RESUMO
1. A SARS vaccine was produced based on recombinant native full-length Spike-protein trimers (triSpike) and efficient establishment of a vaccination procedure in rodents. 2. Antibody-mediated enhancement of SARS-CoV infection with anti-SARS-CoV Spike immune-serum was observed in vitro. 3. Antibody-mediated infection of SARS-CoV triggers entry into human haematopoietic cells via an FcγR-dependent and ACE2-, pH-, cysteine-protease-independent pathways. 4. The antibody-mediated enhancement phenomenon is not a mandatory component of the humoral immune response elicited by SARS vaccines, as pure neutralising antibody only could be obtained. 5. Occurrence of immune-mediated enhancement of SARS-CoV infection raises safety concerns regarding the use of SARS-CoV vaccine in humans and enables new ways to investigate SARS pathogenesis (tropism and immune response deregulation).
Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Facilitadores , Glicoproteínas de Membrana/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/imunologia , Internalização do Vírus , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Antivirais/metabolismo , Linhagem Celular Tumoral , Cisteína Proteases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Monócitos , Peptidil Dipeptidase A/metabolismo , Receptores Fc/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Glicoproteína da Espícula de Coronavírus , VacinasRESUMO
The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.
Assuntos
Proteínas do Nucleocapsídeo/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/metabolismo , Montagem de Vírus , Animais , Chlorocebus aethiops , Proteínas M de Coronavírus , Proteínas do Nucleocapsídeo de Coronavírus , Humanos , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas do Nucleocapsídeo/genética , Glicoproteína da Espícula de Coronavírus , Células Vero , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/genética , Proteínas Viroporinas , Virossomos/metabolismo , Virossomos/ultraestruturaRESUMO
In this post-sequencing era, geneticists can focus on functional genomics on a much larger scale than ever before. One goal is the discovery and elucidation of the intricate genetic networks that co-ordinate transcriptional activation in different regulatory circuitries. High-throughput gene expression measurement using DNA arrays has thus become routine strategy. This approach, however, does not directly identify gene loci that belong to the same regulatory group; e.g., those that are bound by a common (set of) transcription factor(s). Working in yeast, two groups have recently published an elegant method that could circumvent this problem, by combining chromatin immunoprecipitation and DNA microarrays.(1,2) The method is likely to provide a powerful tool for the dissection of global regulatory networks in eukaryotic cells.
