RESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpes virus type 8 (HHV-8) is tightly linked to the development of Kaposi's sarcoma, primary effusion lymphoma (PEL) and some cases of multicentric Castleman's disease. Latency-associated nuclear antigen (LANA) is one of a limited number of KSHV genes consistently expressed in these diseases as well as in KSHV-infected cell lines derived from PEL, and has been shown to play crucial role in persistence of KSHV genomes in the infected cells. In this study, we explored the cellular factors that interact with LANA using yeast two-hybrid screening, and isolated a part of gene encoding human myeloid cell nuclear differentiation antigen (MNDA). MNDA is a hematopoietic interferon-inducible nuclear proteins with a HIN-200 family member with conserved 200-amino acid repeats. Immunoprecipitation assay revealed that LANA interacted with MNDA in a mammalian embryonic kidney cell line. MNDA transcript was undetectable in three PEL cell lines by reverse-transcription polymerase chain reaction, but it was induced by interferon alpha (IFNalpha). Moreover, LANA and MNDA were co-localized in the nuclei of MNDA-expressing PEL cells. Our results suggest that LANA interacts with MNDA in KSHV-infected cells exposed to IFNalpha. Such interaction may modulate IFN-mediated host defense activities.
Assuntos
Antígenos de Diferenciação Mielomonocítica/biossíntese , Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/biossíntese , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos Virais/genética , Antígenos Virais/fisiologia , Sequência de Bases , Divisão Celular , Linhagem Celular , Núcleo Celular/imunologia , Núcleo Celular/virologia , DNA Viral/genética , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/imunologia , Humanos , Interferon Tipo I/farmacologia , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Recombinantes , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/virologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
We have screened a phage peptide library to address whether clones binding to a monoclonal antibody (mAb) could be isolated and if the selected phage particles would be able to elicit an in vivo immune response against the original antigen. A phage peptide library, consisting of seven random amino acids inserted in the minor coat protein (pIII), was screened for specific binding to a rat mAb LAT-27, which is capable of neutralizing human T-cell leukaemia virus type-I (HTLV-I) by binding to its envelope gp46 epitope, (amino acids LPHSNL). Total 37 clones were selected from the library and one clone named 4-2-22 was tested for its immunogenicity in three rabbits. The all rabbit immune sera showed strong binding activity to a gp46 peptide carrying the neutralization sequence, stained gp46-expressing cells and neutralized HTLV-I in vitro as determined by cell fusion inhibition assay. These results show that the selected phage clone was capable of mimicking the epitope recognized by a HTLV-I neutralizing mAb, and it can be used as an immunogen to induce protective immune response against HTLV-I. Thus, the present methodology could be one of the approaches to develop vaccines against infectious agents in a simple and inexpensive way.