DISTRIBUCIÓN INTRACELULAR DE LA PROTEASA VIRAL 3CD DEL FCV DURANTE LA INFECCIÓN

Abstract

Acute gastroenteritis is an important health problem with high mobility and mortality rates in the entire world. It is caused by several pathogens, such as viruses. Among them, the members of the Caliciviridae family, that cause a variety of diseases in animals, are considered the most important cause of outbreaks of viral gastroenteritis in humans, worldwide. These viruses were the first ones identified as responsible for causing gastroenteritis, however, the study of their biology has been delayed because they can not propagate in cell cultures. The use of animal models like the feline caliciviruses (FCV) to understand the biology of the ones that infect humans, has allowed to better understanding its biology and pathogenesis. Caliciviruses are spherical with a single stranded and positive polarity RNA as a genome; that replicate in the cytoplasm of the infected cells and are released by cell lysis. Infection by lythic viruses result in an extensive interaction among viral proteins and genomes with host cell macromolecular pathways, which results in a successful viral replication and cell death. Although caliciviruses replicates in the cell cytoplasm, they depend of molecules located in different cell compartments such as the nucleus, like other RNA viruses. The accessibility to some of the compartmentalized components depend on certain strategies that some viruses have developed, such as some strategies to get some viral components into these compartments or the relocation to certain cell factors to places where viral replication takes place The FCV polymerase protease 3CD or NS6/7 is responsible for the non structural polyprotein processing and RNA replication in the cytoplasm of the host cell. However, we have determined that this viral protein is localized in the perinuclear regions and in certain level in certain proportion in the nucleus of the infected cells. By in silico analysis, it was determined that this protein contains a putative bipartite nuclear localization signal in the carboxy terminal region, conserved in several members of the Caliciviridae family. The transitory expression of a fusion protein: 4EGFP-3CD from FCV into CRFK cells has shown that this protein is expressed in the cytoplasm, however, when cells were super infected with the FCV, this protein showed a nuclear localization in come extent. On the other hand, some evidences generated in our laboratory had demonstrated the interaction of the nucleolin, a multifunctional protein with nucleolar localization, and the 3´ UTR of the FCV genomic RNA. Because this is the place where the protease-polymerase 3CD protein binds to the RNA to start the negative polarity RNA synthesis, we decide to investigate if nucleolin could participate in this process. Initially, we determined that nucleolin is relocated from the nucleolus to the nucleplasm as a consequence or FCV infection, and that both nucleolin and the protease polymerase 3CD interact in the perinuclear region. Furthermore, to determine if nucleolin participates in FCV replication, we performed an siRNA assays to inhibit the expression of a nucleolin. In this assays we have observed that this inhibition resulted in a significant reduction of the non-structural proteins and also a reduction in the viral particle production. Thus, this results indicate that nucleolin interact with FCV 3CD and that this molecule participates in the FCV replication, confirming the functional role of nucleoline in the FCV life cycle.