RESUMO
Positive-strand RNA viruses often use noncanonical strategies to usurp the host translational machinery for their own benefit. These strategies have been analyzed using transient expression assays in the absence of replication, with reporter genes replacing viral genes. A sensitive and convenient reporter assay is the dual luciferase system using Renilla (Renilla reniformis) and firefly (Photinus pyralis) reporter genes. Use of recombinant viral constructs containing the reporter luciferase gene allows us to discern whether a particular RNA sequence or secondary structure elicits an effect on initiation of translation or recoding. This chapter describes a standard luciferase protocol that can be molded to fit any viral sequence, in order to detect cis-acting regulatory elements in viral RNA.
Assuntos
Biossíntese de Proteínas , RNA Viral/genética , Animais , Eletroporação/métodos , Vaga-Lumes , Genes Reporter , Indicadores e Reagentes , Luciferases/genética , Protoplastos/fisiologia , Renilla , Transfecção/métodosRESUMO
Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases. Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins G(N) and G(C), nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates. Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate.
Assuntos
Febre do Vale de Rift/imunologia , Vírus da Febre do Vale do Rift/imunologia , Vacinas Virossomais/imunologia , Replicação Viral , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Citocinas/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Vírus da Leucemia Murina de Moloney/genética , Ratos , Ratos Endogâmicos WF , Febre do Vale de Rift/prevenção & controle , Vírus da Febre do Vale do Rift/fisiologia , Análise de Sobrevida , Linfócitos T/imunologia , Vacinas Virossomais/genética , Proteínas Virais/genética , Virossomos/ultraestruturaRESUMO
Congenital blood disorders are common and yet clinically challenging globin disorders. Gene therapy continues to serve as a potential therapeutic method to treat these disorders. While tremendous advances have been made in vivo, gene delivery protocols and vector prototypes still require optimization. Alternative cis-acting promoter elements derived from VL30 retroelements have been effective in expressing tissue-specific transgene expression in vivo in nonerythroid cells. VL30 promoter elements were isolated from ELM-I-1 erythroid progenitor cells upon erythropoietin (epo) treatment. These promoters were inserted into a VL30-derived expression vector and reintroduced into the ELM-I-1 cells. beta-Galactosidase reporter gene activity from the ELM 5 clone, a BVL-1-like VL30 promoter, was capable of expressing sustained levels of the transgene expression over a 16-week assay period. These findings delineate the potential utility of these retroelement promoters as transcriptionally active, erythroid-specific, long terminal repeat (LTR) components for current globin vector constructs.
Assuntos
Células Precursoras Eritroides/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos/genética , Regiões Promotoras Genéticas/fisiologia , Retroelementos/genética , Animais , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoetina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Óperon Lac , Leucemia Eritroblástica Aguda/patologia , Camundongos , Especificidade de Órgãos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Transfecção , Células Tumorais Cultivadas , beta-Galactosidase/biossínteseRESUMO
Virus-like 30S (VL30) elements are endogenous retro-elements of the mouse retrotransposon family. These elements are transcriptionally responsive in a temporal and tissue-specific manner due to the U3 promoter region of the elements' long terminal repeat (LTR). We have analyzed VL30 promoters from erythroid progenitor cell lines (MEL 585S and ELM-I-1) that contrasted in their response to erythropoietin (epo). Through RT-PCR-generated cDNAs, VL30 promoters were identified and showed homology to the third and fourth U3 subgroups, with GATA-1, Jak2/STAT5, and B10 RRE sites. One clone (ELM5) showed 97% homology to BVL-1, a putative epo-responsive VL30 element. In addition, a novel U3 promoter (MEL/ELM CONSTIT) showed complete sequence homology between both cell lines. Ribonuclease protection confirmed that epo-induced VL30 promoters were activated in ELM-I-1 cells, whereas the conserved VL30 MEL-ELM CONSTIT VL30 promoter showed no enhanced expression in the epo-unresponsive MEL cells. Identification of these U3 promoters suggests that VL30s are conserved and can be transcriptionally activated in an epo-specific manner.