Use of a small DNA virus model to investigate mechanisms of CpG dinucleotide-induced attenuation of virus replication

Figures

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  Fig. 1.. MVM genomic structure and the capsid gene region selected for mutagenesis. (a) Diagram of the MVM genomic structure. Promoters (blue), transcripts (r1-r3) with ORFs (green and orange coloured boxes), mutated region highlighted in yellow. Based on, Fig. 57.10 of Berns and Parrish [65]. (b) SSE was used to calculate the synonymous coding variability observed between MVM and related parvoviruses. The orange region indicates the region chosen for mutations. (c) Distribution of CpGs and TpAs in the MVM genome.

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  Fig. 2.. CpG and TpA dinucleotide distribution in parvovirus genes. Comparison of frequencies of CpG and TpA dinucleotides in MVM NS- and capsid-encoding genes (red and blue filled diamonds) and representative other parvoviruses (circles) with those of mouse (Mus musculus) mRNA sequences (each grey point represents a separate gene). Observed (o) CpG and TpA frequencies were normalized to values expected (e) from their G+C content.

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  Fig. 3.. Effects of V2 sequence modifications on MVM replication kinetics. (a) Multi-step growth curve in the A9 cell line infected with 1 TCID50 unit/cell. Supernatant was harvested at different time points and titred in NB324K cells. Data points represent the mean of three biological replicates. (b) Cross-section of the growth curve at 48 h. Bar heights represent the mean of three biological replicates; error bar show sems.

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  Fig. 4.. Cellular distribution of MVM viral proteins in A9 cells. Immunofluorescence staining of viral NS (green) and VP (red) proteins at 18 h post-infection with 1000 viral copies/cell. Uninfected cells were used as a negative control. n=3, scale bar 100 µm, representative image shown.

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  Fig. 5.. Quantitative analysis of viral protein expression. Quantification of immunofluorescence staining of viral NS and VP proteins using Imaris and Fiji at 18 h post-infection with 1000 viral copies/cell. Uninfected cells were used as a negative control. n=3, +/−sem. (a) Percentage of cells expressing NS or VP. (b) Relative fluorescence intensity of NS or VP compared to WT MVM.

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  Fig. 6.. MVM gene expression and translation. (a) Viral protein expression 18 h post-infection with 1000 viral copies/cell. Uninfected cells were used as a negative control. n=3, representative image shown. (b) In vitro translation of a truncated VP2. RNA was transcribed into mRNA in vitro via a T7 promoter using the synthetic plasmids containing the different MVM inserts. Equal amounts of the RNA were then translated using an in vitro rabbit reticulocyte and measured using immunoblotting. The negative control is a mock translation with no RNA added, while the positive control is an infected cell lysate (VP2 band shown). (c) Quantification of VP2 protein levels (from Fig. 6b) by ImageJ expressed as the ratio to that of the WT gene (y-axis). Bar heights shows the mean of three replicates; error bars show +/−sem.

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  Fig. 7.. MVM R1 mRNA levels post-transfection. MVM R1 mRNA expression 8 and 18 h post-transfection normalized to cellular GAPDH and WT R1 levels. n=3, +/−sem. (a) Plasmid pMM984, viral infectious clone, replication inhibited by Aphidicolin. (b) Plasmid pCAG_MVMp_R3, expression vector containing MVM R3 subclone.

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  Fig. 8.. Cell line-associated differences in MVM replication. Infectivity of MVM 72 h post-infection (1 TCID unit/cell) in different cell lines. n=2, +/−sd. (a) Viral titres of MVM WT virus. (b) Attenuation of CpG-high and codon-optimized mutants of compared to WT. # indicates samples where no virus could be recovered, thus underestimating attenuation.

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  Fig. 9.. Involvement of TLR9 in MVM mutant phenotype. (a) IL-1β induction after TLR9 induction by purified E. coli DNA in the presence or absence of TLR-9 antagonist ODN2088 . n=3, +/−sem. b) Viral genome copies 72 h post-infection with 1000 viral genome copies/cell in the presence or absence of ODN2088. n=3.

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  Fig. 10.. MVM R1 mRNA levels in A549 andA549 B8 (ZAP k/o) cell lines post-transfection. MVM R1 mRNA expression 8 and 18 h post-transfection normalized to cellular GAPDH and MVM WT R1 levels in A549 cells. PlasmidpMM984, viral infectious clone, replication inhibited by Aphidicolin. n=3, +/−sem.

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  Fig. 11.. Cellular distribution of MVM viral proteins in A549 and B8 (ZAP k/o) cells . (a) Immunofluorescence staining of viral NS (green) and VP (red) proteins at 18 h post-infection with 1000 viral copies/cell. Uninfected cells were used as a negative control. (b) Quantification of immunofluorescence staining of viral NS and VP proteins using Imaris and Fiji software separately for the frequency of cells expressing NS or VP (left) or relative fluorescence intensity of NS or VP compared to WT MVM (right). Bar heights represent the mean of three biological replicates; error bars show sems. (c) Replication of WT and CpG-H first half-mutant of MVM in A549 and ZAP k/o cells infected at an m.o.i. of 1. Cell supernatant was harvested at 48 h and infectivity was determined by titration on NB324K cells. Bar heights show the mean of two replicates; error bars show the sd.