Intranuclear Localization of Herpes Simplex Virus Immediate-Early and Delayed-Early Proteins: Evidence that ICP 4 Is Associated with Progeny Virus DNA

Abstract

SUMMARYThe localization of ICP 4, ICP 8, DNA polymerase and alkaline exonuclease within herpes simplex virus type 1 (HSV)-infected cells has been examined by immunofluorescence using specific antibodies to these proteins. Cells were simultaneously counterstained with the DNA-binding fluorochrome 4,6-diaminido-2-phenylindole (DAPI) to reveal the intranuclear distribution of DNA. These studies showed that in the absence of virus DNA replication ICP 4, ICP 8 and DNA polymerase were diffusely distributed throughout the nucleus but during virus DNA replication these proteins accumulated at specific foci within the nucleus. Initially these foci were near the nuclear membrane but with continuing virus DNA replication they increased in size until the whole of the nucleus became affected. The increase in size of these foci was coincident with a redistribution of nuclear DNA and margination of chromatin at the nuclear membrane, as revealed by DAPI staining. The number of foci initially present in an infected cell was dependent on the multiplicity of infection. The distribution of ICP 4, ICP 8 and DNA polymerase within the nucleus was altered by treating the cells with DNase. The majority of alkaline exonuclease was diffusely distributed throughout the nucleus during virus DNA replication and did not localize at specific foci within the nucleus. Autoradiographic examination of the incorporation of [3H]thymidine in cells infected with HSV showed that viral DNA replication occurred in restricted areas within the nucleus that were similar, in terms of number, location and size, to the foci where ICP 4, ICP 8 and DNA polymerase accumulated. Furthermore, in cells blocked in mitosis following infection with HSV, ICP 4, ICP 8 and DNA polymerase, but not alkaline exonuclease, localized in areas outside the condensed chromatin structures. DAPI staining revealed the presence of DNA in these areas and, as such structures were never seen when uninfected cells had entered mitosis, it is suggested that this extrachromosomal DNA is of viral origin. These studies therefore suggest that ICP 4 is associated with progeny virus DNA and that while its intranuclear localization is initially at non-viral sites, as DNA replication proceeds so ICP 4 is recruited into areas of virus DNA transcription and replication.

Evidence that Deletion of Coding Sequences in the 5' End of the Thymidine Kinase Gene of Herpes Simplex Virus Type 1 Affects the Stability of the Gene Products

“…Synthesis of HSV DNA initially occurs near the nuclear membrane (Randall & Dinwoodie, 1986). The accumulation of TK at the nuclear membrane observed in the present work does not imply that this protein is part of a multienzyme complex involved in DNA synthesis.…”

“…The accumulation of TK at the nuclear membrane observed in the present work does not imply that this protein is part of a multienzyme complex involved in DNA synthesis. The localization is different from that of three virus-induced proteins closely associated with DNA synthesis, namely immediate early 175K (ICP 4), the major DNA-binding protein (ICP 8) and DNA polymerase (Randall & Dinwoodie, 1986). Furthermore, the latter two proteins and alkaline exonuclease are able to interact in vitro, but TK seems not to take part in this interaction (Vaughan et al, 1984).…”

Differences in the intracellular localization and fate of herpes simplex virus tegument proteins early in the infection of Vero cells.

“…A number of HSV-1 proteins (see e.g. Liptak et al, 1996) and cellular proteins (Wilcock & Lane, 1991) have been localized to these structures, including the early major DNA-binding protein ICP8 (Randall & Dinwoodie, 1986 ;Knipe et al, 1987 ;Yao & Courtney, 1989). The nuclear co-localization demonstrated in this paper between VP16 and ICP8 (Fig.…”

Characterization of the varicella-zoster virus gene 61 protein

“…Cell monolayers were fixed and permeabilized as described by Randall & Dinwoodie (1986). Incubation with antisera and fiuorescein isothiocyanate-conjugated goat anti-rabbit antibody (Sigma) was carried out at dilutions of 1:20 and 1:80, respectively.…”