Gutierrez-Escolano, A. L. | Search bioRxiv

bioRxiv2024

The Leader of the Capsid protein from Feline calicivirus must be palmitoylated and form oligomers through disulfide bonds for efficient viral replication

Feline calicivirus (FCV), a member of the Vesivirus genus and a model for studying of the members of the Caliciviridae, has been used to understand the biology and viral replication of etiological agents of medical and veterinary importance. The leader of the capsid (LC) protein is exclusive to the Vesivirus genus members whose importance for a successful viral replication has been demonstrated; however, its cell localization, post-translational modifications, and specific functions throughout the viral replication cycle are poorly understood. We have determined that the expression of the LC protein from FCV in a virus-free system is located at the mitochondria and induces apoptosis; furthermore, the in vitro characterization of a purified LC protein showed that it has viroporin characteristics. Here, using an LC-reactive serum, the LC protein expression kinetics, cellular localization, post-translational modifications, and association with cellular PDI proteins in FCV infected cells was determined. We found that the LC protein is present on the membrane of infected cells and in the supernatants, independently of cell lysis, which suggests that it is actively secreted. Moreover, we found that LC protein is palmitoylated during infection and that palmitoylation inhibition alters its levels and subcellular localization. Finally, we determined that the LC protein forms oligomers dependent on disulfide bonds mediated by PDI activity. In addition, it interacts with PDIA3, a disulfide isomerase known as an important factor in the replication of several viruses. The results indicate that the LC protein from FCV might have multiple roles during FCV replication. IMPORTANCEFeline calicivirus (FCV) is a highly transmissible virus that represents a significant cause of upper respiratory infection in domestic and wild cats worldwide. FCV also serves as one of the most valuable models for studying calicivirus biology, as unlike most members of the family, known to cause diseases in animals and humans, it can be easily grown in cell culture. Since there are no efficacious vaccines or antivirals against most caliciviruses, studying their molecular biology and the relationship between viral and cellular components is essential for developing strategies for their prevention and control.

molecular biology↗

bioRxiv2018

The Nonstructural Proteins 3 And 5 From Flavivirus Modulate Nuclear-Cytoplasmic Transport And Innate Immune Response Targeting Nuclear Proteins

Viruses hijack cellular proteins and components to be replicated in the host cell and to evade the immune response. Although flaviviruses have a cytoplasmic replicative cycle, some viral proteins such as the capsid (C) and the RNA dependent RNA polymerase, NS5, can reach the nucleus of the infected cells. Considering the important roles of NS5 in viral replication and in the control of the immune response, and its striking presence in the nucleus, the possible functions of this protein in some mechanisms orchestrated by the nucleus was analyzed. We isolated and identified nuclear proteins that interact with NS5; one of them, the DEAD-box RNA helicase DDX5 is relocated to the cytoplasm and degraded during infection with DENV, which correlates with its function in IFN dependent response. Since DDX5 and many other proteins are relocated from the nucleus to the cytoplasm during flavivirus infection, the integrity and function of the main regulator of the nuclear-cytoplasmic transport, the nuclear pore complex (NPC) was evaluated. We found that during DENV and ZIKV infection nucleoporins (NUPs) such as TPR, Nup153, Nup98, and Nup62 were cleavaged/degraded. The protease NS2B-NS3 induces NUPs degradation and it causes a dramatic inhibition of mature mRNAs export to the cytoplasm but not the export of DDX5 protein, which is dependent on NS5. Here we describe for the first time that the NS3 and NS5 proteins from flavivirus play novel functions hijacking the NPC and some nuclear proteins relevant in triggering immune response pathways, inducing a favorable environment for viral replication.\n\nIMPORTANCEViruses, as intracellular obligate parasites, hijack cellular components to enter and replicate in infected cells. Remarkably, in many cases, viruses hijack molecules with crucial functions for the cells. Here it is described how RNA viruses such as DENV and ZIKV, with a cytoplasmic replicative cycle, use NS3 and NS5, two of their unique non-structural proteins with enzymatic activity, to modulate nuclear-cytoplasmic transport. We found that NS3 disrupts the nuclear pore complex, the main regulator in nuclear-cytoplasmic transport, causing a strong reduction in the amount of mature mRNAs in the cytoplasm and an inhibition in innate immune response. Additionally, NS5 induces the relocation of nuclear proteins to the cytoplasm such as DDX5, involved in immune response, which is later degraded by NS3. These findings allow the understanding of crucial mechanisms that viruses use to deal with the control of the immune response to grant the production of new viral particles.

molecular biology↗