Herpes Simplex Virus DNA Packaging without Measurable DNA Synthesis

Herpes simplex virus (HSV) type 1 (HSV-1) is a complex double-stranded DNA virus which replicates its genome in the nuclei of infected cells (21, 29, 35). Seven viral genes are known to be essential for HSV DNA replication (4, 5, 8, 13, 21, 25, 35), and replication is thought to occur by a rolling circle mechanism, generating head-to-tail concatamers of the 152-kb viral chromosome (4, 29). An additional, inherently recombinogenic replication mechanism may also exist and give rise to branched forms of viral DNA (4, 17, 30). Packaging, which also occurs in the nucleus, involves cleavage of the concatameric viral DNA at the flanking “a” sequences and engulfment of a single full-length copy of the viral chromosome by the maturing viral capsids (19, 29, 31–33). Cleavage at the a sequence appears to be inseparable from packaging and can be readily monitored by Southern blotting since it results in cleavage of the viral SQ BamHI fragment to discrete S and Q fragments. The molecular apparatus responsible for packaging is poorly understood, but one key component is the product of the HSV UL15 gene. This protein has possible homology with the large subunit of the terminase complex responsible for cleavage and packaging of the phage T4 genome (3, 7, 9), and HSV-1 mutants defective in UL15 fail to cleave and package viral DNA (1, 2, 23, 37). At least five other HSV gene products, UL6, UL25, UL28, UL32, and UL33, are also required for the packaging process (references 1, 29, and 37 and references therein). Replication of viral DNA takes place in large globular regions termed replication compartments (8, 25, 28) which initially form adjacent to nuclear subdomains known as ND10 (12, 18). Replication compartments contain all seven virally encoded proteins essential for DNA replication (5, 8, 11, 14–16, 22) but surprisingly also contain the packaging factor UL15 (34). UL15 colocalizes with the DNA synthesis machinery even late in infection, when further levels of nuclear compartmentation become apparent. At these late times, when capsid assembly is thought to be maximal, at least some HSV strains generate dense structures termed assemblons at the periphery of their nuclei (34). Assemblons are recognized by antibodies which react with the major viral capsid protein VP5 and the capsid scaffold protein ICP35 (34). It has been hypothesized that they may represent sites of capsid assembly and maturation, positioned so as to gain access to the pool of actively replicating viral DNA in replication compartments. It has further been proposed that UL15 could actually be an accessory component of the DNA replication machinery (34) and may couple the process of DNA replication to that of genome cleavage and packaging. One of a number of issues which remain unaddressed concerning replication and packaging in HSV-1 is the degree of interdependence between the two events. Does packaging require ongoing DNA synthesis, or can the UL15-dependent packaging machinery capture and process DNA from a previously replicated pool? Similarly, the resolution of branched HSV DNA may be important for production of packaged nucleocapsids capable of further maturation (17, 30). Such events and other kinds of DNA repair generally require DNA synthesis, at least in uninfected cells (36). Determination of the dependence of packaging upon DNA replication would help advance our understanding of the molecular nature of the packaging process. Unfortunately, the inherent complexity of the assembly pathway of HSV and the fact that packaging and replication occur simultaneously and continuously from a point very early in infection make it extremely difficult to dissect the relationship between these two processes. We have recently described a model system to facilitate the study of late events in HSV assembly: the virus strain tsProt.A carries a reversible temperature-sensitive mutation in its maturational protease, which results in accumulation of unpackaged capsids at the nonpermissive temperature of 39°C (10, 24). Following downshift to the permissive temperature of 31°C, these capsids mature, package DNA, and give rise to infectious particles in a single, synchronized wave (6). In the present study, we made use of this system to test the requirement for DNA synthesis in packaging. When DNA replication was inhibited by addition of acyclovir (ACV), we found that packaging and PFU production could subsequently take place at near-normal levels. We conclude that, although DNA packaging and detectable levels of DNA synthesis occur simultaneously during a normal infection, they are not coupled together in an obligate fashion.