RNA binding specificity of Ebola virus transcription factor VP30

<p>The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3′-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3′-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125–80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3′-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2′-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the <i>Paramyxoviridae</i> which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.</p

Geometric mean titres, seropositivity rates, and proportions of seroresponders to rVSVΔG-ZEBOV-GP measured by ZEBOV PsVNA50 in adults.

<p>Geometric mean titres, seropositivity rates, and proportions of seroresponders to rVSVΔG-ZEBOV-GP measured by ZEBOV PsVNA50 in adults.</p

Viral load in saliva for children and adolescents.

<p>rVSV<b>Δ</b>G-ZEBOV-GP (rVSV) RNA copy numbers in saliva presented as log10 rVSV RNA copies/ml from day 2 and 7 (d2 and d7) post-injection in adolescents and children vaccinated with 2 × 10⁷ PFU. The broken line denotes the limit of quantitation, and the dotted line denotes the limit of detection. About 67% (12/18) and 30% (6/20) adolescents and children, respectively, had samples above the limit of quantification at day 7. *P < 0.05; **P < 0.01.PFU, plaque-forming units.</p

Description of viraemia by dose and age.

<p>Description of viraemia by dose and age.</p

Geometric mean titres of ZEBOV antibodies in adults by baseline antibody status measured by whole-virion ELISA.

<p>Geometric mean titres of ZEBOV antibodies in adults by baseline antibody status measured by whole-virion ELISA.</p

Participant flow diagram.

<p>Randomisation and flow of participants over a period of 6 months for adults (cohorts 1 to 5), adolescents (cohort 6; 13–17 years), and children (cohort 7; 6–12 years). Similar dose groups are matched with shading (light grey, 3 × 10⁶ PFU; dark grey, 2 × 10⁷ PFU). GP, glycoprotein; ELISA, enzyme-linked immunosorbent assay; PFU, plaque-forming units; rVSV, recombinant vesicular stomatitis virus.</p

Baseline characteristics of study participants.

<p>Baseline characteristics of study participants.</p

Endpoint geometric mean titres, seropositivity rates, and proportions of seroresponders to rVSVΔG-ZEBOV-GP measured by ZEBOV-GP ELISA in adults.

<p>Endpoint geometric mean titres, seropositivity rates, and proportions of seroresponders to rVSVΔG-ZEBOV-GP measured by ZEBOV-GP ELISA in adults.</p

Reactogenicity to rVSVΔG-ZEBOV-GP vaccine until day 28 post-vaccination.

<p>Reactogenicity to rVSVΔG-ZEBOV-GP vaccine until day 28 post-vaccination.</p

Antibody responses to whole-virion ELISA (AEU/ml) by age group: Comparison of geometric mean concentration of IgG antibodies for children, adolescents, and adults vaccinated with the 2 × 10⁷ PFU dose at day 0, 28, and 56.

<p>P < 0.05 indicates a statistical difference in antibody concentrations between age groups at the measured time points. AEU, arbitrary enzyme-linked immunosorbent assay units; ELISA, enzyme-linked immunosorbent assay; PFU, plaque-forming units; ZEBOV, Zaire Ebola virus.</p