Mutational Analyses of the Influenza A Virus Polymerase Subunit PA Reveal Distinct Functions Related and Unrelated to RNA Polymerase Activity

Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus production, indicating that PA may have a novel role independent of its polymerase activity. To further examine the roles of PA in the viral life cycle, we have now generated and characterized additional mutations in regions flanking the J10 site from residues 497 to 518. All tested di-codon mutations completely abolished or significantly reduced viral infectivity, but they did so through disparate mechanisms. Several showed effects resembling those of J10, in that the mutant polymerase supported normal levels of viral RNA synthesis but nonetheless failed to generate infectious viral particles. Others eliminated polymerase activity, in most cases by perturbing the normal nuclear localization of PA protein in cells. We also engineered single-codon mutations that were predicted to pack near the J10 site in the crystal structure of PA, and found that altering residues K378 or D478 each produced a J10-like phenotype. In further studies of J10 itself, we found that this mutation does not affect the formation and release of virion-like particles per se, but instead impairs the ability of those particles to incorporate each of the eight essential RNA segments (vRNAs) that make up the viral genome. Taken together, our analysis identifies mutations in the C-terminal region of PA that differentially affect at least three distinct activities: protein nuclear localization, viral RNA synthesis, and a trans-acting function that is required for efficient packaging of all eight vRNAs

The Splicing Factor Proline-Glutamine Rich (SFPQ/PSF) Is Involved in Influenza Virus Transcription

The influenza A virus RNA polymerase is a heterotrimeric complex responsible for viral genome transcription and replication in the nucleus of infected cells. We recently carried out a proteomic analysis of purified polymerase expressed in human cells and identified a number of polymerase-associated cellular proteins. 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. As the SFPQ/PSF protein is frequently associated to NonO/p54, we tested the potential implication of the latter in influenza virus replication. However, down-regulation of NonO/p54 by silencing with two independent siRNAs did not affect virus yields. Down-regulation of SFPQ/PSF by siRNA silencing led to a reduction and delay of influenza virus gene expression. 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

Biological characteristics of a cold-adapted influenza A virus mutation residing on a polymerase gene

The biological function of a cold-adapted (ca) mutation residing on the PB2 gene of an influenza A/Ann Arbor/6/60 (A/AA/6/60) ca variant virus in the viral replication cycle at 25° C was studied. The viral polypeptide synthesis of A/AA/6/60 ca variant at 25° C was evident approximately 6 hours earlier than the wild type (wt) virus and yielded twice as many products. The quantitative analysis of viral complementary RNA (cRNA), synthesized in the presence of cycloheximide, revealed that A/AA/6/60 ca variant and a single gene reassortant that contains only the PB2 gene of the ca variant with remaining genes of the wt virus produced equal amount of cRNA at 25° and 33° C, which was an amount approximately four fold greater than the wt virus' cRNA synthesized at 25° C. These results strongly suggest that the ca mutation residing on the PB2 gene of A/AA/6/60 ca variant affects the messenger RNA synthesis at 25° C in the primary transcription.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41692/1/705_2005_Article_BF01310893.pd

Soluble and membrane-bound catechol-O-methyltransferase in normal and malignant mammary gland

Vivir con discapacidad afecta necesariamente al proyecto vital de los estudiantes. Este trabajo describe y compara el nivel de empoderamiento en una muestra de estudiantes con discapacidad y su asociación con la tipología, grado y antigüedad de ésta. Han participado 123 estudiantes universitarios con diferentes tipos de discapacidad (motora, intelectual y sensorial). Los estudiantes cumplimentaron vía online la Escala de Rogers, Chamberlin, Ellison y Crean (Rogers et al., 1997) diseñada para medir el empoderamiento y que incluye cinco factores: Autoestima/Eficacia, Poder/Impotencia, Activismo comunitario/Autonomía, Optimismo/Control sobre el futuro e Ira apropiada. Los resultados indicaron un nivel moderado de empoderamiento, más elevado en los estudiantes con discapacidad motora, con un mayor grado de discapacidad y cuando ésta se produjo de forma sobrevenida. Esto sugiere que esta capacidad puede evolucionar, por lo tanto es importante fomentarla en programas de intervención-acción.Having a disability necessarily affects young people’s life project. This paper describes and compares the level of empowerment in a sample of students with disabilities and its association with typology, degree and time passed from diagnosis of disability. 123 university students with motor, intellectual or sensory disabilities answered an on line questionnaire, the Spanish version of the Rogers, Chamberlin, Ellison and Crean Scale (Rogers et al.1997). It is a scale designed to measure empowerment which includes five factors: Self-efficacy-Self-esteem, Power-Powerlessness, Community activism, Optimism-Control over the future and Righteous anger. Results show moderate levels of empowerment, which were higher among the students presenting a motor disability, a higher degree of disability and an acquired disability. The results suggest that the ability can vary and evolve, and therefore it is important to promote empowerment by implementing programs that encourage in-action