Epitope Mapping of A Viral Propagation-Inhibiting Single-Chain Variable Fragment Against Rabies Lyssavirus Phosphoprotein

Introduction

Rabies lyssavirus (RABV), a member of the genus Lyssavirus and family Rhabdoviridae, is highly neurotropic and causes acute infection of the central nervous system in both humans and animals. It is transmitted by the bites or scratches of rabid animals. From the point of entry, RABV travels retrogradely along nerves to the central nervous system. After viral multiplication in the brain causing acute encephalitis, RABV spreads centrifugally and through cell-to-cell transmission to various peripheral tissues, leading to multiorgan failure. The onset of rabies can be prevented by prompt postexposure prophylaxis. However, if clinical symptoms develop, the mortality rate is ∼100%, with no currently available effective treatment options.

We previously investigated whether intracellularly expressed antibodies, intrabodies, against RABV phosphoprotein (RABV-P), could inhibit RABV propagation in neuronal cells.⁽¹⁾ For the intrabodies, we employed a single-chain variable fragment (scFv), comprising the VH and VL regions of the variable antigen-binding site of an immunoglobulin, connected by a short linker sequence.⁽²⁾ RABV-P was selected as a target because it interacts with various viral or cellular proteins to exert multiple essential functions in viral propagation, including encapsidation of viral genome RNA,⁽³⁾ acting as a cofactor for the RABV large protein⁽⁴⁾ (RABV-L, a viral RNA polymerase), and inhibition of the cellular antiviral system induced by interferons (IFNs).⁽⁵⁾

One of four genetically independent scFv clones derived from an scFv phage-display library, scFv-P19, colocalized with RABV-P in the cytoplasm.⁽¹⁾ Furthermore, scFv-P19, when expressed transiently in mouse neuronal cell lines before RABV infection, severely inhibited RABV propagation and secretion, suggesting that the scFv-based antiviral approach may serve as a candidate for future RABV therapeutic studies if combined with appropriate delivery and application systems; however, more in-depth mechanistic studies are warranted to first elucidate how scFv-P19 elicits its antiviral effects.

In this study, to obtain fundamental information for elucidating the mechanism of RABV inhibition by scFv-P19, we conducted epitope mapping of scFv-P19.

Materials and Methods

Results and Discussion

In this study, we conducted epitope mapping of scFv-P19 to elucidate the mechanism of RABV inhibition by scFv-P19. For epitope mapping, full-length RABV-P (P1), N-terminal-truncated RABV-P isoforms, and C-terminal-truncated RABV-P flanked with FLAG/Strept tags (Fig. 1) were transiently expressed in HEK293T cells and analyzed using IFA and Western blotting (WB). The expression of all RABV-P variants was confirmed using anti-FLAG rabbit polyclonal antibody through IFA (data not shown) and WB (Supplementary Fig. S1). Subsequently, the reactivity of scFv-P19 against each variant was examined using IFA (Fig. 2A) and WB (Supplementary Fig. S1A). IFA showed that scFv-P19 reacted positively with P1, P2, and all C-terminal-truncated RABV-P variants (Fig. 2A).

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FIG. 1.

Schematic representation of the full-length and truncated RABV-P used in this study. Black bars represent full-length and truncated RABV-P with respective C-terminal amino acid positions. Gray circles indicate FLAG/Strept tags fused with proteins. The binding of each RABV-P variant with anti-FLAG antibody and scFv-P19 are shown as reactive (+) or nonreactive (–). RABV-P, rabies lyssavirus phosphoprotein; scFv, single-chain variable fragment.

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FIG. 2.

The detection of full-length and truncated RABV-P using IFA. HEK293T cells transiently expressing full-length/truncated RABV-P- and mock-transfected HEK293T cells subjected to IFA for epitope mapping of scFv-P19. (A) To screen the whole RABV-P, HEK293T cells independently transfected with plasmids encoding full-length RABV-P (P1)/truncated RABV-P (P2–P5 and ΔC20–ΔC80), and mock-transfected HEK293T cells (MT) were stained with scFv-P19 as the primary antibody, anti-myc mouse MoAb as the secondary antibody, and FITC-conjugated goat anti-mouse IgG (H+L) as the tertiary antibody. (B) To narrow down the range of the scFv-P19 epitope, between the start codons of P2 and P3, HEK293T cells independently transfected with plasmids encoding full-length RABV-P (P1)/truncated RABV-P (ΔN26, 33, 40, and 46) and mock-transfected HEK293T cells (MT) were stained likewise. RABV-P stained with scFv-P19, anti-myc mouse MoAb and FITC-conjugated goat anti-mouse IgG (H+L) yielded green fluorescence. Cells were counterstained in red with 0.002% Evans blue. Magnification 100 × . IFA, immunofluorescence analysis.

WB also demonstrated that scFv-P19 recognized P1, P2, and all C-terminal-truncated RABV-P variants (Supplementary Fig. S1A), indicating that the scFv-P19 epitope was located between P2 and P3 initiation codons (amino acids [aa] 19–54). To narrow down the range of the scFv-P19 epitope, four additional N-terminal-truncated RABV-P variants, ΔN26, ΔN33, ΔN40, and ΔN46, flanked with FLAG-/Strept-tag (Fig. 1), were transiently expressed in HEK293T cells and analyzed similarly. IFA (Fig. 2B) and WB (Supplementary Fig. S1B) showed that scFv-P19 reacted with ΔN26, ΔN33, ΔN40, and ΔN46, suggesting that the epitope of scFv-P19 exists in the region, including the aa residues 47–52.

To exert multiple functions of RABV-P, five isoforms (P1–P5) interact with various viral and cellular proteins in infected cells. The function of each isoform is associated with the position of the binding domain with other viral/cellular proteins and its intracellular localization.⁽⁷⁾ P1, which contains an L-binding domain, N-terminal residues 1–19, acts as an essential cofactor for RABV-L to complete RABV genome transcription and replication.⁽⁴⁾ The shorter isoforms (P2–P5) lacking the L-binding domain are involved in other functions, including antagonism of the host cell IFN-dependent immune response.⁽⁵⁾

P1 and P2 are localized primarily in the cytoplasm, whereas P3–P5 are expressed in the nucleus and cytoplasm. Nucleocytoplasmic localization is dominated by the interaction of distinct nuclear trafficking signals,⁽⁷⁾ nuclear localization sequence/nuclear export sequences (NES) within each isoform, and cellular proteins involved in nuclear import/export. The cytoplasmic localization of P1 and P2 is thought to be based on the N-terminal NES located at aa residues 49–58, which is found only in P1 and P2. A recent study revealed experimental evidence that RABV-P isoforms play a critical role in rabies pathogenesis.⁽⁸⁾

In this study, we revealed that the scFv-P19 epitope partially overlapped with the N-terminal NES. This is consistent with our previous IFA finding, which revealed that scFv-P19 is colocalized with RABV-P in the cytoplasm.⁽¹⁾ Moreover, in this study we observed that aa residues 47–52, and the adjacent N-terminal NES, were relatively well conserved in RABV-P, which is the least conserved among all RABV proteins (Supplementary Fig. S2).

Our results also suggest that scFv-P19 might affect the nuclear trafficking of isoforms P1 and P2 leading to RABV inhibition. To explore this possibility, a detailed investigation of the intracellular localization of RABV-P isoforms with/without of scFv-P19, as well as quantitative analysis of IFN signaling, are required. Considering that intrabodies flanked with signaling sequences (i.e., ER retention signal) have previously been used to alter the intra- and extracellular localization of target proteins,⁽⁹⁾ scFv-P19 with certain modifications might provide potential therapeutic applications in future studies.

In addition, scFv-P19 can be an effective tool for investigating the nuclear trafficking of RABV-P. Considering that recent studies^(10,11) suggest that RABV-P may interact with more cellular proteins than previously reported, some of these interactions might be disrupted by scFv-P19 in future studies.

In conclusion, scFv-P19 targets aa 47–52 at the N-terminus, which partially overlaps with the N-terminal NES of RABV-P isoforms, P1 and P2. Thus, scFv-P19 may serve as an effective tool not only for scFv-based therapeutic studies against RABV, but also for investigating the roles of RABV-P isoforms in rabies pathogenesis.