RESUMO
RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.
Assuntos
Capuzes de RNA/genética , Infecções por Vírus de RNA/genética , Proteínas Recombinantes de Fusão/genética , Regiões 5' não Traduzidas/genética , Animais , Bovinos , Linhagem Celular , Cricetinae , Cães , Humanos , Vírus da Influenza A/metabolismo , Camundongos , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Fases de Leitura Aberta/genética , Capuzes de RNA/metabolismo , Infecções por Vírus de RNA/metabolismo , Vírus de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica/genética , Proteínas Virais/metabolismo , Replicação Viral/genéticaRESUMO
Interactions of host cell factors with RNA sequences and structures in the genomes of positive-strand RNA viruses play various roles in the life cycles of these viruses. Our understanding of the functional RNA elements present in norovirus genomes to date has been limited largely to in vitro analysis. However, we recently used reverse genetics to identify evolutionarily conserved RNA structures and sequences required for norovirus replication. We have now undertaken a more detailed analysis of RNA structures present at the 3' extremity of the murine norovirus (MNV) genome. Biochemical data indicate the presence of three stable stem-loops, including two in the untranslated region, and a single-stranded polypyrimidine tract [p(Y)] of variable length between MNV isolates, within the terminal stem-loop structure. The well-characterized host cell pyrimidine binding proteins PTB and PCBP bound the 3'-untranslated region via an interaction with this variable sequence. Viruses lacking the p(Y) tract were viable both in cell culture and upon mouse infection, demonstrating that this interaction was not essential for virus replication. However, competition analysis with wild-type MNV in cell culture indicated that the loss of the p(Y) tract was associated with a fitness cost. Furthermore, a p(Y)-deleted mutant showed a reduction in virulence in the STAT1(-/-) mouse model, highlighting the role of RNA structures in norovirus pathogenesis. This work highlights how, like with other positive-strand RNA viruses, RNA structures present at the termini of the norovirus genome play important roles in virus replication and virulence.
Assuntos
Regiões 3' não Traduzidas , Sequência de Bases , Genoma Viral , Norovirus , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , Animais , Linhagem Celular , Evolução Molecular , Regulação Viral da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Norovirus/genética , Norovirus/patogenicidade , Fases de Leitura Aberta , Pirimidinas , RNA Viral/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Replicação Viral/genéticaRESUMO
Four bacterial isolates were recovered from the blood cultures of four patients, two of whom were from Hong Kong and two of whom were from Canada. The two Hong Kong strains were isolated from a 48-year-old man with intestinal obstruction and secondary sepsis (strain HKU16T) and from a 39-year-old man with acute appendicitis (strain HKU17), while the two Canadian strains were isolated from a 74-year-old man with biliary sepsis (strain CA1) and from a 66-year-old woman with metastatic carcinoma and sepsis (strain CA2). While the first three patients survived, the last patient died 2 weeks after the episode of bacteremia. All four isolates are strictly anaerobic, nonsporulating, gram-positive coccobacilli that were unidentified by conventional phenotypic tests and commercial identification systems. They grow on sheep blood agar as nonhemolytic pinpoint colonies after 48 h of incubation at 37 degrees C in an anaerobic environment. All are catalase positive and motile, with flagella. They produce acid from arabinose, glucose, mannose, and xylose. They do not produce indole or reduce nitrate. They are sensitive to penicillin, vancomycin, and metronidazole but resistant to cefotaxime. 16S rRNA gene sequence analysis showed 16.0%, 16.8%, and 21.0% base differences from Clostridium propionicum, Clostridium neopropionicum, and Atopobium minutum, respectively. The G+C content of strain HKU16T is 40.2% +/- 2.2%. Based on their phylogenetic affiliation, unique G+C content, and phenotypic characteristics, we propose a new genus and species, Catabacter hongkongensis gen. nov., sp. nov., to describe the bacterium, for which HKU16 is the type strain, and suggest that it be assigned to a new family, Catabacteriaceae. The gastrointestinal tract was probably the source of the bacterium for at least three of the four patients. The isolation of a catalase-positive, motile, nonsporulating, anaerobic gram-positive bacillus in clinical laboratories should raise the possibility of C. hongkongensis. Further studies should be performed to ascertain the epidemiology and other disease associations of this bacterium.