RESUMO
The small RNA segment of some hantaviruses (family Bunyaviridae) encodes two proteins: the nucleocapsid protein and, in an overlapping reading frame, a non-structural (NSs) protein. The hantavirus NSs protein, like those of orthobunya- and phleboviruses, counteracts host innate immunity. Here, for the first time, the NSs protein of a hantavirus (Tula virus) has been observed in infected cells and shown to localize in the perinuclear area. Transiently expressed NSs protein showed similar localization, although the kinetics was slightly different, suggesting that to reach its proper location in the infected cell, the NSs protein does not have to cooperate with other viral proteins.
Assuntos
Núcleo Celular/metabolismo , Infecções por Hantavirus/metabolismo , Orthohantavírus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Orthohantavírus/genética , Infecções por Hantavirus/virologia , Humanos , Dados de Sequência Molecular , Transporte Proteico , Transfecção , Proteínas não Estruturais Virais/genéticaRESUMO
BACKGROUND: The competitiveness of two Tula hantavirus (TULV) isolates, TULV/Lodz and TULV/Moravia, was evaluated in interferon (IFN) -competent and IFN-deficient cells. The two isolates differ in the length of the open reading frame (ORF) encoding the nonstructural protein NSs, which has previously been shown to inhibit IFN response in infected cells. RESULTS: In IFN-deficient Vero E6 cells both TULV isolates survived equally well. In contrast, in IFN-competent MRC5 cells TULV/Lodz isolate, that possesses the NSs ORF for the full-length protein of 90 aa, survived for more consequent passages than TULV/Moravia isolate, which contains the ORF for truncated NSs protein (66-67 aa). CONCLUSION: Our data show that expression of a full-length NSs protein is beneficial for the virus survival and competitiveness in IFN-competent cells and not essential in IFN-deficient cells. These results suggest that the N-terminal aa residues are important for the full activity of the NSs protein.
Assuntos
Infecções por Hantavirus/virologia , Interferons/metabolismo , Orthohantavírus/química , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Orthohantavírus/genética , Orthohantavírus/crescimento & desenvolvimento , Infecções por Hantavirus/imunologia , Humanos , Interferons/deficiência , Interferons/genética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Alinhamento de Sequência , Inoculações SeriadasRESUMO
Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.
Assuntos
Orthohantavírus/genética , Orthohantavírus/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biblioteca Gênica , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Camundongos , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Técnicas do Sistema de Duplo-HíbridoRESUMO
The S RNA genome segment of hantaviruses carried by Arvicolinae and Sigmodontinae rodents encodes the nucleocapsid (N) protein and has an overlapping (+1) open reading frame (ORF) for a putative nonstructural protein (NSs). The aim of this study was to determine whether the ORF is functional. A protein corresponding to the predicted size of Tula virus (TULV) NSs was detected using coupled in vitro transcription and translation from a cloned S segment cDNA, and a protein corresponding to the predicted size of Puumala virus (PUUV) NSs was detected in infected cells by Western blotting with an anti-peptide serum. The activities of the interferon beta (IFN-beta) promoter, and nuclear factor kappa B (NF-kappaB)- and interferon regulatory factor-3 (IRF-3) responsive promoters, were inhibited in COS-7 cells transiently expressing TULV or PUUV NSs. Also IFN-beta mRNA levels in IFN-competent MRC5 cells either infected with TULV or transiently expressing NSs were decreased. These data demonstrate that Tula and Puumala hantaviruses have a functional NSs ORF. The findings may explain why the NSs ORF has been preserved in the genome of most hantaviruses during their long evolution and why hantavirus-infected cells secrete relatively low levels of IFNs.