Ebolavirus species-specific interferon antagonism mediated by VP24

Members of the Ebolavirus genus demonstrate a marked differences in pathogenicity in humans with Ebola (EBOV) being the most pathogenic, Bundibugyo (BDBV) less pathogenic, and Reston (RESTV) is not known to cause a disease in humans. The VP24 protein encoded by members of the Ebolavirus genus blocks type I interferon (IFN-I) signaling through interaction with host karyopherin alpha nuclear transporters, potentially contributing to virulence. Previously, we demonstrated that BDBV VP24 (bVP24) binds with lower affinities to karyopherin alpha proteins relative to EBOV VP24 (eVP24), and this correlated with a reduced inhibition in IFN-I signaling. We hypothesized that modification of eVP24-karyopherin alpha interface to make it similar to bVP24 would attenuate the ability to antagonize IFN-I response. We generated a panel of recombinant EBOVs containing single or combinations of point mutations in the eVP24-karyopherin alpha interface. Most of the viruses appeared to be attenuated in both IFN-I-competent 769-P and IFN-I-deficient Vero-E6 cells in the presence of IFNs. However, the R140A mutant grew at reduced levels even in the absence of IFNs in both cell lines, as well as in U3A STAT1 knockout cells. Both the R140A mutation and its combination with the N135A mutation greatly reduced the amounts of viral genomic RNA and mRNA suggesting that these mutations attenuate the virus in an IFN-I-independent attenuation. Additionally, we found that unlike eVP24, bVP24 does not inhibit interferon lambda 1 (IFN-λ1), interferon beta (IFN-β), and ISG15, which potentially explains the lower pathogenicity of BDBV relative to EBOV. Thus, the VP24 residues binding karyopherin alpha attenuates the virus by IFN-I-dependent and independent mechanisms

Role of protein phosphatase 1 in dephosphorylation of Ebola virus VP30 protein and its targeting for the inhibition of viral transcription

The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional but not replication activity of EBOV polymerase; to support transcription, VP30mustbein a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation because expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03, that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome and completely suppressed replication of EBOV in cultured cells. Finally, mutations of Thr143 and Thr146 of VP30 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29-46, suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for the development of antivirals against EBOV and other filovirus species

Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

<div><p>Rift Valley fever virus (RVFV; genus <i>Phlebovirus</i>, family <i>Bunyaviridae</i>) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever.</p> </div

rMP12-PTNSs and rMP12-SFSNSs inhibit IFN-β mRNA synthesis.

<p>MEF cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs or rMP12-SFSNSs at a m.o.i of 3. Total RNA was harvested at 7 hpi. Northern blotting was performed with strand-specific RNA probes to detect mouse IFN-β or ISG56 mRNA, or RVFV anti-sense S-segment/N mRNA, respectively. The 18S rRNA was shown as loading control. Representative data from three independent experiments are shown.</p

Replication of rMP12-PTNSs and rMP12-SFSNSs in cell culture.

<p>(A) VeroE6 cells, (B) MEF cells or (C) MRC-5 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs, or rMP12-SFSNSs at a m.o.i of 0.01. Culture supernatants were collected at 72 hpi (A and B), or indicated time points (C) and virus titer was determined by plaque assay with VeroE6 cells. Means+standard deviations of three independent experiments are shown in the graph. Asterisk represents statistical significance (Unpaired t-test, **p<0.01, vs. MP-12).</p

Efficacy and immunogenicity of rMP12-PTNSs or rMP12-SFSNSs in mice.

<p>Five-week-old CD1 mice were mock-vaccinated with PBS (n = 10) or vaccinated subcutaneously with 1×10⁵ pfu of MP-12 (n = 20), rMP12-NSsR173A (n = 10), rMP12-PTNSs (n = 9) or rMP12-SFSNSs (n = 10). Sera were collected at 42 days post vaccination, and mice were challenged with 1×10³ pfu of wt RVFV ZH501 strain (i.p) at 45 days post vaccination. Mice were observed for 21 days post-challenge. (A) Kaplan-Meier survival curves of vaccinated mice after wt RVFV challenge. (B) Neutralizing antibody titers of vaccinated mice (PRNT₈₀). Asterisk represents statistical significance (Mann-Whitney U-test, *p<0.05, **p<0.01 vs. rMP12-NSsR173A). (C) Anti-N IgG titer measured by IgG ELISA. Y-axis shows endpoint titers of sera. Asterisk represents statistical significance (Mann-Whitney U-test, *p<0.05, **p<0.01 vs. rMP12-NSsR173A). (D) Anti-NSs IgG level measured by IgG ELISA. Y-axis shows OD405 nm of sera at 1∶100 dilutions, and cut-off at 0.204 is shown as dotted line.</p

Host general transcriptional suppression by RVFV MP-12 mutants.

<p>(A) Flow cytometry analysis was performed in 293 cells. 293 cells were mock-infected or infected with MP-12, rMP12-C13type, rMP12-PTNSs or rMP12-SFSNSs at a m.o.i of 3 and treated with 0.5 mM EU at 8 hpi for 3 hours. Mock-infected cells were co-treated with ActD (5 µg/ml) at 8 hpi for 3 hours. Incorporated EU was stained with Alexa Fluor 647-azide, and RVFV antigens were stained with anti-RVFV antibodies and detected by Alexa Fluor 488 anti-mouse IgG. Subsequently, cells were analyzed by flow cytometry. Representative data from two independent experiments are shown. X-axis: signal intensity for RVFV antigen, Y-axis: signal intensity for EU. (B) Relative fluorescence intensity of EU-positive cells is shown as a histogram.</p