In vitro synthesis of DNA in nuclei isolated from human lung cells infected with herpes simplex type II virus

Abstract

An isolated nuclei system prepared from herpes type II-and mock-infected human embryonic lung cells is able to synthesize cellular and viral DNA in the same proportion as in vivo at various times after infection. Incorporation of [3H ]TTP in the in vitro reaction mixture requires Mg2+ and ATP. Overall in vitro DNA synthesis in nuclei isolated from herpes-infected cells is semiconservative as demonstrated by bromodeoxyuridine-substituted DNA density-transfer experiments, but exhibits a significant fraction of repair-type replication. Relative rates of total DNA synthesis in vitro and in vivo are the same any time after infection. Isolated nuclei synthesize cell and viral DNA for a length of time and at a rate dependent upon the incubation temperature, but there are differences in the length of time of linear in vitro DNA synthesis between herpesand mockinfected cells. The temperature optima for in vitro DNA synthesis differ significantly for herpesand mock-infected cells, and are the same for cells abortively infected with herpes type II as for mock-infected cells.

DNA polymerase in nuclei isolated from herpes simplex virus type-2-infected cells. Characterization of the reaction product and inhibition by substrate analogs

“…All assays were performed in duplicate or triplicate. which permitted linear incorporation for at least 5 rain plus hyperbolic incorporation up to 1 h. A decreasing rate of incorporation in HSV-infected nuclei has been consistently reported by others [9,10,27] and has been shown to be associated with replicative DNA synthesis [13]. The reasons for limited synthesis are unknown but the need to avoid high Mg 2+ concentrations suggests that the DNA product may be degraded by the HSV-induced DNAase(s) which are activated by Mg 2+ [28].…”

“…The use of isolated nuclei should be very valuable in this regard because the nuclear matrix is intimately involved in DNA replication [2] and in herpes virogenesis [3]. In addition, much of the replication complex -including the natural template and DNA polymerases [4] -is functional [5][6][7][8][9][10][11][12][13].…”

Anatomy of Herpes Simplex Virus DNA. VIII. Properties of the Replicating DNA

“…(iii) Both pseudorabies virus DNA (15) and HSV DNA (17) replicate in a semiconservative fashion. Newly replicated DNAs contain relatively small oligonucleotide fragments, which are repaired and/or ligated on maturation (1,3,7).…”

“…Newly replicated DNAs contain relatively small oligonucleotide fragments, which are repaired and/or ligated on maturation (1,3,7). Replication of HSV DNA in isolated nuclei is accompanied by extensive repair synthesis (17). (iv) Frenkel et al (5,6) concluded that at least one site of initiation of viral DNA synthesis occurs at or near the terminus of the S component.…”

Replication of herpes simplex virus type I DNA in permeabilized infected cells

“…A number of infected cell preparations supporting the synthesis of herpes simplex virus (HSV) DNA have been described, including disrupted whole infected cells (7), isolated nuclei (7,8,9,10), nuclear monolayers (11), soluble chromatin (12,13,14), and ill-defined complexes released from infected cell nuclei (15,16). Most of these, however, fail to synthesize unit size viral DNA or to support semi-conservative replication, and none has been shown to incorporate precursors into the large concatemers thought to be the primary templates for HSV DNA replication (17,18).…”