Schematic representation of BTV NS3 and putative amino acid sequences of mutant viruses.

<p>(A) Schematic representation of BTV NS3. Numbers indicate amino acid position according to the NS3 amino acid sequence The two cytoplasmic domains (IC), the two transmembrane domains (TM; yellow), the extracellular domain (EC; orange), the calpactin S100A10/p11 binding site (blue), the late domain motifs (LD; green), the VP2 binding domain (VP2 BD: red), and the conserved N-linked glycosylation site at position 150 (NLG) are indicated. (B) NS3 is an integral membrane protein that interacts to cellular release factors calpactin S100A10/p11 and Tsg101, whereas the C-terminal domain binds VP2 on the outside of the virus particle. (C) The putative amino acid sequence in the regions of the <i>Sty</i>I, <i>Bsi</i>WI sites and StyI-filled or BsiWI-filled are shown in single letter code. Changed amino acids by a 4-basepairs insertion are underlined. (D) Single and double AUG→GCC mutations are shown. Dots and dashes indicate no change and absence of expression, respectively.</p

Replication kinetics of AUG mutant viruses on mammalian and insect cells.

<p>Virus growth and virus release of AUG mutant viruses grown on BSR cells and KC cells infected in duplicate with wtBTV1/8 (S10), mutAUG1, mutAUG2 and mutAUG1+2 viruses and harvested at indicated time points post infection. Virus titers were determined in supernatant and in cells by endpoint dilution, and the mean values are expressed as ¹⁰logTCID₅₀/ml. (A) released virus from BSR cells (upper left panel) or (B) BSR cell-associated virus (upper right panel). (C) released virus from KC cells (lower left panel) or (D) KC cell-associated virus (lower right panel).</p

Sequence analysis and plaque phenotype of AUG mutant viruses.

<p>(A) Sequence analysis of Seg-10 amplicons from AUG mutant viruses shown in DNASTAR Lasergene Seqman assembly software. The mutated codons are indicated in rectangles (B) Plaque morphology of BSR cells infected with wtBTV1/8(S10), mutAUG1, mutAUG2 and mutAUG1+2 virus grown under 1% methylcellulose overlay medium are shown. At 2 dpi cells were fixed and immunostained with anti-VP7 Mab.</p

Detection of viral proteins of AUG mutant viruses.

<p>Westernblot analysis of expression of NS3 and VP5 in cell lysates from BSR cells infected with wtBTV1/8(S10) (lane 1), mutAUG1 (lane 2), mutAUG2 (lane 3), mutAUG1+2 (lane 4) and mock-infected cells (lane 5) using antibodies against BTV NS3 or VP5.</p

Analysis of revertant viruses Sty-rev and BsiWI-rev.

<p>(A) anti-VP7 immunostained cells after 2 or 13 days post transfection with BTV1 Seg-1-9 and mutated Seg-10 StyI-filled resulting in revertant virus StyI-rev1 showing CPE at 13 dpt. (B) sequence analysis of Seg-10 amplicons from StyI-rev1 passages at different time points after transfection. In single letter code, putative translation of all three frames is shown of which the middle is the ORF of NS3 with the insertion of Alanine (A) in StyI-rev1. Here the analysis is shown by use of a sequence primer located downstream of the <i>Sty</i>I site. Note the mixed sequence at 8 dpt upstream of the filled site due the introduction of the point deletion at the <i>Sty</i>I site in a subpopulation of the fragments. (C) Comparison of the amino acid sequences of NS3 of wtBTV1/8(S10), the 4-basepairs insertions, and the rescued revertant viruses Sty-rev1, 2 and BsiWI-rev1. Amino acids changes in the regions of the 4-basepairs insertion of the revertant viruses Sty-rev1, 2 and BsiWI-rev1 are in bold.</p

Stability of Seg-10 mutant viruses.

<p>(A) Stability of all Seg-10 deletion mutants was examined during three successive passages. Complete Seg-10 was amplified by RT-PCR, and Seg-10 stability was examined by gel electrophoresis. wtBTV1 was used as control. (B) Stability of Seg-10 of mutant virus ΔD(S2del) was confirmed for more than ten passages, by complete Seg-10 amplification using RT-PCR, gel electrophoresis and sequencing. (C) Stability of variants of Seg-10 mutant viruses with Seg-2 insertion during three successive passages. Seg-10 of ΔD(S2) and ΔD(S2reposition) were stable during three passages, whereas Seg-10 of ΔD(S2inverted) was not. Amplicons of the original Seg-10 mutant and Seg-10 mutant with additional inserted viral sequences are indicated by a dot and asterisks, respectively.</p

dsRNA of Seg-10 deletion mutant viruses.

<p>dsRNA was isolated from cells infected with passage 4 of all Seg-10 deletion mutant viruses. Black dots indicate the segments 1–10 of BTV1, with Seg-5 and Seg-6 almost at the same position in the gel. A black dot also indicates the band with the expected size of Seg-10 based on the deletion, for the different mutant viruses. White dots indicate Seg-10 bands of mutant viruses, different from deletion Seg-10 of the expected size. All mutant viruses contain a band with the size of the original deletion Seg-10. All Seg-10 deletion mutant viruses contain Seg-10 variants, except for ΔB and ΔH. Note that the ladder used is made of dsDNA, so the height in the gel of the dsRNA cannot be used to determine the exact size of the band.</p

Overview of Seg-10 deletion mutants with insertions.

<p>Stability of Seg-10 deletion mutants during virus growth is indicated. For unstable mutants, changes in Seg-10 are indicated and specified for segment number of origin and nucleotide numbering (between brackets) of the respective segment. The location of the insertion is indicated by the nucleotide number of full length Seg-10.</p><p>* BTV mutant with the ΔA deletion in Seg-10 was not viable.</p

Phenotype and growth of wild type, AUG1+2 and ΔD(S2)del virus on BSR cells.

<p>(A) BSR cells, 1dpi, infected with MOI 0.1. CPE is clearly visible in BSR cells infected with BTV1. Upper row: Typical BTV1 CPE is indicated (arrows). Cells infected with the double ATG mutant (AUG1+2) also show CPE, but delayed. The ΔD(S2del) virus shows no CPE and infected cells look comparable to uninfected cells. Lower row: Infected monolayers were immunostained with αVP7 MAb. For BTV1 all cells are positive, AUG1+2 shows less positive cells and ΔD(S2del) only shows immunostaining of single cells or small groups of cells. (B) Virus titers of infected cells were examined in medium and cell fractions at time points up to 54 hpi. Virus titers in cell fractions are not significantly different for both viruses, except for 22 hpi. However, virus release in medium is significantly delayed and reduced for ΔD(S2del) virus compared to BTV1. Error bars represent SEM and asterisks indicate a significant difference in virus titer between ΔD(S2del) virus compared to BTV1 with p<0.05.</p

Stability of ΔD(S2delGFP) mutant virus.

<p>(A) ΔD(S2del) virus with the GFP sequence inserted (ΔD(S2delGFP)) was generated. GFP expression was obvious during several successive virus passages in BSR cells, as shown for passage 6 and 7 (p6, p7). GFP expression was less obvious after subsequent passages, as shown for passages 8 and 9 (p8, p9). (B) Genetic stability of Seg-10 of ΔD(S2delGPF) during ten passages was studied by RT-PCR amplification of Seg-10. The original Seg-10 of ΔD(S2delGFP) mutant virus was identified (.), but in subsequent passages additional smaller amplicons became more prominent (*). The middle small band has a deletion in the GFP sequence, the smallest amplicon has a larger deletion in the GFP sequence, and in the largest of the small amplicons, the Seg-2 insertion is also deleted, but a Seg-6 sequence is inserted instead.</p