RESUMO
Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. By transfection of various combinations of cDNA encoding wild-type and serial deletion mutants of each P protein subunit and co-immunoprecipitation with subunit-specific antibodies, the subunit-subunit contact sites on all three of the P proteins were determined. Results indicate that binary complexes are formed between PB1-PB2 and PB1-PA but not between PB2-PA. Therefore, we concluded that PB1 is the core subunit for assembly of the virus RNA polymerase. The C-terminal 158 amino acids of PB1 bound to the N-terminal 249 amino acids of PB2, while the N-terminal 140 amino acids of PB1 bound to the C-terminal two-thirds of PA. PB2-PA binding was not detected when they were expressed in the absence of the PB1 subunit.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Orthomyxoviridae/enzimologia , RNA Polimerase Dependente de RNA , Proteínas Virais/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Transformada , Chlorocebus aethiops , Expressão Gênica , Coelhos , Deleção de Sequência , TransfecçãoRESUMO
Influenza virus nucleoprotein (NP) is associated with the genome RNA, forming ribonucleoprotein cores. To identify the amino acid sequence involved in RNA binding, we performed Northwestern blot analysis with a set of N- and C-terminal deletion mutants of NP produced in Escherichia coli. The RNA binding region has been mapped between amino acid residues 91 and 188, a stretch of residues that contains a sequence that is not only highly conserved among NPs from A-, B-, and C-type influenza viruses but also similar to the RNA binding domain of a plant virus movement protein.
Assuntos
Vírus da Influenza A/metabolismo , Nucleoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Deleção de Sequência , Proteínas do Core Viral/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Núcleo Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Immunoblotting , Vírus da Influenza A/genética , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , Nucleoproteínas/biossíntese , Nucleoproteínas/isolamento & purificação , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Plasmídeos , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/isolamento & purificaçãoRESUMO
This paper describes the sequence of 257 nucleotides from the 3' end of RNA 2 of barley stripe mosaic virus ( BSMV , strain Argentina Mild) including an internal oligo (A) tract localized at a distance of 236 nucleotides from the 3' end, and the 3' terminal tRNA-like structure accepting tyrosine. This sequence is shown to be the same with RNAs 1,2 and 3 of another BSMV strain, Norwich , for at least the first 106 nucleotides from the 3' end. The 3' extremity of BSMV RNA bears some resemblance to tRNATyr from yeast in its primary structure. The possible secondary structures of the tRNA-like sequence in BSMV genome are discussed.
Assuntos
Genes Virais , Vírus do Mosaico/genética , RNA de Transferência/genética , RNA Viral/genética , Sequência de Bases , Endonucleases , Endorribonucleases , Hordeum , Conformação de Ácido Nucleico , RNA de Transferência/isolamento & purificação , RNA Viral/isolamento & purificação , Ribonuclease H , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
Barley stripe mosaic virus (BSMV) is a representative of the hordeiviruses which has a functionally fragmented RNA genom. 3'-terminal nucleotide sequence of the noncoding region of BSMV RNA 2 has been determined. This region contains the internal olygo(A) sequence (n = 21) and the tyrosine accepting tRNA-like structure at 3'-extreme end. tRNA-like sequence can fold into the two distinct secondary structure which strikingly similar to such a structures which has been proposed recently for cucumo- and bromoviruses.