MBP-eVP35 IID and sFab H3, and D

MBP-eVP35 IID and sFab H3, and D. using an approach that provides a framework for generating engineered Fabs targeting other viral proteins. Keywords: Marburg KW-2478 virus, VP35, IFN antagonist, synthetic antibodies, phage display, mass spectrometry, X-ray crystallography Graphical Abstract Marburg and Ebola are members of the family of non-segmented negative strand RNA viruses (NNSVs) and are the cause of sporadic but deadly outbreaks of severe viral hemorrhagic fever (VHF)1C2. The 2005 Marburg outbreak in Angola, the 2014C2016 Ebola virus outbreak in West Africa, the 2017 Marburg virus outbreak in Uganda, and the ongoing 2018 Ebola virus outbreak in the Democratic Republic of Congo highlight the global impact on human health and underscore the critical need for prophylactic and therapeutic treatments for filoviral infections (https://www.cdc.gov/ncezid/dhcpp/vspb/outbreaks.html). Currently, there are no approved effective treatments available for filoviral infections, KW-2478 although there are several anti-filoviral interventions that are in development, including antibody-based therapies, siRNAs, phosphorodiamidate morpholino oligomers, small molecule antivirals, as well as immunomodulatory approaches3. Filovirus disease is characterized by uncontrolled viral replication coupled to potent immune suppression by viral proteins. Like other NNSVs, Marburg virus (MARV) contains a single-stranded genome of ~19 kilobases, which encodes for seven proteins and is encapsidated by nucleoprotein (mNP). Genome replication is carried out by the viral RNA-dependent RNA polymerase (RdRp) complex that is comprised of Marburg viral protein 35 (mVP35), mNP, and the large protein (mL) polymerase, which is the only enzymatic subunit. Marburg VP30 (mVP30) is also essential for viral replication, but its exact role is unclear and, unlike its Ebola counterpart, may KW-2478 be dispensable for viral transcription4C6. mVP35 functions as a potent immune antagonist and is a cofactor required for viral RNA synthesis that bridges the interaction between mNP and mL, analogous to phosphoprotein (P) from other NNSVs6C7. mVP35 contains a coiled-coil motif at the N-terminus, which is required for oligomerization of mVP358C9, and a C-terminal interferon (IFN) inhibitory domain (mIID), which binds dsRNA and is essential for innate immune inhibition10C11. In addition, mVP35 contains an N-terminal peptide that binds mNP and maintains mNP in a non-oligomeric and RNA-free state12C15. How mVP35 simultaneously coordinates interactions between mNP and mL to facilitate viral RNA synthesis remains elusive. Here we describe a strategy to generate molecular tools to further characterize the Marburg viral replication cycle in order to define novel therapeutic approaches. To this end, we engineered an antibody that specifically targets VP35 protein, which is important for MARV replication. Our approach utilized phage display technology to identify synthetic antibody fragments (sFabs) that bind viral replication components with high affinity and specificity. We characterized selected anti-mVP35 sFabs for their ability to differentiate between viral proteins from different species. In addition, we used structural studies of KW-2478 the sFab/mVP35 complex to identify the critical binding interface residues and to reveal the likely mechanism by which the sFab inhibits viral RNA synthesis. The pipeline described here for the generation and characterization of sFabs targeting mVP35 should be able to probe Rabbit polyclonal to FOXRED2 viral protein function generally and to validate potential targets for therapeutic development. Results Selection of sFabs specific for mVP35. To isolate antibodies targeting mVP35, we utilized a previously established synthetic antibody library with chemical diversity introduced in four complementarity-determining regions (CDRs) of the light chain (CDRL3) and heavy chain (CDRH1, CDRH2, CDRH3) of a single human framework engineered for KW-2478 stability and minimal immunogenicity16C17. We performed four rounds of selection for binding to recombinant Marburg virus VP35 (mVP35) interferon (IFN) inhibitory domain (IID) protein and screened individual.