Comparison of the substrate properties of 20/65 and 20/75 (5′-mismatched) duplexes

<p><b>Copyright information:</b></p><p>Taken from "Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase"</p><p>Nucleic Acids Research 2005;33(7):2259-2268.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083429.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Molecules containing 65mer or 75mer hairpin template strands () were prepared as described in Materials and Methods, and incubated with vaccinia DNA polymerase in the presence of dNTPs

Comparison of the substrate properties of 20/65 and 30/65 (3′-mismatched) duplexes

<p><b>Copyright information:</b></p><p>Taken from "Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase"</p><p>Nucleic Acids Research 2005;33(7):2259-2268.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083429.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Molecules containing 20mer or 30mer primer strands () were prepared as described in Materials and Methods, and incubated with vaccinia DNA polymerase in the presence of dNTPs. The 20mer strand and 30mer strand substrates are indicated. Also indicated are the 27mer, 25mer and 22mer bands that bear a 3′-terminal dCMP (‘C’, upper panel, at right)

Effect of partial sequence homology located on the 5′ side of a 30/75 mismatched substrate

<p><b>Copyright information:</b></p><p>Taken from "Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase"</p><p>Nucleic Acids Research 2005;33(7):2259-2268.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083429.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Molecules containing 75mer hairpin template strands () and either capable (30/75h) or incapable (30/75) of a limited amount of branch migration were incubated with vaccinia DNA polymerase in the presence of dNTPs

Comparison of the substrate properties of 30/65 (3′-mismatched) and 30/75 (3′ and 5′-mismatched) duplexes

<p><b>Copyright information:</b></p><p>Taken from "Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase"</p><p>Nucleic Acids Research 2005;33(7):2259-2268.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083429.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Molecules containing 65mer or 75mer hairpin template strands () were prepared as described in Materials and Methods, and incubated with vaccinia DNA polymerase in the presence of dNTPs

Tracking the appearance of virus-encoded mCherry proteins.

<p>(A) EGFPcro BSC-40 cells were infected at a MOI = 5 with VACV-pE/L-mCherry-cro, and the red and green fluorescence then tracked over time, collecting images 5 minutes apart, across 10 different fields (only a single representative field is shown). These are stills, the complete time-lapse movie is found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005824#ppat.1005824.s006" target="_blank">S3 Video</a>. (B) EGFPcro BSC-40 cells were co-infected at a total MOI = 5 with VACV-pE/L-mCherry(t) and VACV-mCherry-cro viruses, and tracked using live cell microscopy to detect the appearance of recombinant mCherry-cro protein. The panels show different stills taken from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005824#ppat.1005824.s007" target="_blank">S4 Video</a>. Note the delay in the appearance of a mCherry-cro signal compared to panel (A). The scale bar = 25 μm.</p

Recombination between pE/L-mCherry(t) and pE/L-mCherry-lacZ viruses.

<p>(A) The figure shows the two parent viruses, the predicted recombinants, and the <i>Hin</i>dIII fragments that should be detected by the pE/L oligonucleotide probe (blue bar). (B) Southern blot analysis of cells co-infected with the pE/L-mCherry(t) and pE/L-mCherry-lacZ viruses. BSC-40 cells were infected with each of the parental viruses at MOI = 5, or co-infected with the two viruses at a combined MOI = 5, and the DNA was extracted 24 h post-infection. The samples were then Southern blotted using a biotin-labeled probe. Although faint, two bands at 2.2 and 0.9 kbp are seen that indicate the presence of recombinant genomes. Collectively they comprise about 2% of the DNA.</p

Live-Cell Imaging of Vaccinia Virus Recombination

<div><p>Recombination between co-infecting poxviruses provides an important mechanism for generating the genetic diversity that underpins evolution. However, poxviruses replicate in membrane-bound cytoplasmic structures known as factories or virosomes. These are enclosed structures that could impede DNA mixing between co-infecting viruses, and mixing would seem to be essential for this process. We hypothesize that virosome fusion events would be a prerequisite for recombination between co-infecting poxviruses, and this requirement could delay or limit viral recombination. We have engineered vaccinia virus (VACV) to express overlapping portions of mCherry fluorescent protein fused to a cro DNA-binding element. In cells also expressing an EGFP-cro fusion protein, this permits live tracking of virus DNA and genetic recombination using confocal microscopy. Our studies show that different types of recombination events exhibit different timing patterns, depending upon the relative locations of the recombining elements. Recombination between partly duplicated sequences is detected soon after post-replicative genes are expressed, as long as the reporter gene sequences are located <i>in cis</i> within an infecting genome. The same kinetics are also observed when the recombining elements are divided between VACV and transfected DNA. In contrast, recombination is delayed when the recombining sequences are located on different co-infecting viruses, and mature recombinants aren’t detected until well after late gene expression is well established. The delay supports the hypothesis that factories impede inter-viral recombination, but even after factories merge there remain further constraints limiting virus DNA mixing and recombinant gene assembly. This delay could be related to the continued presence of ER-derived membranes within the fused virosomes, membranes that may once have wrapped individual factories.</p></div

Virus design strategy.

<p>(<b>a</b>) Cloned synthetic DNA fragments used to assemble HPXV. Nine different clones were synthesized spanning all but the first and last 40 bp in GenBank entry DQ792504, each overlapping the adjacent fragment by ~1 kbp. All of the <i>Aar</i>I and <i>Bsa</i>I restriction sites were eliminated from fragments 1A to 7, inclusive, using silent mutations and the same strategy was used to add <i>Ava</i>I and <i>Stu</i>I sites in Frag_2. To facilitate virus recovery a gene encoding a YFP-gpt fusion protein was inserted into Frag_3, at the site of the HPXV thymidine kinase locus. An additional HPXV095 fragment spans the thymidine kinase locus and was subsequently used to delete and replace the YFP-gpt marker using homologous recombination. (<b>b</b>) Synthetic hairpin telomeres. Because the HPXV genome was not sequenced to the ends, we substituted two hairpin sequences based upon those reported for VACV strain WR (green coloured nucleotides). These are called “fast” and “slow” forms based upon their electrophoretic properties. The nucleotides coloured in black come from the HPXV genome sequence and provide an element essential for telomere resolution.</p

Size and location of each synthetic DNA fragment within HPXV (Accession #DQ792504).

<p>Size and location of each synthetic DNA fragment within HPXV (Accession #DQ792504).</p

Virulence and vaccine properties of scHPXV.

<p><b>a</b>. Virulence studies. Immune competent BALB/c mice (5 per group) were inoculated intranasally with the indicated viruses. scHPXV was tested using doses ranging from 10⁵−10⁷ pfu per mouse. Some weight loss due to illness was seen in animals inoculated with 5×10³ pfu VACV strain WR (¢), or 1×10⁷ pfu of VACV strain DPP15 (<b>Δ</b>), but no illness was detected in any of the animals infected with HPXV clones. <b>b</b>. VACV challenge studies. Four weeks after exposure to the indicated agent as shown in panel <b>a</b> (or mock treatment with buffered saline), the mice were exposed to a lethal dose of VACV strain WR (1×10⁶ pfu) by the same route (day 0 in <b>b</b> is day 28 in graph <b>a</b>). The animals were monitored for signs of disease and euthanized if the weight loss exceeded 25% of the initial body weight. The scHPXV strain provided good protection at the two highest pre-challenge doses (1×10⁶ and 1×10⁷ pfu). <b>c</b>. Disease course in HPXV vaccinated mice. The mice were inspected to detect four signs of disease (ruffled fur, hunched posture, difficulty breathing, and reduced mobility) and a clinical score calculated from the sum of the individual scores averaged across all five (or surviving) mice per cohort. Little or no signs of illness were detected in mice first vaccinated with 1×10⁶ or 1×10⁷ pfu scHPXV <b>d</b>. Kaplan-Meyer analysis of survivorship. All of the mock-vaccinated (i.e. saline-treated) mice succumbed to VACV strain WR challenge, whereas treatment of animals vaccinated with either VACV strain WR, VACV strain DPP15, or all doses of scHPXV (10⁵−10⁷ pfu per mouse) protected 100% of the animals.</p