Glutamine starvation inhibits snakehead vesiculovirus replication via inducing autophagy associated with the disturbance of endogenous glutathione pool.

Autophagy is a degradation cellular process which also plays an important role in virus infection. Glutamine is an essential substrate for the synthesis of glutathione which is the most abundant thiol-containing compound within the cells and plays a key role in the antioxidant defense and intracellular signaling. There is an endogenous cellular glutathione pool which consists of two forms of glutathione, i.e. the reduced form (GSH) and the oxidized form (GSSG). GSH serves as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging free radicals and other reactive oxygen species (ROS) which can lead to autophagy. Under physiological conditions, the concentration of GSSG is only about 1% of total glutathione, while stress condition can result in a transient increase of GSSG. In our previous report, we showed that the replication of snakehead fish vesiculovirus (SHVV) was significant inhibited in SSN-1 cells cultured in the glutamine-starvation medium, however the underlying mechanism remains enigmatic. Here, we revealed that the addition of L-Buthionine-sulfoximine (BSO), a specific inhibitor of the GSH synthesis, could decrease the γ-glutamate-cysteine ligase (GCL) activity and GSH levels, resulting in autophagy and significantly inhibition of the replication of SHVV in SSN-1 cells cultured in the complete medium. On the other hand, the replication of SHVV was rescued and the autophagy was inhibited in the SSN-1 cells cultured in the glutamine-starvation medium supplemented with additional GSH. Furthermore, the inhibition of the synthesis of GSH had not significantly affected the generation of reactive oxygen species (ROS). However, it significantly decreased level of GSH and enhanced the level of GSSG, resulting in the decrease of the value of GSH/GSSG, indicating that it promoted the cellular oxidative stress. Overall, the present study demonstrated that glutamine starvation impaired the replication of SHVV in SSN-1 cells via inducing autophagy associated with the disturbance of the endogenous glutathione pool.

Higher antiviral response of RIG-I through enhancing RIG-I/MAVS-mediated signaling by its long insertion variant in zebrafish.

As an intracellular pattern recognition receptor (PRR), the retinoic acid-inducible gene-I (RIG-I) is responsible for the recognition of cytosolic viral nucleic acids and the production of type I interferons (IFNs). In the present study, an insertion variant of RIG-I with 38 amino acids inserted in the N-terminal CARD2 domain, as well as the typical type, named as RIG-Ia and RIG-Ib respectively were identified in zebrafish. RIG-Ia and RIG-Ib were all up-regulated following the infection of a negative ssRNA virus, the Spring Viremia of Carp Virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, indicating the RLR may have a role in the recognition of both viruses and bacteria. The over-expression of RIG-Ib in cultured fish cells resulted in significant increase in type I IFN promoter activity, and in protection against SVCV infection, whereas the over-expression of RIG-Ia had no direct effect on IFN activation nor antiviral response. Furthermore, it was revealed that both RIG-Ia and RIG-Ib were associated with the downstream molecular mitochondrial antiviral signaling protein, MAVS, and interestingly RIG-Ia when co-transfected with RIG-Ib or MAVS, induced a significantly higher level of type I IFN promoter activity and the expression level of Mx and IRF7, implying that the RIG-Ia may function as an enhancer in the RIG-Ib/MAVS-mediated signaling pathway.

The E3-ligase TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors.

Innate immunity conferred by the type I interferon is critical for antiviral defense. To date only a limited number of tripartite motif (TRIM) proteins have been implicated in modulation of innate immunity and anti-microbial activity. Here we report the complementary DNA cloning and systematic analysis of all known 75 human TRIMs. We demonstrate that roughly half of the 75 TRIM-family members enhanced the innate immune response and that they do this at multiple levels in signaling pathways. Moreover, messenger RNA levels and localization of most of these TRIMs were found to be altered during viral infection, suggesting that their regulatory activities are highly controlled at both pre- and posttranscriptional levels. Taken together, our data demonstrate a very considerable dedication of this large protein family to the positive regulation of the antiviral response, which supports the notion that this family of proteins evolved as a component of innate immunity.

CaBP1, a calcium binding protein of the thioredoxin family, is a resident KDEL protein of the ER and not of the intermediate compartment.

A cDNA encoding rat CaBP1 has been isolated and sequenced. The deduced polypeptide chain consists of 440 amino acids including two internal thioredoxin-like domains and a C-terminal KDEL retention/retrieval signal. Regarding the high degree of identity to the hamster protein P5, CaBP1 is considered to be the homologous rat protein. Previous work has suggested that CaBP1 is a resident luminal protein of the intermediate compartment (Schweizer, A., Peter, F., Nguyen Van, P., Söling, H.D. and Hauri, H.P. (1993) Eur. J. Cell Biol. 60, 366-370). Our conclusion that CaBP1 is a resident protein of the endoplasmic reticulum and not of the intermediate compartment is based on three different approaches: subcellular fractionation, indirect immunofluorescence and overexpression of CaBP1. Subcellular fractionation of Vero cells in a velocity controlled step gradient led to copurification of CaBP1-containing vesicles and several marker proteins for the ER including calreticulin and alpha-SSRP. The intermediate compartment, as defined by a monoclonal antibody against the marker protein p53 (ERGIC-53), could be separated from these ER markers. Double immunofluorescence analysed by laser scanning microscopy showed no significant colocalization between CaBP1 and p53, but between CaBP1 and calreticulin. In addition experiments, Vero cells were infected with VSV tsO45. At 15 degrees C the VSV-G protein accumulated in punctuate structures representing the intermediate compartment, while CaBP1 maintained its original reticular localization. Even after high-level overexpression in COS cells, CaBP1 was not detected in the intermediate compartment, but was efficiently retained in the ER as judged by light microscopy.