Quantifying RNA binding sites transcriptome-wide using DO-RIP-seq.

RNA-binding proteins (RBPs) and noncoding RNAs orchestrate post-transcriptional processes through the recognition of specific sites on targeted transcripts. Thus, understanding the connection between binding to specific sites and active regulation of the whole transcript is essential. Many immunoprecipitation techniques have been developed that identify either whole transcripts or binding sites of RBPs on each transcript using cell lysates. However, none of these methods simultaneously measures the strength of each binding site and quantifies binding to whole transcripts. In this study, we compare current procedures and present digestion optimized (DO)-RIP-seq, a simple method that locates and quantifies RBP binding sites using a continuous metric. We have used the RBP HuR/ELAVL1 to demonstrate that DO-RIP-seq can quantify HuR binding sites with high coverage across the entire human transcriptome, thereby generating metrics of relative RNA binding strength. We demonstrate that this quantitative enrichment of binding sites is proportional to the relative in vitro binding strength for these sites. In addition, we used DO-RIP-seq to quantify and compare HuR's binding to whole transcripts, thus allowing for seamless integration of binding site data with whole-transcript measurements. Finally, we demonstrate that DO-RIP-seq is useful for identifying functional mRNA target sets and binding sites where combinatorial interactions between HuR and AGO-microRNAs regulate the fate of the transcripts. Our data indicate that DO-RIP-seq will be useful for quantifying RBP binding events that regulate dynamic biological processes.

DO-RIP-seq to quantify RNA binding sites transcriptome-wide.

Post-transcriptional processes orchestrate gene expression through dynamic protein-RNA interactions. These interactions occur at specific sites determined by RNA sequence, secondary structure, or nucleotide modifications. Methods have been developed either to quantify binding of whole transcripts or to identify the binding sites, but there is none proven to quantify binding at both the whole transcript and binding site levels. Here we describe digestion optimized RNA immunoprecipitation with deep sequencing (DO-RIP-seq) as a method that quantitates at the whole transcript target (RIP-Seq-Like or RSL) level and at the binding site level (BSL) using continuous metrics. DO-RIP-seq methodology was developed using the RBP HuR/ELAVL1 as a test case (Nicholson et al., 2016). DO-RIP-seq employs treatment of cell lysates with a nuclease under optimized conditions to yield partially digested RNA fragments bound by RNA binding proteins, followed by immunoprecipitations that capture the digested RNA-protein complexes and assess non-specific or background interactions. Analyses of sequenced cDNA libraries made from the bound RNA fragments yielded two types of enrichment scores; one for RSL binding events and the other for BSL events (Nicholson et al., 2016). These analyses plus the extensive read coverage of DO-RIP-seq allows seamless integration of binding site and whole transcript information. Therefore, DO-RIP-seq is useful for quantifying RBP binding events that are regulated during dynamic biological processes.

Mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop.

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.

Viral entry inhibitors block dengue antibody-dependent enhancement in vitro.

Severe dengue virus (DENV) disease symptoms, including dengue hemorrhagic fever and dengue shock syndrome, have been correlated with the presence of pre-existing antibodies that enhance rather than neutralize infections in Fc receptor bearing cells. These antibodies can originate from previous infection with a different serotype of dengue, or from waning antibody titers that occur in infants and young children as they are weaned from breast milk that contains protective dengue-specific antibodies. Despite the apparent importance of this antibody dependent enhancement (ADE) effect, there has been no description of any specific inhibitors of this process. We explored DENV entry inhibitors as a potential strategy to block ADE. Two different peptide entry inhibitors were tested for the ability to block antibody-mediated DENV-2 infection of human, FcRII bearing K562 cells in vitro. Both peptides were able to inhibit ADE, showing that entry inhibitors are possible candidates for the development of specific treatment for severe DENV infection.

Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient.

BACKGROUND: Antibodies produced in response to infection with any of the four serotypes of dengue virus generally provide homotypic immunity. However, prior infection or circulating maternal antibodies can also mediate a non-protective antibody response that can enhance the course of disease in a subsequent heterotypic infection. Naturally occurring human monoclonal antibodies can help us understand the protective and pathogenic roles of the humoral immune system in dengue virus infection. RESULTS: Epstein-Barr Virus (EBV) transformation of B cells isolated from the peripheral blood of a human subject with previous dengue infection was performed. B cell cultures were screened by ELISA for antibodies to dengue (DENV) envelope (E) protein. ELISA positive cultures were cloned by limiting dilution. Three IgG1 human monoclonal antibodies (HMAbs) were purified and their binding specificity to E protein was verified by ELISA and biolayer interferometry. Neutralization and enhancement assays were conducted in epithelial and macrophage-like cell lines, respectively. All three HMAbs bound to E from at least two of the four DENV serotypes, one of the HMAbs was neutralizing, and all were able to enhance DENV infection. CONCLUSIONS: HMAbs against DENV can be successfully generated by EBV transformation of B cells from patients at least two years after naturally acquired DENV infections. These antibodies show different patterns of cross-reactivity, neutralizing, and enhancement activity.