Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.

"Região cis-atuante reguladora da transcrição do gene que codifica celobiohidrolase I em Trichoderma reesei: expressão basal e induzida / Cis regulatory region of gene transcription which codify cellobiohydrolase I in Trichoderma reesei: induced and basal expression\"

A enzima celobiohidrolase I (CBHI) do fungo multicelular Trichoderma reesei catalisa a liberação de celobiose a partir das extremidades redutoras das cadeias de celulose. Esta enzima é um dos membros do sistema de celulases extracelular necessário para a hidrólise completa da celulose até glicose. A expressão do gene cbhl é controlada pela fonte de carbono energético usado no meio de cultura. O crescimento em presença de celulose - não de glicose ou glicerol - resulta na indução do transcrito de cbhl em pelo menos 1200 vezes. Esta indução parece requerer a expressão basal do sistema das celulases, as quais são necessárias para catalisar a formação do indutor solúvel a partir da celulose. A expressão do transcrito de cbhl também é controlada pelo estado metabólico da mitocôndria; os transcritos das celulases são regulados sob condições tidas como repressoras da respiração mitocondrial...