MODULATING CHROMATIN STRUCTURE AND GENE EXPRESSION DURING AFRICAN SWINE FEVER VIRUS INFECTION – NEW STRATEGIES FOR AN EFFICIENT VACCINE RATIONAL DESIGN

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Modulating chromatin structure and gene expression during African swine fever virus infection : new strategies for an efficient vaccine rational design

Publication . Frouco, Gonçalo Daniel dos Santos; Ferreira, Fernando António da Costa; Martins, Carlos Lopes Vieira

African swine fever virus (ASFV) is a nucleo-cytoplasmic large DNA virus which infects all members of the family Suidae, causing a fatal disease of domestic swine and wild boar. Since no effective vaccine or treatment is available, ASF is considered a global threat for pig husbandry. The ASFV genome encodes among others, enzymes required for virion assembly, genome transcription and replication, including a putative histone-like protein, pA104R. In bacteria, these proteins perform topological modification of the chromosome (twisting, bending and folding), playing important structural and regulatory functions. Since ASFV has a large genome, a viral histone-like protein may be important for packaging its genome within the virion particle and/or for viral replication and transcriptional events. In this study, the ASFV-pA104R activity was characterized and its DNA-binding activities were evaluated. pA104R binds both to ssDNA and dsDNA, although having higher affinity to ds-DNA, over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. The arginine residue located in pA104R’s DNA-binding domain, at position 69, also revealed to be important for an efficient DNA-binding. Additionally, since pA104R together with the viral type II topoisomerase, pP1192R, displayed DNA-supercoiling activity, a synergistic effect between these viral is proposed. The expression of pA104R was observed in the late phase of infection in infected cells with the Vero-adapted ASFV isolate Ba71V, co-localizing with cell nucleus and viral factories. siRNA experiments showed that the knockdown of A104R induce a reduction of viral progeny, copy numbers of viral genomes and transcription of a late viral gene, revealing that pA104R plays a critical role in viral DNA replication and gene expression. Results obtained on these studies prompted us to pursue the objective to generate a defective infectious single cycle (DISC) ASFV lacking the A104R gene. Recombinant virus was successfully obtained, however the complementary cell line previously developed did not support its replication. The antiviral activity of four HDACi against ASFV was also evaluated in this study. The results showing the abrogation of viral replication by NaPB open new insights on its use as an antiviral strategy to control ASFV spreading. Overall our data strongly support that pA104R plays an important role on ASFV replication opening a new window for the design of ASF control measures through the development of efficient and safe vaccines and antivirals.

2018-01-30Tese de doutoramento

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Fundação para a Ciência e a Tecnologia

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SFRH/BD/89426/2012