Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 May;77(10):5821-8.
doi: 10.1128/jvi.77.10.5821-5828.2003.

Determination of minimum herpes simplex virus type 1 components necessary to localize transcriptionally active DNA to ND10

Qiyi Tang  1 , Luge Li, Alexander M Ishov, Valerie Revol, Alberto L Epstein, Gerd G Maul
Affiliations

Determination of minimum herpes simplex virus type 1 components necessary to localize transcriptionally active DNA to ND10

Qiyi Tang et al. J Virol. 2003 May.

Abstract

DNA viruses such as herpes simplex virus type 1 (HSV-1) appear to start their replicative processes at specific nuclear domains known as ND10. In analyses to determine the minimum viral components needed for transcript accumulation at ND10, we find that a specific viral DNA sequence, OriS, and the viral immediate-early proteins ICP4 and ICP27 are sufficient for a reporter gene placed in cis to the OriS sequence to transcribe at ND10. A chromatin immunoprecipitation assay demonstrated expected critical intermediates in retaining the minimal genome at ND10 for the HSV-1 replication origin through direct or indirect binding to the host protein Daxx. Coimmunoprecipitation assays with antibodies to Daxx and ICP4, ICP27, and ICP8 showed that the respective proteins interact, possibly forming a complex. A potential complex between the origin, early viral DNA-binding protein ICP8 and Daxx did not result in transcription at ND10. Thus, the deposition of transcriptionally active HSV-1 genomes at ND10 is most likely a consequence of retention at ND10 through the interaction of viral genome-bound ICP4 and ICP27 with Daxx. Such a complex might be more likely immobilized at the outside of ND10 by the PML-interacting Daxx than at other nuclear sites.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Localization of viral genomes or their transcripts relative to ND10 (PML labeling). Labeled components are indicated in the upper corners of the panels with their respective colors. (A) HEp-2 cells were infected with HSV-1 amplicons and prepared for in situ hybridization at 3 h p.i. Amplicon DNA was not found at ND10. (B) Same as panel A, but labeling β-Gal transcripts with most cells showed elevated but diffuse nuclear signals of RNA. (C) HEp-2 cell coinfected with amplicons and the ICP0 deletion mutant 1403 revealed amplicon-derived β-Gal transcripts at ND10.
FIG. 2.
FIG. 2.
Individual amplicon sequences were deleted as shown. Amplicon DNA was transfected, and after 3 h the cells were infected with HSV-1 1403. (A) The location of β-Gal transcripts relative to ND10 was determined by in situ hybridization. A “+” indicates transcript association with ND10. (B) pASK/E-derived transcripts are associated with ND10 (Daxx). (C) Transcripts from pASK/E, deleted in the packaging signal “a” were found adjacent to ND10 (Daxx). (D and E) Transcripts from pASK/E deleted in the origin were located throughout the nucleoplasm (D), but in fewer cells in aggregates not associated with ND10 (Daxx) (E). (F) Transcripts from pASK/E, deleted in both origin and the packaging signal, did not associate with ND10 (Daxx).
FIG. 3.
FIG. 3.
ChIP analysis of Daxx+/+ and Daxx−/− cells after infection with amplicons and helper virus. Cells were infected or mock infected for the times indicated. Anti-Daxx antibodies precipitated the origin of replication only in Daxx-containing cells.
FIG. 4.
FIG. 4.
Coimmunoprecipitation analysis of HSV-1-infected Daxx+/+ and Daxx−/− MEF infected with HSV-1 1403. (A) Immunoprecipitation was carried out on infected cell lysates with anti-mouse (M, lane 5) and anti-rabbit antibodies (R, lane 6) as controls and with anti-ICP8 monoclonal and rabbit anti-Daxx antibodies. After SDS-PAGE of the immunoprecipitate, Western blots were probed with ICP4, ICP8, and ICP27 monoclonal antibodies. The input in lanes 1 to 4 represents ca. 10% of lysate used for immunoprecipitation. (B) Same as panel A but showing immunoprecipitation with ICP27 and ICP4 monoclonal antibodies. (C) Diagram showing proteins coimmunoprecipitated (arrow origin) by immunoprecipitated proteins (arrow tip). Double-headed arrows indicate reverse coimmunoprecipitation.
FIG. 5.
FIG. 5.
Cells transfected to express ICP8 were infected with amplicons at 24 h after transfection, fixed at 16 h p.i., and tested for the location of β-Gal transcripts relative to ND10, as identified by anti-Daxx antibodies. (A) Combined colors of RNA (green), ICP8 (red), and Daxx (blue); (B) Daxx only; (C) RNA only; (D) same as panel A, but depicting a cell with dispersed ICP8.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Andrews, N. C., and D. V. Faller. 1991. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 19:2499. - PMC - PubMed
    1. Bell, P., R. Brazas, D. Ganem, and G. G. Maul. 2000. Hepatitis delta virus replication generates complexes of large hepatitis delta antigen and antigenomic RNA that affiliate with and alter nuclear domain 10. J. Virol. 74:5329-5336. - PMC - PubMed
    1. Bell, P., P. M. Lieberman, and G. G. Maul. 2000. Lytic but not latent replication of Epstein-Barr virus is associated with PML and induces sequential release of nuclear domain 10 proteins. J. Virol. 74:11800-11810. - PMC - PubMed
    1. Boutell, C., S. Sadis, and R. D. Everett. 2002. Herpes simplex virus type 1 immediate-early protein ICP0 and is isolated RING finger domain act as ubiquitin E3 ligases in vitro. J. Virol. 76:841-850. - PMC - PubMed
    1. Burkham, J., D. M. Coen, C. B. Hwang, and S. K. Weller. 2001. Interactions of herpes simplex virus type 1 with ND10 and recruitment of PML to replication compartments. J. Virol. 75:2353-2367. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources