RESUMO
Replication and transmission of avian influenza virus in humans poses a pandemic threat. The molecular determinants that facilitate this process are not well understood. We used DBA/2 mice to identify viral factors that mediate the difference in pathogenesis between a virulent (H7N3) and a non-virulent (H7N9) avian influenza virus from North America. In vitro and in vivo characterization of reassortant viruses identified the PB2 and PA polymerase genes as major determinants of H7N3 pathogenesis. Analysis of individual residues in the PB2 and PA genes identified position 358 (E358V) in PB2 and positions 190 (P190S) and 400 (Q400P) in PA that reduced the virulence of H7N3 virus. The E358V and P190S substitutions also caused reduced inflammation after infection. Our results suggest that specific residues in the polymerase proteins PB2 and PA are important for replication and virulence of avian influenza viruses in a mammalian host.
Assuntos
Vírus da Influenza A Subtipo H7N3/genética , Vírus da Influenza A Subtipo H7N3/patogenicidade , Infecções por Orthomyxoviridae/virologia , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Aminoácidos , Animais , Sequência Conservada , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Camundongos , Camundongos Endogâmicos DBA , Infecções por Orthomyxoviridae/mortalidade , RNA Polimerase Dependente de RNA/química , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade , Índice de Gravidade de Doença , Carga Viral , Proteínas Virais/química , Fatores de Virulência/genética , Replicação ViralRESUMO
The obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole and employs a type III secretion mechanism to translocate host-interactive proteins. These proteins most likely contribute to pathogenesis through modulation of host cell mechanisms crucial for the establishment and maintenance of a permissive intracellular environment. Using a surrogate Yersinia type III secretion system (T3SS), we have identified the conserved gene product CT847 as a chlamydial T3SS substrate. Yeast two-hybrid studies using CT847 as bait to screen a HeLa cell cDNA library identified an interaction with mammalian Grap2 cyclin D-interacting protein (GCIP). Immunoblot analyses of C. trachomatis-infected HeLa cells showed that GCIP levels begin to decrease (as compared with mock-infected HeLa cells) between 8 h and 12 h post infection. GCIP was virtually undetectable in 24 h time point material. This decrease was inhibited by proteasome inhibitors lactacystin and MG-132, and the T3SS inhibitor Compound 1. CT847 was detectible in purified reticulate body but not elementary body lysates, and reverse transcription polymerase chain reaction (RT-PCR) expression analyses indicate a mid-cycle expression pattern. Both of these findings are consistent with CT847 contributing to the observed effect on GCIP. Given the established roles of GCIP, we believe that we have discovered a novel C. trachomatis antihost protein whose activity is relevant to chlamydial pathogenesis.
