Intracellular Crosslinking of Filoviral Nucleoproteins with Xintrabodies Restricts Viral Packaging

Abstract

Viruses assemble large macromolecular repeat structures that become part of the infectious particles or virions. Ribonucleocapsids (RNCs) of negative strand RNA viruses are a prime example where repetition of nucleoprotein (NP) along the genome creates a core polymeric helical scaffold that accommodates other nucleocapsid proteins including viral polymerase. The RNCs are transported through the cytosol for packaging into virions through association with viral matrix proteins at cell membranes. We hypothesized that RNC would be ideal targets for crosslinkers engineered to promote aberrant protein–protein interactions, thereby blocking their orderly transport and packaging. Previously, we had generated single-domain antibodies (sdAbs) against Filoviruses that have all targeted highly conserved C-terminal regions of NP known to be repetitively exposed along the length of the RNCs of Marburgvirus (MARV) and Ebolavirus (EBOV). Our crosslinker design consisted of dimeric sdAb expressed intracellularly, which we call Xintrabodies (X- for crosslinking). Electron microscopy of purified NP polymers incubated with purified sdAb constructs showed NP aggregation occurred in a genus-specific manner with dimeric and not monomeric sdAb. A virus-like particle (VLP) assay was used for initial evaluation where we found that dimeric sdAb inhibited NP incorporation into VP40-based VLPs whereas monomeric sdAb did not. Inhibition of NP packaging was genus specific. Confocal microscopy revealed dimeric sdAb was diffuse when expressed alone but focused on pools of NP when the two were coexpressed, while monomeric sdAb showed ambivalent partition. Infection of stable Vero cell lines expressing dimeric sdAb specific for either MARV or EBOV NP resulted in smaller plaques and reduced progeny of cognate virus relative to wild-type Vero cells. Though the impact was marginal at later time-points, the collective data suggest that viral replication can be reduced by crosslinking intracellular NP using relatively small amounts of dimeric sdAb to restrict NP packaging. The stoichiometry and ease of application of the approach would likely benefit from transitioning away from intracellular expression of crosslinking sdAb to exogenous delivery of antibody. By retuning sdAb specificity, the approach of crosslinking highly conserved regions of assembly critical proteins may well be applicable to inhibiting replication processes of a broad spectrum of viruses.

Single-Domain Antibodies As Therapeutics against Human Viral Diseases

“…Although the penetration of sdAbs through the cell membrane remains a problematic issue, many sdAbs have sufficient inherent stability to be functional in antigen-binding, even in the absence of the disulfide bond as occurring in the reducing environment of the cytoplasm. When expressed as intrabodies, they can bind antigens to inhibit viral replication ( 35 , 41 ). Furthermore, the high binding specificity of sdAbs can only rarely be achieved by small-molecule drugs.…”

“…Furthermore, Darling et al advanced an alternative approach to generate a dimeric intracellularly expressed sdAb against Filoviruses which targeted highly conserved C-terminal regions of nucleoprotein (NP). This dimeric sdAb can restrict viral packaging and inhibit Marburg and Ebola replication ( 41 ). As such, sdAbs represent a rich source of functional intrabodies of potential therapeutic importance.…”

Human transbodies that interfere with the functions of Ebola virus VP35 protein in genome replication and transcription and innate immune antagonism

“…Each molecule of the HuscFv contains six complementarity-determining regions (CDRs), and each CDR comprises several residues that can cooperate in capturing the target, which in this study consisted of several highly conserved critical residues pivotal for the function of VP35-IID 12 . Two recent studies, one employing V H H as cross-linkers of highly conserved regions of filoviral nucleoprotein to inhibit viral replication 48 and the other evaluating the anti-MARV V H H epitopes as highly conserved 49 , antecede that targeting vital conserved regions of intracellular viral antigens may be less prone to selection of escape mutants. Escape mutants of GP were identified in EBOV-infected Rhesus monkeys treated with antibody cocktails (MB-003) 50 .…”

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

“…Infection with Ebola and Marburg virus, which are both enveloped RNA filoviruses [ 207 ], poses a significant threat due to their high lethality rates of up to 90% in some cases of outbreaks [ 208 ]. As a strategy to allow only empty and non-infectious particles to form by inhibiting packaging, viral proteins from Ebola and Marburg virus were cross-linked inside the cell via a dimeric intrabody [ 209 ].…”