The Structure of Herpes Simplex Virus Type 1 DNA as Probed by Micrococcal Nuclease Digestion

Abstract

SUMMARYMicrococcal nuclease digestion was used to probe the structures in which herpes simplex virus type I (HSV-0 DNA is found during virus replication. Parental DNA, progeny DNA and DNA in nucleocapsids were analysed. Parental DNA was examined after infection of Vero cells with 32p. or 3H-thymidinelabelled HSV-L Progeny DNA was examined after HSV-I-infected Vero cells were pulse-labelled with 3H-thymidine during HSV-I DNA synthesis. In both cases, nuclei were isolated and digested with micrococcal nuclease. Digestion products were analysed by agarose or polyacrylamide gel electrophoresis (PAGE). Most parental DNA remained as intact molecules. However, a small amount was degraded into fragments which were heterogeneous in size or the size of nucleosomal cell DNA. These two classes of fragments were also produced upon digestion of progeny DNA. The heterogeneous fragments and nucleosomal fragments comprised major and minor fractions, respectively, of digested progeny DNA. When digested DNA from HSV-1-infected cells was transferred from composite polyacrylamide-agarose gels to diazobenzyloxymethyl paper, nucleosomal fragments hybridized to 32P-labelled HSV-l DNA as well as to 32P-labelled Vero cell DNA. Therefore, nucleosomal fragments contained HSV-I DNA sequences. HSV-I DNA in nucleocapsids was analysed by micrococcal nuclease digestion after nucleocapsids were disrupted with pH 9"3 buffer, pyridine, Sarkosyl or NaC1/urea. Only fragments of heterogeneous size were produced. Thus, HSV-~ DNA is found predominantly in structures other than nucleosomes during virus replication.

Ultrastructural Localization of the Herpes Simplex Virus Major DNA-binding Protein in the Nucleus of Infected Cells

“…The absence of DBP from nucleosomal DNA (i.e. non-viral DNA, see Mouttet et al, 1979;Leinbach & Summers, 1980) (Fig. 10c, d) is consistent with its disappearance from peripheral nuclear regions after detergent treatment.…”

Chromatin Structure of Epstein-Barr Virus

“…Nucleosome-free molecules are observed during productive replication in superinfected Raji cells (Shaw et al, 1979). Also, herpes simplex virus nucleocapsid DNA lacks nucleosomes (Leinbach & Summers, 1980). Presumably, EBV DNA in the nucleocapsid is folded on another basic, perhaps virus-encoded, protein structure to permit the compact packaging of the DNA.…”

“…Packaged EBV DNA is not thought to have nucleosome structure (Shaw et al, 1979) since, in superinfected Raji cells, DNA undergoing replication prior to packaging does not give a nucleosome ladder on micrococcal nuclease digestion. Further evidence that the packaged EBV DNA is unlikely to have any nucleosome structure comes from the observation that herpes simplex virus encapsidated DNA does not have detectable nucleosomes (Leinbach & Summers, 1980). An intensity of EBV-specific hybridization similar to that displayed by the micrococcal nuclease ladder was seen when total DNA from B95-8 cells was simply cut with a restriction enzyme, blotted and probed (data not shown).…”

Herpes Simplex Virus 1 U L 31 and U L 34 Gene Products Promote the Late Maturation of Viral Replication Compartments to the Nuclear Periphery

“…3). We saw no staining of HSV RCs with the H1 antibody, and HSV DNA is not thought to bind H1 during lytic infection (15). Cells infected for 6 h showed formation of multiple RCs, as detected by staining for ICP8 (Fig.…”