27 research outputs found
Functional Analysis of Rift Valley Fever Virus NSs Encoding a Partial Truncation
<div><p>Rift Valley fever virus (RVFV), belongs to genus <em>Phlebovirus</em> of the family <em>Bunyaviridae</em>, causes high rates of abortion and fetal malformation in infected ruminants as well as causing neurological disorders, blindness, or lethal hemorrhagic fever in humans. RVFV is classified as a category A priority pathogen and a select agent in the U.S., and currently there are no therapeutics available for RVF patients. NSs protein, a major virulence factor of RVFV, inhibits host transcription including interferon (IFN)-Ξ² mRNA synthesis and promotes degradation of dsRNA-dependent protein kinase (PKR). NSs self-associates at the C-terminus 17 aa., while NSs at aa.210β230 binds to Sin3A-associated protein (SAP30) to inhibit the activation of IFN-Ξ² promoter. Thus, we hypothesize that NSs function(s) can be abolished by truncation of specific domains, and co-expression of nonfunctional NSs with intact NSs will result in the attenuation of NSs function by dominant-negative effect. Unexpectedly, we found that RVFV NSs truncated at aa. 6β30, 31β55, 56β80, 81β105, 106β130, 131β155, 156β180, 181β205, 206β230, 231β248 or 249β265 lack functions of IFNβΞ² mRNA synthesis inhibition and degradation of PKR. Truncated NSs were less stable in infected cells, while nuclear localization was inhibited in NSs lacking either of aa.81β105, 106β130, 131β155, 156β180, 181β205, 206β230 or 231β248. Furthermore, none of truncated NSs had exhibited significant dominant-negative functions for NSs-mediated IFN-Ξ² suppression or PKR degradation upon co-expression in cells infected with RVFV. We also found that any of truncated NSs except for intact NSs does not interact with RVFV NSs even in the presence of intact C-terminus self-association domain. Our results suggest that conformational integrity of NSs is important for the stability, cellular localization and biological functions of RVFV NSs, and the co-expression of truncated NSs does not exhibit dominant-negative phenotype.</p> </div
Characterization of NSs encoding alanine substitutions at the C-terminus.
<p>(A) Schematics of NSs mutations. Acidic residues, Glu (E) at aa.263β255 and Asp (D) at aa.257β259, were substituted with alanine, and recombinant MP-12 encoding NSs mutations were recovered (NSs-E253-255A/D257β259, NSs-D257-259A and NSs-E253-255A). Underline indicates the amino acids substituted. (B) VeroE6 cells were mock-infected or infected with indicated viruses at an moi of 3. Cells were collected at 16 hpi, and the abundance of PKR (anti-PKR antibody), NSs and N (anti-RVFV antibody), and Ξ²-actin (anti-actin antibody) were analyzed by Western blot. (B) A549 cells were mock-infected or infected with indicated viruses at an moi of 3. Total RNA was extracted at 7 hpi, and IFN-Ξ² mRNA, ISG56 mRNA and RVFV anti-viral-sense S-RNA/N mRNA were detected by Northern blot with specific RNA probe. Representative data from at least 3 independent experiments are shown.</p
Co-expression of truncated NSs in RVFV-infected cells.
<p>293 cells were mock-infected or infected with rMP12-NSs-Flag at an moi of 3, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT (control) or NSs with indicated truncations. Cells were collected at 16 hpi, and PKR (anti-PKR antibody), NSs (anti-RVFV antibody), NSs-Flag (anti-Flag antibody), CAT-myc (anti-myc antibody) and Ξ²-actin (anti-actin antibody) were detected by Western blot. (B) A549 cells were mock-infected or infected with rMP12-NSs-Flag at an moi of 3, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT or NSs with indicated truncations. Total RNA was extracted at 7 hpi, and IFN-Ξ² mRNA, ISG56 mRNA and RVFV anti-viral-sense S-RNA/N mRNA were detected by Northern blot with specific RNA probe. Representative data from at least 3 independent experiments are shown.</p
Association of MP-12 NSs and truncated NSs.
<p>293 cells were mock-infected or infected with rMP12-NSs-SF at an moi of 3, and cells were immediately transfected with in vitro synthesized RNA encoding MP-12 NSs, CAT, or truncated NSs. After incubation for 6 hours, newly synthesized proteins were then labeled with [<sup>35</sup>S] methionine/cycteine. Using cell lysates, SF-tagged proteins were precipitated with Strep-Tactin beads. Then, co-precipitated proteins were analyzed by separating on 10% SDS-PAGE gel and followed by autoradiography. Part of lysates before precipitation were used for Western blot using anti-NSs antibody (input control).</p
Functions of truncated NSs (A) VeroE6 cells were mock-infected or infected with indicated viruses at an moi of 3.
<p>Cells were collected at 16 hpi, and the abundance of PKR (anti-PKR antibody), NSs and N (anti-RVFV antibody) and Ξ²-actin (anti-actin antibody) were analyzed by Western blot. (B) A549 cells were mock-infected or infected with indicated viruses at an moi of 3. Total RNA was extracted at 7 hpi, and IFN-Ξ² mRNA, ISG56 mRNA and RVFV anti-viral-sense S-RNA/N mRNA were detected by Northern blot with specific RNA probe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045730#pone.0045730-Ikegami6" target="_blank">[66]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045730#pone.0045730-Narayanan1" target="_blank">[67]</a>. Representative data from at least 3 independent experiments are shown.</p
Co-expression of truncated NSs in RVFV-infected cells.
<p>293 cells were mock-infected or infected with rMP12-NSs-Flag at an moi of 3, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT (control) or NSs with indicated NSs mutants. Cells were collected at 16 hpi, and NSs-Flag/NSs (a-RVFV antibody), NSs-Flag (a-Flag antibody), PKR (anti-PKR antibody), CAT-myc (anti-myc antibody) and Ξ²-actin (anti-actin antibody) were detected by Western blot. (B) A549 cells were mock-infected or infected with rMP12-NSs-Flag at an moi of 3, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT or NSs with indicated NSs mutants. As a control to induce IFN-Ξ² and ISG56 mRNA synthesis, A549 cells were infected with rMP12-C13type (C13type) at an moi of 3. Total RNA was extracted at 7 hpi, and IFN-Ξ² mRNA, ISG56 mRNA and RVFV anti-viral-sense S-RNA/N mRNA were detected by Northern blot with specific RNA probe. Representative data from at least 3 independent experiments are shown. (C) A549 cells were infected with MP-12 at moi of 0.01, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT or NSs of NSΞ249β265 or NSs-E253-255A. Culture supernatants were harvested at 72 hpi, and virus titers were measured by plaque assay. P-values of unpaired Student's t-test are shown (*; p<0.05, ns; not significant).</p
Schematics of truncated NSs design.
<p>MP-12 NSs (top) encodes SAP30-binding domain (aa.210β230)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045730#pone.0045730-LeMay2" target="_blank">[26]</a> and C-terminus self-association domain (aa.249β265)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045730#pone.0045730-Yadani1" target="_blank">[65]</a>. The rMP12-C13type (C13type) encodes an in-frame truncation of aa.16β198, and the N-terminus and C-terminus fragments are linked with Asp (D) and Val (V). Other 11 NSs mutants encode in-frame 25 or 17 amino acid truncations.</p
NSs expression of MP-12 and NSΞ249
<p>β<b>265.</b> 293 cells were mock-transfected (mock) or transfected with in vitro transcribed RNA encoding CAT (control) or NSs of MP-12 or NSΞ249β265. At 16 hpi, cells were fixed with methanol for 5 min, and stained with anti-RVFV antibody (1:500) at 37<sup>Β°</sup>C for 1 hr and Alexa Fluor 594, goat anti-mouse IgG (H+L) at 37<sup>Β°</sup>C for 1 hr. After washing with PBS, cells were stained with DAPI, and observed under fluorescent microscope.</p
Risk analysis of inter-species reassortment through a Rift Valley fever phlebovirus MP-12 vaccine strain
<div><p>Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Arabian Peninsula. The causative agent, Rift Valley fever phlebovirus (RVFV), belongs to the genus <i>Phlebovirus</i> in the family <i>Phenuiviridae</i> and causes high rates of abortions in ruminants, and hemorrhagic fever, encephalitis, or blindness in humans. Viral maintenance by mosquito vectors has led to sporadic RVF outbreaks in ruminants and humans in endemic countries, and effective vaccination of animals and humans may minimize the impact of this disease. A live-attenuated MP-12 vaccine strain is one of the best characterized RVFV strains, and was conditionally approved as a veterinary vaccine in the U.S. Live-attenuated RVF vaccines including MP-12 strain may form reassortant strains with other bunyavirus species. This study thus aimed to characterize the occurrence of genetic reassortment between the MP-12 strain and bunyavirus species closely related to RVFV. The Arumowot virus (AMTV) and Gouleako goukovirus (GOLV), are transmitted by mosquitoes in Africa. The results of this study showed that GOLV does not form detectable reassortant strains with the MP-12 strain in co-infected C6/36 cells. The AMTV also did not form any reassortant strains with MP-12 strain in co-infected C6/36 cells, due to the incompatibility among N, L, and Gn/Gc proteins. A lack of reassortant formation could be due to a functional incompatibility of N and L proteins derived from heterologous species, and due to a lack of packaging via heterologous Gn/Gc proteins. The MP-12 strain did, however, randomly exchange L-, M-, and S-segments with a genetic variant strain, rMP12-GM50, in culture cells. The MP-12 strain is thus unlikely to form any reassortant strains with AMTV or GOLV in nature.</p></div
Minigenome assays with expressions of N and L proteins derived from Arumowot virus (AMTV) or Rift Valley fever phlebovirus (RVFV).
<p>BHK/T7-9 cells were transfected with plasmids expressing M-segment minigenome RNA from either RVFV or AMTV, and those expressing N or L proteins derived from either AMTV or RVFV. The RVFV-M-rLuc and AMTV-M-rLuc represent the M-segment minigenomes derived from RVFV and AMTV, respectively. Cell lysates were collected at 72 hpt, and the ratio of <i>Renilla</i> luciferase (rLuc) activities to firefly luciferase (fLuc) activities derived from pT7-IRES-fLuc (control plasmid) was shown as percentage: i.e., the same type of minigenome without N and L expression was set as 100%. Bars represent means plus standard errors. Asterisks on error bars represent statistically significant increases compared to samples expressing minigenome only (One-way ANOVA **p < 0.01).</p