Jacqueline Cotte-Laffitte

The Human Cytomegalovirus Protein TRS1 Inhibits Autophagy via Its Interaction with Beclin 1

ABSTRACT 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.

Rotavirus Infection Induces an Increase in Intracellular Calcium Concentration in Human Intestinal Epithelial Cells: Role in Microvillar Actin Alteration

ABSTRACT Rotaviruses, which infect mature enterocytes of the small intestine, are recognized as the most important cause of viral gastroenteritis in young children. We have previously reported that rotavirus infection induces microvillar F-actin disassembly in human intestinal epithelial Caco-2 cells (N. Jourdan, J. P. Brunet, C. Sapin, A. Blais, J. Cotte-Laffitte, F. Forestier, A. M. Quero, G. Trugnan, and A. L. Servin, J. Virol. 72:7228–7236, 1998). In this study, to determine the mechanism responsible for rotavirus-induced F-actin alteration, we investigated the effect of infection on intracellular calcium concentration ([Ca2+]i) in Caco-2 cells, since Ca2+ is known to be a determinant factor for actin cytoskeleton regulation. As measured by quin2 fluorescence, viral replication induced a progressive increase in [Ca2+]i from 7 h postinfection, which was shown to be necessary and sufficient for microvillar F-actin disassembly. During the first hours of infection, the increase in [Ca2+]i was related only to an increase in Ca2+ permeability of plasmalemma. At a late stage of infection, [Ca2+]i elevation was due to both extracellular Ca2+ influx and Ca2+ release from the intracellular organelles, mainly the endoplasmic reticulum (ER). We noted that at this time the [Ca2+]i increase was partially related to a phospholipase C (PLC)-dependent mechanism, which probably explains the Ca2+ release from the ER. We also demonstrated for the first time that viral proteins or peptides, released into culture supernatants of rotavirus-infected Caco-2 cells, induced a transient increase in [Ca2+]i of uninfected Caco-2 cells, by a PLC-dependent efflux of Ca2+from the ER and by extracellular Ca2+ influx. These supernatants induced a Ca2+-dependent microvillar F-actin alteration in uninfected Caco-2 cells, thus participating in rotavirus pathogenesis.

The Herpes Simplex Virus 1 Us11 Protein Inhibits Autophagy through Its Interaction with the Protein Kinase PKR

ABSTRACT 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.

Rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration.

ABSTRACT Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. In vivo, rotavirus exhibits a marked tropism for the differentiated enterocytes of the intestinal epithelium. In vitro, differentiated and undifferentiated intestinal cells can be infected. We observed that rotavirus infection of the human intestinal epithelial Caco-2 cells induces cytoskeleton alterations as a function of cell differentiation. The vimentin network disorganization detected in undifferentiated Caco-2 cells was not found in fully differentiated cells. In contrast, differentiated Caco-2 cells presented Ca2+-dependent microtubule disassembly and Ca2+-independent cytokeratin 18 rearrangement, which both require viral replication. We propose that these structural alterations could represent the first manifestations of rotavirus-infected enterocyte injury leading to functional perturbations and then to diarrhea.

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

ABSTRACT 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.

Correction: "The human cytomegalovirus protein TRS1 inhibits autophagy via its interaction with Beclin 1" [86, 5 (2012) (2571-2584)] DOI:10.1128/JVI.05746-11

Volume 86, no. 5, p. 2571–2584, 2012, https://doi.org/10.1128/JVI.05746-11. Page 2575, Fig. 2A: Due to an error in figure file processing, the Western blots did not have the correct actin loading controls in the lower panel. All of the original films have been retrieved, and the corresponding actin loading controls have been replaced. Figure 2A should appear as shown below. This panel has a space between the blots for mock conditions and those for supernatant conditions (as in the original Fig. 2A), since the lanes were not adjacent in the original immunoblot.