RESUMO
The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.
Assuntos
DNA Viral/genética , Dependovirus/genética , Regulação Viral da Expressão Gênica , Saccharomyces cerevisiae/virologia , Vírion/genética , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dependovirus/crescimento & desenvolvimento , Dependovirus/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Vírus Auxiliares/genética , Vírus Auxiliares/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/crescimento & desenvolvimento , Vírion/metabolismo , Replicação ViralRESUMO
Stimulation of the CD28 costimulatory receptor can lead to an increased surface lipid raft expression in T lymphocytes. Here, we demonstrate that CD28 itself is recruited to lipid rafts in both Jurkat and peripheral blood T lymphocytes. This recruitment of CD28 is triggered by engagement with either anti-CD28 mAbs or a natural ligand of CD28, B7.2 (CD86). All detectable tyrosine-phosphorylated CD28 is in the lipid raft fractions, as is all of the CD28 associated with phosphatidylinositol 3-kinase, which is recruited to CD28 by tyrosine phosphorylation. Targeting the CD28 cytoplasmic domain to lipid rafts results in its tyrosine phosphorylation, indicating that tyrosine phosphorylation of CD28 may occur after translocation to lipid rafts. Studies with Jurkat cells deficient in Lck and CD45 demonstrate that movement of CD28 into lipid rafts does not require Lck and CD45 and can occur despite reduction of CD28 tyrosine phosphorylation to below the levels of detection. Analysis of murine CD28 mutants reveals a correlation between translocation to lipid rafts and costimulation of IL-2 production. Taken together with the known importance of lipid rafts in T cell activation, these observations suggest that translocation to lipid rafts may play an important role in CD28 signaling.