RESUMO
In this study, overlapping cDNA clones covering the entire S RNA molecule of Junin virus, an arenavirus that causes Argentine haemorrhagic fever, were generated. The complete sequence of this 3400 nucleotide RNA was determined using the dideoxynucleotide chain termination method. The nucleocapsid protein (N) and the glycoprotein precursor (GPC) genes were identified as two non-overlapping open reading frames of opposite polarity, encoding primary translation products of 564 and 481 amino acids, respectively. Intracellular processing of the latter yields the glycoproteins found in the viral envelope. Comparison of the Junin virus N protein with the homologous proteins of other arenaviruses indicated that amino acid sequences are conserved, the identity ranging from 46 to 76%. The N-terminal half of GPC exhibits an even higher degree of conservation (54 to 82%), whereas the C-terminal half is less conserved (21 to 50%). In all comparisons the highest level of amino acid sequence identity was seen when Junin virus and Tacaribe virus sequences were aligned. The nucleotide sequence at the 5' end of Junin virus S RNA is not identical to that determined of the other sequenced arenaviruses. However, it is complementary to the 3'-terminal sequences and may form a very stable panhandle structure (delta G-242.7 kJ/mol) involving the complete non-coding regions upstream from both the N and GPC genes. In addition, a distinct secondary structure was identified in the intergenic region, downstream from the coding sequences; Junin virus S RNA shows a potential secondary structure consisting of two hairpin loops (delta G -163.2 and -239.3 kJ/mol) instead of the single hairpin loop that is usually found in other arenaviruses. The analysis of the arenavirus S RNA nucleotide sequences and their encoded products is discussed in relation to structure and function.
Assuntos
Arenaviridae/genética , Arenavirus do Novo Mundo/genética , RNA Viral/genética , Sequência de Aminoácidos , Animais , Arenaviridae/classificação , Arenavirus do Novo Mundo/classificação , Sequência de Bases , Northern Blotting , Capsídeo/química , Capsídeo/genética , Linhagem Celular , Clonagem Molecular , Códon/química , Glicoproteínas/química , Glicoproteínas/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Precursores de Proteínas/química , Precursores de Proteínas/genética , RNA Viral/química , Células Vero , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
The cloned genes for the nucleocapsid proteins N of Junín and LCM (lymphocytic choriomeningitis) arenaviruses were inserted into the SV40-derived expression vector designated pKG4. When BHK-21 (baby hamster kidney fibroblasts) and CV-1 (African green monkey kidney fibroblasts) cell lines were transfected using these constructions, the transient expression yielded a polypeptide that could not be distinguished either by size nor by immunoreactivity from the N protein synthesized during the viral infection. The immunofluorescence analysis showed a pattern of intracellular localization similar to that observed in virus infected cells, i.e. varying from a diffuse cytoplasmic staining to granules, either distributed throughout the cytoplasm or concentrated in the perinuclear region. The association of the N protein with basophilic granules is similar to that observed in the cytopathic effect caused by arenaviruses, and could be related to the physicochemical properties of this polypeptide containing numerous basic amino acid sequences, that would allow for the interaction with cellular RNAs.
Assuntos
Arenavirus do Novo Mundo/genética , Capsídeo/biossíntese , Vírus da Coriomeningite Linfocítica/genética , Proteínas Recombinantes de Fusão/biossíntese , Transfecção , Proteínas do Core Viral/biossíntese , Animais , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Efeito Citopatogênico Viral , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Regulação Viral da Expressão Gênica , Vetores Genéticos , Mesocricetus , Vírus 40 dos SímiosRESUMO
The cloned genes for the nucleocapsid proteins N of Junín and LCM (lymphocytic choriomeningitis) arenaviruses were inserted into the SV40-derived expression vector designated pKG4. When BHK-21 (baby hamster kidney fibroblasts) and CV-1 (African green monkey kidney fibroblasts) cell lines were transfected using these constructions, the transient expression yielded a polypeptide that could not be distinguished either by size nor by immunoreactivity from the N protein synthesized during the viral infection. The immunofluorescence analysis showed a pattern of intracellular localization similar to that observed in virus infected cells, i.e. varying from a diffuse cytoplasmic staining to granules, either distributed throughout the cytoplasm or concentrated in the perinuclear region. The association of the N protein with basophilic granules is similar to that observed in the cytopathic effect caused by arenaviruses, and could be related to the physicochemical properties of this polypeptide containing numerous basic amino acid sequences, that would allow for the interaction with cellular RNAs.