Site-Directed Mutagenesis of Large DNA Palindromes: Construction and In Vitro Characterization of Herpes Simplex Virus Type 1 Mutants Containing Point Mutations That Eliminate the oriL or oriS Initiation Function

Replication of the 152-kb double-stranded DNA (dsDNA) herpes simplex virus type 1 (HSV-1) genome is a pivotal event in the dual life cycle of the virus in that the commencement of viral DNA synthesis signals the commitment of the infected cell to the production of new infectious virus and, in most cases, cell lysis. In contrast, the absence of viral DNA synthesis in sensory neurons is a hallmark of latent infection and cell survival. The correlation between the levels of viral DNA synthesis and the establishment, maintenance, and reactivation of latency reflects the complex relationship between viral DNA synthesis and the regulation of viral gene expression. Specifically, pharmacological or genetic inhibition of viral DNA synthesis has been reported to result in marked decreases in immediate-early (IE) and early (E) transcript levels in primary sympathetic neurons but not in Vero cells (39). A similar response to the inhibition of viral DNA synthesis has been reported to occur in vivo during acute viral replication and reactivation from latency in mouse trigeminal ganglia (32, 33). Viral DNA synthesis is also required for the efficient expression of HSV-1 late (L) genes in all cell types tested (28). Viral DNA synthesis initiates at defined sequences in viral genomes termed origins of replication. Viral origins are commonly composed of core palindromic sequences and auxiliary sequences (17). Core sequences are essential for origin function and typically contain a DNA-unwinding element, often consisting of AT-rich sequences, and an origin recognition element. Auxiliary sequences contain transcription factor binding sites which are commonly associated with promoter/regulatory elements of genes transcribed divergently from the core origin. Both core and auxiliary sequences serve as sites for the assembly of protein-DNA complexes that initiate the process of DNA synthesis. In addition, viral origins can also be involved in nuclear matrix attachment (1, 30, 37), mediated in part by AT-rich sequences located in the core origin (5). Moreover, because they are frequently located within the promoter/regulatory elements of flanking genes, origins are thought to be involved in the transcriptional regulation of these genes (24). HSV-1 contains three origins of DNA replication, namely, one copy of oriL located in the center of the UL region of the genome and two copies of oriS located in the “c” repeats flanking the US region of the genome (Fig. ​(Fig.1A).1A). Both oriL and oriS reside within the promoter/enhancer regions of divergently transcribed genes, contain palindromic sequences that serve as core elements, and share >90% sequence homology in these core elements (60). Specifically, the oriL core origin consists of a perfect 144-bp palindrome with a 20-bp AT-rich sequence at the apex (Fig. ​(Fig.1B)1B) which is thought to unwind during the initiation of DNA synthesis. Contiguous with both the 5′ and 3′ ends of the AT-rich apex are consensus, functional glucocorticoid response element (GRE) half-sites (26). In contrast to oriL, the oriS core origin consists of a 90-bp sequence which includes a 45-bp imperfect palindrome, an 18-bp AT-rich apical sequence, and degenerate GRE half-sites immediately adjacent to the apex (Fig. ​(Fig.1B).1B). The virus-encoded origin binding protein (OBP), six other viral E proteins (Fig. ​(Fig.1A),1A), and an unknown number of cellular proteins are required to initiate DNA synthesis at all three viral origins (2, 10, 11, 38). As a consequence of the sequence homology of their respective palindromes, oriL and oriS share two types of OBP binding sites, sites I and III. OBP has a high affinity for site I, which is essential for origin-dependent DNA replication, and a lower affinity for site III, which is required for efficient initiation (27). A third OBP binding site, site II, present solely in oriS, is bound by OBP with an affinity intermediate between those of sites I and III. Although only 6 of the 71 nucleotides present in shared oriL and oriS sequences differ, 5 of these 6 nucleotides occur near the AT-rich apex, of which 4 are located within GRE half-sites (Fig. ​(Fig.1B).1B). Thus, despite extensive DNA sequence homology in shared core sequences, a comparison of the complete nucleotide sequences of the oriL and oriS core elements demonstrates that the two origins differ significantly in their structure, sequence, and type and number of OBP binding sites, suggesting that the two origins perform distinct functions in the HSV-1 life cycle. FIG. 1. Genomic locations and sequence alignment of HSV-1 oriL and oriS. (A) Diagram of the HSV-1 genome showing the UL region flanked by inverted repeat sequences, ab and b′a′, and the US region flanked by inverted repeat sequences, a′c′ ... Functional characterizations of HSV-1 origins using genetic approaches have been carried out almost exclusively with oriS because (i) the oriS but not the oriL palindrome is stable during propagation in standard strains of Escherichia coli (45, 50, 59, 60), (ii) oriS but not oriL sequences are readily changed by site-directed mutagenesis, and (iii) oriS but not oriL is easily sequenced by standard automated DNA sequencing procedures. To date, the major limitation of studies with oriS has been the difficulties associated with the introduction of identical mutations into both copies of oriS in the viral genome. The only published virus lacking both copies of oriS described to date (R7711) (29) was constructed using a complex scheme involving insertions and deletions of origin sequences, resulting in a virus that lacks not only both origins but also three viral genes, UL23, US11, and US12. This virus also contains the ICP27 promoter in place of the ICP22 promoter and the simian virus 40 (SV40) promoter in place of one of the two ICP4 promoters (29). Although studies with this virus demonstrated that oriS is not essential for productive infection in vitro, the multiple alterations in R7711 render it unsuitable for further functional characterization. Thus, the effects of the absence of oriS function on viral replication at the molecular level remain unknown. Unlike oriS, oriL is highly deletion-prone during the propagation of oriL-containing plasmids in standard strains of E. coli (45, 50, 59, 60). Intact copies of oriL are also far less amenable to DNA sequencing and site-directed mutagenesis (25, 26). Indeed, the only reported cloned copy of oriL containing specific point mutations required nearly 2 years to generate and sequence (26). For these reasons, considerably less is known about the functions of oriL or the roles of individual cis-acting elements within oriL core sequences in oriL function. Despite the difficulties associated with mutagenesis and the insertion of mutations into the HSV-1 genome, a definitive functional characterization of the roles of viral origins in the dual life cycle of HSV-1 will necessitate the isolation of viruses mutated in specific origin elements. In this study, we describe a method of introducing site-specific mutations into oriL and the effects of mutations in OBP binding site I on oriL function. We show that single point mutations in site I that eliminate OBP binding in only one arm of the oriL palindrome are unstable upon propagation in bacteria, whereas these point mutations remain stable when introduced into both arms of the palindrome (thus conserving the palindromic sequence of the oriL core). Mutant viruses containing a single point mutation in both copies of site I in oriL or a single point mutation in site I sequences in both copies of oriS were generated, as were the corresponding rescuant viruses. In vitro experiments comparing these four viruses with wild-type virus for viral growth, DNA replication, and transcription of origin-flanking genes confirmed and extended the results of previous studies of HSV-1 origin function, and we recommend the use of the mutagenic procedure for genetic analyses of large DNA palindromes in other eukaryotic systems.