Multiple consecutive initiation of replication producing novel brush-like intermediates at the termini of linear viral dsDNA genomes with hairpin ends

Abstract

Linear dsDNA replicons with hairpin ends are found in the three domains of life, mainly associated with plasmids and viruses including the poxviruses, some phages and archaeal rudiviruses. However, their replication mechanism is not clearly understood. In this study, we find that the rudivirus SIRV2 undergoes multiple consecutive replication reinitiation events at the genomic termini. Using a strand-displacement replication strategy, the multiple reinitiation events from one parental template yield highly branched intermediates corresponding to about 30 genome units which generate exceptional 'brush-like' structures. Moreover, our data support the occurrence of an additional strand-coupled bidirectional replication from a circular dimeric intermediate. The multiple reinitiation process ensures rapid copying of the parental viral genome and will enable protein factors involved in viral genome replication to be specifically localised intracellularly, thereby helping the virus to avoid host defence mechanisms.

Figure 1.

Multimeric replication intermediates from SIRV2-infected cells. (A). P2-probe binding site on the SIRV2 genome. The size between P2-binding site and the right terminus is shown. (B). Presence of a single probe binding site in the monomeric SIRV2 genome. DNA extracted from SIRV2 virions that were purified by cesium chloride gradient ultracentrifugation was used for DNA spreading and FISH. (C–F). Representative RIs from SIRV2-infected cells. Infected cells were directly lysed on the slide before DNA spreading and FISH. Panels from left to right: entire fiber DNA shown in blue visualized by an anti-DNA antibody (viral DNA); FISH signal in green revealed by site-specific probe DIG-P2 (probe); entire fiber and probe signals are superimposed (overlay); and interpretation of the topology of each RI (interpretation). In the Interpretation panel, single black lines indicate a monomeric dsDNA genome (1n) and double black lines represent the genome dimer (2n) whereas daughter DNA is depicted in grey. The P2-probe binding site is shown by green dots; arrowheads indicate two adjacent probe signals in an internal segment. Bars, 6 μm.

Figure 2.

Large viral replication intermediates detected by PFGE during the SIRV2 life cycle. The viral RI sizes are determined for the early/mid phases (A) and late phase (B) of the life cycle. A size marker is placed at one side of each blot. (C). SIRV2 replication intermediates reach the size of >1.2 Mb. MidRange I PFG Marker was used in (A) and (B), while Yeast Chromosome PFG Marker was used in (C). W, wells; M, viral genome monomer.

Figure 3.

Strand-displacement replication of SIRV2. (A) HindIII digestion map of SIRV2. The HindIII recognition sites are indicated with short and vertical lines with the relevant sites labeled in nucleotides above the bar. Red arrows indicate the locations of the two terminal probes P1 and P2. (B) RIs revealed at both genomic termini. Left panel, 2D-AGE pattern of the left terminal fragment revealed with probe P1; middle panel, 2D-AGE pattern of the right terminal fragment (P2); right panel, diagrammatic illustrations of the gels: L, linear arc; 1n, unit-length linear fragment; 1.5n, 2n, 4n, fragments of 1.5, 2 and 4 times the unit-length, respectively; arc a1, bubble-like form intermediates ranging in size from 1n to around 1.5n; X, X-shaped molecules; m, 1n fragments derived from melted 2n species. Dimensions of the 2D-AGE (first and second) are indicated. (C) Illustration of early intermediates from strand-displacement replication. SIRV2 genome is depicted as black rods. Light blue rectangle indicates the probe position. The four HindIII recognition sites at the left side of the genome are indicated by vertical lines (dotted for the first site). The dark blue dot illustrates the Rep protein while the red arrowed line indicates the newly synthesized DNA strand. Arc a1 is the semi-bubble-like form as depicted in B, arcs a2 and a3 are described in the text.

Figure 4.

Strand-coupled replication of SIRV2 genome. (A) HindIII digestion map of SIRV2 as in Figure 3A. The locations of the left terminal probes P1 and the internal probe P3 are indicated with red arrows. (B) 2D patterns of S1 nuclease treated plugs. Plugs were treated with or without S1 nuclease after HindIII digestion and before 2D gel electrophoresis. Dimensions of the 2D-AGE (first and second) are indicated. (C) Illustration of strand-coupled replication intermediates for the left terminal fragment. The dark blue dot indicates the Rep protein while the dark red arrowed line indicates the leading strand and light red arrowed lines depict the lagging strand. The gray triangles indicate the position of the genomic termini. Species 1n, 2n, 4n and X are as depicted in Figure 3; arc b, bubble form intermediates ranging in size from 2n to 4n; arc c, Y-shaped molecules; arc d, diffused molecules containing long ssDNA.

Figure 5.

SIRV2 replication dynamics. The left terminal probe P1 was used for all 3 time points, 30, 60 and 90 min post-infection. Species 1n, 2n, 4n and X are as depicted in Figure 4; arcs are as depicted in Figures 3 and 4. Dimensions of the 2D-AGE (first and second) are indicated. Arc a1, intermediates from strand-displacement replication ranging in size between 1n to about 1.5n; arc b, bubble form intermediates from strand-coupled replication ranging in size from 2n to 4n.

Figure 6.

Multiple replication reinitiation events during SIRV2 DNA replication. (A) Model of multiple replication reinitiation. The parental DNA is depicted in black and the daughter DNA is presented in red; the blue dots indicate the qPCR target in ITRs (ends) while the yellow dots indicate the center. (B) The ends:center ratio in SIRV2-infected cultures at different times post-infection. Results from three independent biological replicates are shown. The line indicates the average ratio at each time point and the error bars indicate the standard error. E:C, ends:center ratio.

Figure 7.

Model of the genome replication mechanism of SIRV2. (A) Strand-displacement replication initiated from the left end of the genome. (B) Rolling-circle mode of strand-displacement replication initiated from the right end. (C) Multiple replication reinitiation leading to the brush-like replication. (D) Strand-coupled replication. The parental DNA is shown in black and the newly synthesized DNA is depicted in shades of red. The green dots indicate the position of the probe DIG-P2 at the right end of the genome.