Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.Poxviruses comprise a large family of complex enveloped DNA viruses that infect vertebrates and insects and includes the agent responsible for human smallpox (1). In contrast to the nuclear location exploited for genome replication by many other DNA viruses, the 130- to 230-kbp linear double-stranded DNA genomes of poxviruses are synthesized within discrete, specialized regions of the cytoplasm known as virus factories or virosomes. Furthermore, most, if not all, proteins required for DNA replication are virus-encoded (2). Poxvirus genomes, as shown for the prototype species vaccinia virus (VACV), have covalently closed hairpin termini, so that the DNA forms a continuous polynucleotide chain (3). The hairpin is a 104-nucleotide (nt) A+T-rich incompletely base-paired structure that exists in two inverted and complementary forms. As expected for a genome with such covalently closed ends, VACV replicative intermediates are head-to-head or tail-to-tail concatemers (4, 5). The concatemers exist only transiently because they are cleaved by a virus-encoded Holliday junction resolvase into unit length genomes with hairpin ends before incorporation into virus particles (6, 7). Because VACV genomes and concatemers resemble the replicative intermediates of the much smaller parvoviruses, the rolling hairpin model of replication originally proposed for the latter family was extended to poxviruses and has become the current scheme for poxvirus genome replication (2). In the rolling hairpin model, DNA synthesis occurs by strand displacement without discontinuous synthesis of the lagging strand and accordingly implies no role for Okazaki fragments. However, in contrast to parvoviruses, poxviruses have not been shown to encode an endonuclease that introduces a nick to provide a free 3′-OH for priming DNA synthesis. On the other hand, all poxviruses do encode a primase fused to a helicase, which is essential for VACV DNA replication (8). The presence of the essential primase–helicase and the requirement for the viral ligase or the host DNA ligase 1 (9) imply that poxvirus DNA replication is RNA-primed and could involve discontinuous synthesis of the lagging strand.Although not followed up on for nearly 4 decades, early studies on poxvirus DNA replication described putative Okazaki fragments of about 1,000 nt in length and RNA primers on the 5′-ends of newly made chains of VACV DNA (10, 11). Additionally, the specific activity of ³H]thymidine incorporated during a short pulse under conditions of synchronous VACV DNA synthesis was highest in fragments from the ends of the genome, suggesting that replication originated close by (12). In agreement with the apparent terminal localization of origins, variable size double-stranded DNA loops were described at one end of replicating VACV DNA (13). Collectively, these data are compatible with a discontinuous or semidiscontinuous mode of genome replication, with origins located near the termini. Subsequently, however, the existence of specific VACV origins came into question by the demonstration that any circular DNA replicates in VACV or Shope fibroma virus-infected cell cytoplasm, and that replication is not enhanced by inclusion of any VACV DNA sequence (14, 15). Moreover, all VACV proteins known to be required for genome replication are also required for origin-independent plasmid replication (16). In contrast to a circular plasmid, however, efficient replication of a linear minichromosome with covalently closed ends requires a specific 150-bp telomere segment derived from the poxvirus genome, whereas a smaller 65-bp segment is insufficient (17). Those findings are compatible with the location of a replication origin within the terminal 150 bp. However, the latter region contains the consensus resolution sequence that is required for the conversion of the concatemer junction into the covalently closed ends of mature genomes (18, 19). Therefore, the possibility remained that the concatemer resolution sequence is required for the continuation of minichromosome replication, whereas initiation potentially could occur at any position. Thus, the existence of distinct origins of replication near the ends of the VACV genome remained an open question.Genome replication in most cellular life forms is bidirectional and semidiscontinuous, resulting in a switch in the direction of the Okazaki fragments at the origins of replication and transition from continuous to discontinuous synthesis. This asymmetry in the distribution of RNA-primed nascent strands has been used to map initiation points at nucleotide accuracy and to generate high-resolution maps of replication origins (20–22). We used the same basic strategy but applied directional deep sequencing to map the 5′-ends of short nascent fragments to plus or minus strands of the genome instead of hybridization or primer extension and sequencing gels used in the original classical studies. A major advantage of deep sequencing for determination of origins is that no prior information on their location is required, as documented by recent studies with yeast showing that the transition points in the direction of Okazaki fragments identified by directional deep sequencing were in good agreement with the position of replication origins identified by other methods (23–25). In addition, the ability to computationally discriminate viral and host DNA provided by deep sequencing is important for the analysis of virus DNA replication. This next generation sequencing approach provided evidence for VACV replication origins at the nucleotide level.