantly decreased in rhesus monkey rotavirus (RRV)-infected cells. We found that the decrease in enzyme activity is not dependent of the Ca(2+)- and cAMP-dependent signalling events triggered by the virus. The LPH biosynthesis, stability, and expression of the protein at the BBM of infected cells were not modified. We provide evidence that in RRV-infected cells the kinetic of lactase enzymatic activity present at the BBM was modified. Both BBM(control) and BBM(RRV) have identical K(m) values, but hydrolyse the substrate at different rates. Thus, the BBM(RRV) exhibits almost a 1.5-fold decreased V(max) than that of BBM(control) and is therefore enzymatically less active than the latter. Our study demonstrate conclusively that the impairment of lactase enzymatic activity at the BBM of the enterocyte-like Caco-2 cells observed during rotavirus infection results from an inhibitory action of the secreted non-structural rotavirus protein NSP4.

Rotavirus induces apoptosis in fully differentiated human intestinal Caco-2 cells.

Rotaviruses, which are the main cause of viral gastroenteritis in young children, induce structural and functional damages in infected mature enterocytes of the small intestine. To investigate a relationship between rotavirus infection and cell death by apoptosis, we used the human intestinal Caco-2 cell line. We demonstrated by several methods including TUNEL and ELISA detection of cytoplasmic histone-associated DNA fragments that the infection of fully differentiated Caco-2 cells by the RRV rotavirus strain induces apoptosis. Rotavirus infection leads to the loss of mitochondrial membrane potential and the release of cytochrome C from mitochondria. We showed that rotavirus-induced apoptosis was dependent of the multiplicity of infection and increased with time from 4 h to 24 h of infection. Flow cytometric analysis showed that DNA fragmentation occurs in productively infected cells, suggesting that rotavirus induces apoptosis by a direct mechanism. We also demonstrated that non-replicative RRV particles are not sufficient to induce apoptosis and viral gene expression seems required. Intracellular calcium plays a role in RRV-induced apoptosis because treatment with an intracellular calcium ion chelator (BAPTA-AM) partially inhibited apoptosis.

A cyclic AMP protein kinase A-dependent mechanism by which rotavirus impairs the expression and enzyme activity of brush border-associated sucrase-isomaltase in differentiated intestinal Caco-2 cells.

We undertook a study of the mechanism by which rhesus monkey rotavirus (RRV) impairs the expression and enzyme activity of brush border-associated sucrase isomaltase (SI) in cultured, human, fully differentiated, intestinal Caco-2 cells. We provide evidence that the RRV-induced defects in the expression and enzyme activity of SI are not related to the previously observed, RRV-induced, Ca2+ -dependent, disassembly of the F-actin cytoskeleton. This conclusion is based on the facts that: (i) the intracellular Ca2+ blocker, BAPTA/AM, which antagonizes the RRV-induced increase in [Ca2+](i), fails to inhibit the RRV-induced decrease in SI expression and enzyme activity; and (ii) Jasplakinolide (JAS) treatment, known to stabilize actin filaments, had no effect on the RRV-induced decrease in SI expression. Results reported here demonstrate that the RRV-induced impairment in the expression and enzyme activity of brush border-associated SI results from a hitherto unknown mechanism involving PKA signalling. This conclusion is based on the observations that (i) intracellular cAMP was increased in RRV-infected cells and (ii) treatment of RRV-infected cells with PKA blockers resulted in the reappearance of apical SI expression, accompanied by the restoration of the enzyme activity at the brush border. In addition, in RRV-infected cells a twofold increase of phosphorylated form of cytokeratin 18 was observed after immunopurification and Western Blot analysis, which was antagonized by exposing the RRV-infected cells to the PKA blockers.