Protein expression by recombinant viruses.

<p><b>A.</b> Western blot analysis of proteins synthesised in BHK-21 cells infected with rMP12 (M), rMP12:S-Swap (S), rMP12ΔNSs:eGFP (MΔ) or rMP12:S-SwapΔNSs:eGFP (SΔ) at MOI of 1. Cell lysates, prepared at the indicated h p.i., were fractionated by SDS-PAGE, and after transfer, membranes were reacted with rabbit antibodies specific for N, NSs, Gn or eGFP as indicated. Anti-tubulin antibodies were used as a loading control. <b>B.</b> eGFP fluorescence in BHK-21 infected with rMP12ΔNSs:eGFP or rMP12:S-SwapΔNSs:eGFP as above. <b>C.</b> Western blot analysis of infected mosquito cells. <i>A.albopictus</i> C6/36, U4.4 or <i>A. aegypti</i> Ae cells were infected with recombinant viruses (MOI of 1) and lysates prepared at different times post infection. Fractionated proteins were probed with the indicated antibodies. <b>D.</b> eGFP fluorescence in mosquito cells infected with rMP12ΔNSs:eGFP or rMP12:S-SwapΔNSs:eGFP as above. Note that eGFP fluorescence in parts B and D was recorded first and then the same cells were harvested for the western blotting.</p

Summary of qRT-PCR analysis.

a<p>, significantly different, p<0.05 (see Methods for details).</p>b<p>, not significantly different,</p

Growth properties of recombinant viruses.

<p><b>A.</b> Viral growth curves in BHK-21 and A549 cells infected with rMP12 or rMP12:S-Swap (MOI of 0.01 or 5 as indicated). <b>B.</b> The effect of multiplicity of infection on viral yield in BHK-21 cells. Cells were infected with rMP12 or rMP12:S-Swap at multiplicities from 0.0005 to 5 PFU/cell. Viral supernatants were harvested at 72 h p.i. and titrated by plaque assay. Graphs are presented for one representative experiment. <b>C.</b> Viral growth curves in mosquito cells. <i>A. albopictus</i> C6/36 and U4.4, and <i>A. aegypti</i> Ae, cells were infected with rMP12, rMP12:S-Swap, rMP12ΔNSs:eGFP or rMP12:S-SwapΔNSs:eGFP at MOI of 1; BHK-21 cells were similarly infected as a control.</p

Creation of rMP12:S-Swap.

<p><b>A.</b> Schematic of the transcription and replication products of the S segments of rMP12 and rMP12:S-Swap. The sites at which oligonucleotides 1 and 2 anneal are indicated. <b>B.</b> Agarose gel showing RT-PCR products to confirm structure of S segment. BHK-21 cells were infected with rMP12 (MP12) or rMP12:S-Swap (SWAP) viruses at an MOI of 1. Total cellular RNA was extracted at 48 h p.i., and S-segment RT-PCR was performed. As a control, PCR on the appropriate cDNA-containing plasmids was performed with the same primers. <b>C.</b> Titres of recombinant virus stocks from multiple independent preparations were determined by plaque assay in BHK-21 cells. The mean titre and standard error of n = 4 preparations of each recombinant virus stock are shown (* p>0.05) <b>D.</b> Comparison of plaque sizes of rMP12, rMP12:S-Swap, rMP12ΔNSs:eGFP or rMP12:S-SwapΔNSs:eGFP on BHK-21 cells. Cell monolayers were fixed at 96 h p.i. with 4% paraformaldehyde and stained with Giemsa solution.</p

Oligonucleotides used in reverse transcription and qRT-PCR reactions.

a<p>From Rift Valley fever virus strain MP-12 segment S, DQ380154.</p>b<p>From Rift Valley fever virus strain MP-12 segment M, DQ380208.</p><p>Tagged oligonucleotides were used for strand specific reverse transcription. The TAG sequence is underlined.</p

Serial passage of mosquito cells infected with rMP12 or rMP12:S-Swap.

<p><i>A. albopictus</i> C6/36, U4.4 cells or <i>A. aegypti</i> Ae were infected with rMP12 or rMP12:S-Swap at a MOI of 0.01. Cell monolayers were passaged (split ratio 1∶5) every 5–7 days (when 100% confluency was observed). Cell extracts were prepared from each passage, proteins fractionated SDS-PAGE, transferred to a membrane, and blots probed with anti-N, anti-NSs and anti-tubulin antibodies as indicated. C3/36 cells infected with rMP12:S-Swap died after passage 3.</p

Chimera 3D Model of RVFV N protein monomeric structure with mutated residues highlighted (PDB: 3LYF) [18].

<p><b>(A)</b> RVFV N protein with highlighted point mutations (top image) and corresponding surface view (lower image). <b>(B)</b> Alternative orientation of the RVFV N protein point mutations at a 90-degree offset to (A). <b>(C)</b> N protein with highlighted N-terminal arm position 1–14 in red, 15–31 in blue. <b>(D)</b> Mutant RVFV N protein with the full delN1-31 arm removed and surface view.</p

Activity of RVFV N protein mutants in a VLP assay.

<p>BSR-T7/5 CL21 cells were transfected with pTM1-L, pTVT7-GM:hRen, pTM1-M, pTM1-FF-Luc and mutant or WT pTM1-N as described. Negative control cells were transfected with full complement of plasmids without pTM1-N (Con). After 48 hours, the supernatant was harvested and Benzonase treated. The treated supernatant was applied to BSR-T7/5 CL21 cells transfected 24 hours prior with pTM1-N and pTM1-L. <i>Rluc</i> activity was measured at 24 h post-infection. Experiments were carried out in triplicate and repeated three times.</p

Summary of known RVFV N protein functions.

<p>Compiled predicted and known functions of RVFV N protein from studies focused on uncovering the RNA binding properties of RVFV N. Functional information was determined through varied methods, including analysis of RVFV N crystal structure and mutagenesis studies [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006155#pntd.0006155.ref010" target="_blank">10</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006155#pntd.0006155.ref018" target="_blank">18</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006155#pntd.0006155.ref019" target="_blank">19</a>].</p

Effect of N protein mutations on RVFV-derived minigenome activity.

<p>BSR-T7/5 cells were transfected with pTM1-N (wildtype [WT] N, mutant N) or empty plasmid as negative control (Con) expressing-plasmids, pTM1-L, pTVT7-GM:hRen as well as pTM1-FF-Luc as a transfection control. <b>(A)</b> Values of triplicate experiments presented were calculated by dividing <i>Rluc</i> activity by <i>FFluc</i> luciferase activity to normalise differences in transfection efficiency; * denotes p<0.05, ** for p<0.001 using Student’s T-test. <b>(B)</b> Western blot of cell lysate probed with RVFV anti-N antibody (top panel) and anti-actin (bottom panel) as loading control.</p