Effect of NSs4KR mutations on known functions of NSs.

<p>(A) Induction of apoptosis in infected cells as measured by DNA fragmentation. A549 cells were mock-infected or infected with wtBUNV, rBUN4KR, or rBUNdelNSs2. Cells were harvested at 12, 24, and 48 hpi and analysed for DNA fragmentation. (B) Regulation of the viral polymerase in a minireplicon assay. Increasing amounts of plasmids (1.6–160 ng) expressing wt NSs or NSs4KR or a nonrelated protein (GFP) were added to a minireplicon assay expressing the viral minigenome (expressing <i>Renilla</i> luciferase), viral polymerase and nucleoprotein. Values shown are from quadruplicate measurements and represent <i>Renilla</i> luciferase activity over firefly luciferase activity, normalised to the control reaction. (C) Generation of defective RNAs. wtBUNV, rBUNdelNSs2, and rBUN4KR viruses were serially passaged at a fixed volume in BHK cells. Virions from the virus stock (P0) and passage 8 (P8; or P7 for rBUNdelNSs2) were pelleted and their extracted RNA was analysed by Northern blotting using a probe to detect genome-sense L RNA. All lanes are from the same exposure of the same Northern blot. Positions of size markers (in kb) are indicated to the left, and the amount of RNA and PFU-equivalents (log(pfu)) loaded are indicated below the blot. (D) Titres of the viruses at each passage.</p

Effect of ubiquitin ligase E3 and E1 inhibitors on NSs degradation.

<p>Cells were infected at 5 PFU/cell and harvested at the indicated times post infection (top of blots) and cell lysates were analysed by immunoblotting using antibodies against the proteins indicated to the right of the panels. (A) Effect of E3 inhibitor. A549 cells were mock-infected or infected with wtBUNV and left untreated (−) or treated with MG132 (10 μM; MG) or MLN4924 (1 µM; MLN) from 5 hpi until harvest. (B) Effect of E1 inhibitor. BHK cells were mock-infected (m) or infected with wtBUNV and left untreated (−) or treated with PYR-41 (50 µM) from 8 hpi.</p

Virulence in mice.

<p>Assessment of wtBUNV and rBUN4KR virulence in mice. Strain 129 mice were infected i.c. with 1000 PFU of wtBUNV or rBUN4KR virus. The mice were monitored twice daily and sacrificed when moribund.</p

Degradation of NSs.

<p>BHK cells were infected at 5 PFU/cell and harvested at the indicated times post infection (top of blots) and cell lysates were analysed by immunoblotting using antibodies against the proteins indicated to the right of the panels (tub  =  tubulin). (A) Effect of proteasome inhibitors. Cells were mock-infected or infected with wtBUNV and either left untreated or treated with MG132 (10 μM) or Epoxomicin (200 nM) from 5 hpi until harvest. (B) Expression of 4KRNSs. Cells were mock-infected or infected with wtBUNV or rBUN4KR. (C) Schematic overview of the NSs proteins of recombinant viruses expressing NSs proteins containing a single lysine at one of the four positions (K1-K4). (D) Expression of NSs containing single lysine residues. Cells were mock-infected or infected with wtBUNV, rBUN4KR, or rBUN-K1, -K2, -K3 and -K4. m =  mock-infected; wt  =  wtBUNV; 4KR  =  rBUN4KR.</p

Biofoundry-Scale DNA Assembly Validation Using Cost-Effective High-Throughput Long-Read Sequencing

Biofoundries are automated high-throughput facilities specializing in the design, construction, and testing of engineered/synthetic DNA constructs (plasmids), often from genetic parts. A critical step of this process is assessing the fidelity of the assembled DNA construct to the desired design. Current methods utilized for this purpose are restriction digest or PCR followed by fragment analysis and sequencing. The Edinburgh Genome Foundry (EGF) has recently established a single-molecule sequencing quality control step using the Oxford Nanopore sequencing technology, along with a companion Nextflow pipeline and a Python package, to perform in-depth analysis and generate a detailed report. Our software enables researchers working with plasmids, including biofoundry scientists, to rapidly analyze and interpret sequencing data. In conclusion, we have created a laboratory and software protocol that validates assembled, cloned, or edited plasmids, using Nanopore long-reads, which can serve as a useful resource for the genetics, synthetic biology, and sequencing communities

PO activity in U4.4 cell-conditioned medium.

<p>(<b>A</b>) PO activity in conditioned medium without immune challenge (Control) or after the addition of <i>E. coli</i> or purified SFV virions. One unit (U) of PO activity was defined as <b>Δ</b>A₄₉₀ = 0.001 after 30 minutes incubation (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002977#s4" target="_blank">Materials and Methods</a>). Each bar represents the mean from 10 reactions; error bars show standard deviation. This experiment was repeated three times with similar results. (<b>B</b>) SFV virion viability after a 1 h incubation at 28°C in unconditioned culture medium or medium conditioned by U4.4 cells for 48 h. Viability was then determined by titration of SFV on BHK-21 cells. PFU: plaque forming units. Each bar represents the mean from triplicate incubations; error bars show standard deviation. This experiment was repeated three times with similar results. (<b>C</b>) Staining for intracellular PO activity in U4.4 cells. Arrow indicates a U4.4 cell that melanised after fixation and incubation with the PO substrate dopamine. Note the larger size of this cell and its rounded morphology relative to surrounding cells that have not melanised.</p

Spread of SFV in mosquito and vertebrate cells.

<p>(<b>A</b>) The addition of glutathione (GSH) to medium enhances the spread of SFV. U4.4 cells were infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F (Egf1.0F) or SFV4(3H)-<i>FFLuc</i>-Egf1.0R (Egf1.0R) at an MOI of 0.005 followed by determination of <i>FFLuc</i> activity at 48 h p.i. + GSH: 0.5 mM GSH; − GSH: negative control. Each bar represents the mean from triplicate cultures; error bars show standard deviation. This experiment was repeated three times with similar results. (<b>B</b>) Egf1.0 has no effect on SFV spread in BHK-21 cells. Cells were infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F (Egf1.0F) or SFV4(3H)-<i>FFLuc</i>-Egf1.0R (Egf1.0R) at an MOI of 0.005 followed by determination of <i>FFLuc</i> activity at 24 h and 48 h p.i. Each bar represents the mean from triplicate cultures; error bars show standard deviation. This experiment was repeated three times with similar results.</p

Expression of Egf1.0 increases mortality of <i>Ae. aegypti</i> and replication of SFV <i>in vivo</i>.

<p>(<b>A</b>) <i>Ae. aegypti</i> were fed blood containing SFV4(3H)-<i>FFLuc</i>-Egf1.0F or SFV4(3H)-<i>FFLuc</i>-Egf1.0R. Uninfected blood meals served as a control. Mosquito mortality was then monitored daily post-bloodmeal. Combined survival data from three independent experiments (cohorts of 22–25 infected mosquitoes per virus or control mosquitoes in each experiment) are shown. Error bars show standard deviation. (<b>B</b>) SFV genome copy number as determined by real time qPCR. Total RNA was extracted 3 days post-bloodmeal from mosquitoes infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F or SFV4(3H)-<i>FFLuc</i>-Egf1.0R. Viral genome RNA levels from 10 mosquitoes for each virus are shown. Values at 0 represent uninfected mosquitoes. Horizontal bar indicates average genome copy number from infected mosquitoes. This experiment was repeated three times with similar results.</p

Recombinant SFV expresses Egf1.0 and inhibits PO activity in U4.4 cell-conditioned medium.

<p>(<b>A</b>) Immunoblots showing Egf1.0 expression and secretion from mosquito cells. U4.4 cells were infected with SFV4(3F)-<i>ZsGreen</i>-Egf1.0F or SFV4(3F)-<i>ZsGreen</i>-Egf1.0R at an MOI of 10 followed by preparation of cell lysate and medium samples at 48 h p.i. as indicated in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002977#s4" target="_blank">Materials and Methods</a>. The left blot was probed with an anti-SFV nsP3 antibody with individual lanes labeled as follows: U4.4 cells infected with SFV4(3F)-<i>ZsGreen</i>-Egf1.0R (R = cell lysate, Rm = conditioned medium), U4.4 cells infected with SFV4(3F)-<i>ZsGreen</i>-Egf1.0F (F = cell lysate, Fm = conditioned medium), or uninfected cells (U = cell lysate, Um = conditioned medium). Black star identifies the nsP3-ZsGreen protein, only detected in lysates from SFV-infected cells. Black diamond indicates bovine serum albumin (non-specifically detected because of high abundance). The right blot shows the same samples probed with an anti-Egf1.0 antibody. A control lane (C) was added to this blot (purified, recombinant Egf1.0). Note that Egf1.0 is only detected in the control lane, and F and Fm lanes. Black arrow indicates uncut Egf1.0; open arrow identifies a band corresponding to the predicted C-terminal domain of Egf1.0 after PAP cleavage. Molecular mass markers indicated to the left. (<b>B</b>) PO activity in conditioned medium from uninfected U4.4 cells (Control), cells infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F (Egf1.0F), SFV4(3H)-<i>FFLuc</i>-Egf1.0R (Egf1.0R), cells infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F with <i>E. coli</i> added to the medium (Egf1.0F+<i>E. coli</i>), cells infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0R with <i>E. coli</i> added to the medium (Egf1.0R+<i>E. coli</i>), or medium from uninfected cells with <i>E. coli</i> added (<i>E. coli</i>). PO activity was measured as outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002977#ppat-1002977-g002" target="_blank">Fig. 2A</a>; 1 ml of conditioned medium was taken at 48 h p.i. from 2.6×10⁵ U4.4 cells infected at an MOI of 10, or uninfected (Control). Each bar represents the mean from 10 reactions; error bars show standard deviation. This experiment was repeated three times with similar results.</p

Viruses used in study.

<p>(<b>A</b>) SFV (prototype strain SFV4). (<b>B</b>) SFV(3H)-<i>FFLuc</i>-Egf1.0F and SFV(3H)-<i>FFLuc</i>-Egf1.0R, encoding Firefly luciferase (<i>FFLuc</i>) as part of the non-structural polyprotein (inserted between duplicated nsP2 cleavage sites at the nsP3/4 junction), and from a duplicated subgenomic promoter the melanisation inhibitor Egf1.0 in sense (F virus; top) or (as negative control) antisense orientation (R virus; bottom). (<b>C</b>) SFV(3F)-<i>ZsGreen</i>-Egf1.0F and SFV(3F)-<i>ZsGreen</i>-Egf1.0R, expressing ZsGreen inserted into the C-terminal region of nsP3, and from a duplicated subgenomic promoter the melanisation inhibitor Egf1.0 in sense (F virus; top) or (as negative control) antisense orientation (R virus; bottom).</p