RESUMO
Antiretroviral therapy (ART) suppresses HIV-1 viremia and prevents progression to AIDS. Nonetheless, chronic inflammation is a common problem for people living with HIV-1 on ART. One possible cause of inflammation is ongoing transcription from HIV-1 proviruses, whether or not the sequences are competent for replication. Previous work has shown that intron-containing RNA expressed from the HIV-1 provirus in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells, activates type 1 interferon. This activation required HIV-1 rev and was blocked by the XPO1 (CRM1)-inhibitor leptomycin. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with shRNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the IFN-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with non-targetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant inhibitory CARD-deletion or phosphomimetic point mutations, indicates that IFIH1 filament formation, dephosphorylation, and association with MAVS, are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1-knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 was revealed by formaldehyde crosslinking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is required for innate immune activation by intron-containing RNA from the HIV-1 provirus, and potentially contributes to chronic inflammation in people living with HIV-1.
RESUMO
Ebolavirus (EBOV) is the etiology of Ebola hemorrhagic fever (EHF). A major EHF outbreak in 2014-2015 in West Africa claimed >11,000 lives. A licensed vaccine is not available for EHF, although several vaccines have undergone clinical trials. We developed a human adenovirus (Ad) serotype 5-based candidate EHF vaccine based on controlled expression of the EBOV (Makona strain) glycoprotein (GP) as the immunogen. Two clones, AdGP72 and AdGP75, and a control Ad515 vector, were generated and tested for protein expression in vitro and immunogenicity in mice. Eight groups of mice were immunized with three doses of buffer, Ad515, AdGP72, and AdGP75, by two different dose regimens. Three different antigens (AdGP75-infected Vero E6 cell extract and two baculovirus expressed EBOV GP antigens, namely, GP alone or GP with EBOV VP40) were used to evaluate the immune response. Expression studies indicated that full-length GP was cleaved into its component subunits when expressed in mammalian cells through the Ad vectors. Moreover, in coimmunoprecipitation studies, EBOV GP was found to be associated with VP40 when expressed in baculoviruses. The candidate vaccines were immunogenic in mice, as evaluated by enzyme-linked immunosorbent assay using mammalian- or baculovirus-derived antigens. Further characterization and development of the candidate vaccines are warranted.
