The influenza A viruses are members of the Orthomyxoviridae family and cause yearly epidemics and occasional more severe pandemics of respiratory infections. Although influenza in humans is usually resolved without complications, the infection can be more severe in the young infants, in the elderly and in persons with previous medical conditions. Thus, influenza leads to 250.000-500.000 deaths per year and these numbers can be much larger in a pandemic. The genome of influenza A viruses is fragmented into 8 single-stranded RNA molecules of negative polarity that together with the heterotrimeric polymerase complex (PA, PB1 and PB2) and the nucleoprotein (NP), form ribonucleoprotein complexes (RNPs). These RNPs are responsible for the replication and transcription of the virus genes in the nucleus of the infected cells. Although at present we have effective influenza antivirals available targeting virus dissemination, the appearance of resistant mutant viruses poses a serious limitation for their widespread use. Antivirals drugs that target cellular proteins may play major roles in combating virus resistance. In the present work we propose two different strategies to identify cellular proteins with potential antiviral activity. The first strategy is based in the characterisation of the complexes formed intracellularly by the influenza polymerase in human cells. Several influenza polymerase-associated proteins were identified by MALDI-MS. Here we characterise the role of one such host factors, SFPQ/PSF, during virus infection. Down-regulation of SFPQ/PSF by silencing with two independent siRNAs reduced the virus yield by 2-5 log in low-multiplicity infections, while the replication of unrelated viruses as VSV or Adenovirus was almost unaffected. Immunofluorescence analyses showed a good correlation between SFPQ/PSF and NP levels in infected cells. Analysis of virus RNA accumulation in silenced cells showed that production of mRNA, cRNA and vRNA is reduced by more than 5-fold but splicing is not affected. Likewise, the accumulation of viral mRNA in cicloheximide-treated cells was reduced by 3-fold. In contrast, down-regulation of SFPQ/PSF in a recombinant virus replicon system indicated that, while the accumulation of viral mRNA is reduced by 5-fold, vRNA levels are slightly increased. In vitro transcription of recombinant RNPs generated in SFPQ/PSF-silenced cells indicated a 4-5-fold reduction in polyadenylation but no alteration in cap snatching. These results indicate that SFPQ/PSF is a host factor essential for influenza virus transcription that increases the efficiency of viral mRNA polyadenylation and open the possibility to develop new antivirals targeting the accumulation of primary transcripts, a very early step during infection. The second strategy is based in the screen for new anti-influenza inhibitors targeted to viral RNA replication or transcription in a recombinant virus replicon system. We proposed to use collections of compounds that have been previously studied for the therapy of human pathologies and hence have known toxicology and pharmacological profiles. This approach could allow the rapid progress to the market of new effective influenza antivirals. The compounds that showed activity in the basic screen were tested for their anti-influenza activity in cultured infected cells. Two compounds inhibited the influenza virus strains used in cell cultures from different origins: Ribavirin, a previously described anti-influenza compound, and Montelukast sodium. Subtoxic concentrations of Montelukast sodium inhibited the viral proteins synthesis. The identification of the target of this compound could reveal new cellular pathways important for influenza virus infection.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-07-2014