RESUMEN
UNLABELLED: The leader (L) and 2A proteins of cardioviruses are the primary antihost agents produced during infection. For encephalomyocarditis virus (EMCV), the prototype of the genus Cardiovirus, these proteins interact independently with key cellular partners to bring about inhibition of active nucleocytoplasmic trafficking and cap-dependent translation, respectively. L and 2A also bind each other and require this cooperation to achieve their effects during infection. Recombinant L and 2A interact with 1:1 stoichiometry at a KD (equilibrium dissociation constant) of 1.5 µM. The mapped contact domains include the amino-proximal third of 2A (first 50 amino acids) and the central hinge region of L. This contact partially overlaps the L segment that makes subsequent contact with Ran GTPase in the nucleus, and Ran can displace 2A from L. The equivalent proteins from Theiler's murine encephalomyelitis virus (TMEV; BeAn) and Saffold virus interact similarly in any subtype combination, with various affinities. The data suggest a mechanism whereby L takes advantage of the nuclear localization signal in the COOH region of 2A to enhance its trafficking to the nucleus. Once there, it exchanges partners in favor of Ran. This required cooperation during infection explains many observed codependent phenotypes of L and 2A mutations. IMPORTANCE: Cardiovirus pathogenesis phenotypes vary dramatically, from asymptomatic, to mild gastrointestinal (GI) distress, to persistent demyelination and even encephalitic death. Leader and 2A are the primary viral determinants of pathogenesis, so understanding how these proteins cooperate to induce such a wide variety of outcomes for the host is of great important and interest to the field of virology, especially to those who use TMEV as a murine model for multiple sclerosis.
Asunto(s)
Virus de la Encefalomiocarditis/fisiología , Mapeo de Interacción de Proteínas , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Células HeLa , Humanos , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Alineación de Secuencia , Replicación ViralRESUMEN
The leader (L) protein of encephalomyocarditis virus (EMCV) shuts off host cell nucleocytoplasmic trafficking (NCT) by inducing hyperphosphorylation of nuclear pore proteins. This dramatic effect by a nonenzymatic protein of 6 kDa is not well understood but clearly involves L binding to cellular Ran GTPase, a critical factor of active NCT. Exogenous GDP and GTP are inhibitory to L-Ran binding, but the guanine-nucleotide exchange factor RCC1 can relieve this inhibition. In the presence of RCC1, L binds Ran with a KD (equilibrium dissociation constant) of ≈ 3 nM and reaches saturation within 20 min. The results of fluorescently tagged nucleotide experiments suggest that L-Ran interactions affect the nucleotide-binding pocket of Ran.
Asunto(s)
Infecciones por Cardiovirus/enzimología , Infecciones por Cardiovirus/virología , Proteínas de Ciclo Celular/metabolismo , Virus de la Encefalomiocarditis/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Virales/metabolismo , Proteína de Unión al GTP ran/metabolismo , Infecciones por Cardiovirus/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Virus de la Encefalomiocarditis/genética , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Interacciones Huésped-Patógeno , Humanos , Cinética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , Proteínas Virales/química , Proteínas Virales/genética , Proteína de Unión al GTP ran/química , Proteína de Unión al GTP ran/genéticaRESUMEN
Maltose metabolism during the conversion of transitory (leaf) starch to sucrose requires a 4-alpha-glucanotransferase (EC 2.4.1.25) in the cytosol of leaf cells. This enzyme is called DPE2 because of its similarity to the disproportionating enzyme in plastids (DPE1). DPE1 does not use maltose; it primarily transfers a maltosyl unit from one maltotriose to a second maltotriose to make glucose and maltopentaose. DPE2 is a modular protein consisting of a family 77 glycosyl hydrolase domain, similar to DPE1, but unlike DPE1 the domain is interrupted by an insertion of approximately 150 amino acids as well as an N-terminal extension that consists of two carbohydrate binding modules. Phylogenetic analysis shows that the DPE2-type enzyme is present in a limited but highly diverse group of organisms. Here we show that DPE2 transfers the non-reducing glucosyl unit from maltose to glycogen by a ping-pong mechanism. The forward reaction (consumption of maltose) is specific for the beta-anomer of maltose, while the reverse reaction (production of maltose) is not stereospecific for the acceptor glucose. Additionally, through deletion mutants we show that the glycosyl hydrolase domain alone provides disproportionating activity with a much higher affinity for short maltodextrins than the complete wild-type enzyme, while absence of the carbohydrate binding modules completely abolishes activity with large complex carbohydrates, reflecting the presumed function of DPE2 in vivo.