RESUMO
The HA (haemagglutinin) of influenza viruses must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. We previously showed using FRET that deletion of the two raft-targeting features of HA, S-acylation at the cytoplasmic tail and the hydrophobic amino acids VIL (Val-Ile-Leu) in the outer part of the TMR (transmembrane region), lead to reduced raft association. In addition, exchange of VIL, but not of the S-acylation sites severely retards transport of HA through the Golgi. In the present study, we have further characterized the ill-defined signal in the TMR. A sequence comparison suggests that the leucine residue of VIL might be part of a CCM (cholesterol consensus motif) that is known to bind cholesterol to seven-transmembrane receptors. The signal also comprises a lysine residue and a tryptophan residue on one and a tyrosine residue on another TMR helix and is conserved in group 2 HAs. Mutations in the CCM retard Golgi-localized processing of HA, such as acquisition of Endo H (endoglycosidase H)-resistant carbohydrates in the medial Golgi and proteolytic cleavage in the TGN (trans-Golgi network). The delay in transport of HA to and from the medial Golgi varied with the mutation, suggesting that different transport steps are affected. All mutants analysed by FRET also showed reduced association with rafts at the plasma membrane. Thus the raft-targeting signal of HA encompasses not only hydrophobic, but also aromatic and positively charged, residues. We speculate that binding to cholesterol might facilitate intracellular transport of HA and association with rafts.
Assuntos
Hemaglutininas Virais/metabolismo , Vírus da Influenza A Subtipo H7N1/metabolismo , Microdomínios da Membrana/metabolismo , Rede trans-Golgi/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Colesterol , Cricetinae , Cricetulus , Hemaglutininas Virais/genética , Vírus da Influenza A Subtipo H7N1/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Microdomínios da Membrana/genética , Microdomínios da Membrana/virologia , Mutação , Transporte Proteico/genética , Rede trans-Golgi/genética , Rede trans-Golgi/virologiaRESUMO
No disponible
Assuntos
Humanos , Masculino , Feminino , Coinfecção/imunologia , Coinfecção/microbiologia , Influenza Humana/epidemiologia , Influenza Humana/imunologia , Influenza Humana/microbiologia , /imunologia , /isolamento & purificação , /metabolismo , Vírus da Influenza A Subtipo H7N1/isolamento & purificação , Vírus da Influenza A Subtipo H7N1/metabolismo , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/microbiologiaRESUMO
Highly pathogenic avian influenza (HPAI) H7N1 viruses caused a series of epizootics in Italy between 1999 and 2001. The emergence of these HPAI viruses coincided with the deletion of the six amino acids R(225)VESEV(230) at the C terminus of NS1. In order to assess how the truncation of NS1 affected virus replication, we used reverse genetics to generate a wild-type low-pathogenic avian influenza (LPAI) H7N1 virus with a 230aa NS1 (H7N1(230)) and a mutant virus with a truncated NS1 (H7N1(224)). The 6aa truncation had no impact on virus replication in duck or chicken cells in vitro. The H7N1(230) and H7N1(224) viruses also replicated to similar levels and induced similar immune responses in ducks or chickens. No significant histological lesions were detected in infected ducks, regardless of the virus inoculated. However, in chickens, the H7N1(230) virus induced a more severe interstitial pneumonia than did the H7N1(224) virus. These findings indicate that the C-terminal extremity of NS1, including the PDZ-binding motif ESEV, is dispensable for efficient replication of an LPAI virus in ducks and chickens, even though it may increase virulence in chickens, as revealed by the intensity of the histological lesions.
Assuntos
Galinhas/virologia , Patos/virologia , Vírus da Influenza A Subtipo H7N1/genética , Vírus da Influenza A Subtipo H7N1/metabolismo , Influenza Aviária/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Embrião de Galinha , Galinhas/imunologia , Patos/imunologia , Vírus da Influenza A Subtipo H7N1/imunologia , Influenza Aviária/genética , Influenza Aviária/imunologia , Influenza Aviária/virologia , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Proteínas não Estruturais Virais/imunologia , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
Avian influenza viruses A/duck/Mongolia/47/2001 (H7N1) (47/01) and A/duck/Mongolia/867/2002 (H7N1) (867/02) were defined as low-pathogenic avian influenza viruses (LPAIVs) using an intravenous pathogenicity test in chickens. On the other hand, the intracerebral pathogenicity indices of 47/01 and 867/02 were 1.30 and 0.00, respectively. A series of reassortant viruses were generated between 47/01 and 867/02, and their intracerebral pathogenicity was compared in one-day-old chicks to identify the protein(s) responsible for the intracerebral pathogenicity of 47/01. The results indicate that the amino acids at positions 50 and 98 of the nucleoprotein are related to the pathogenicity of 47/01 in chicks by intracerebral inoculation. A significant association was found between mortality of the chicks inoculated intracerebrally with 47/01 and virus replication in the lungs and/or brain. These results indicate that the NP of avian influenza viruses may be responsible for intracerebral pathogenicity in the host.
