Search tips
Search criteria 


Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
J Virol. 1995 February; 69(2): 935–947.
PMCID: PMC188662

Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27.


Previous work has shown that the herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 localizes to the cell nucleus and that certain mutant ICP27 polypeptides localize preferentially in nucleoli. To map the signals in ICP27 which mediate its nuclear localization, we identified the portions of ICP27 which can direct a cytoplasmic protein, pyruvate kinase (PK), to nuclei. Our results demonstrate that ICP27 contains multiple nuclear localization signals (NLSs) that function with differing efficiencies. First, ICP27 possesses a strong NLS, mapping to residues 110 to 137, which bears similarity to the bipartite NLSs found in Xenopus laevis nucleoplasmin and other proteins. Second, ICP27 possesses one or more weak NLSs which map to a carboxyl-terminal portion of the protein between residues 140 and 512. Our PK-targeting experiments also demonstrate that ICP27 contains a relatively short sequence, mapping to residues 110 to 152, that can function as a nucleolar localization signal (NuLS). This signal includes ICP27's strong NLS as well as 15 contiguous residues which consist entirely of arginine and glycine. This latter sequence is very similar to an RGG box, a putative RNA-binding motif found in a number of cellular proteins which are involved in nuclear RNA processing. To confirm the results of the PK-targeting experiments, we mutated the ICP27 gene by deleting sequences encoding either the strong NLS or the RGG box. Deletion of the strong NLS (residues 109 to 138) resulted in an ICP27 molecule that was only partially defective for nuclear localization, while deletion of the RGG box (residues 139 to 153) resulted in a molecule that was nuclear localized but excluded from nucleoli. Recombinant HSV-1s bearing either of these deletions were unable to replicate efficiently in Vero cells, suggesting that ICP27's strong NLS and RGG box carry out important in vivo functions.

Full Text

The Full Text of this article is available as a PDF (895K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ackermann M, Braun DK, Pereira L, Roizman B. Characterization of herpes simplex virus 1 alpha proteins 0, 4, and 27 with monoclonal antibodies. J Virol. 1984 Oct;52(1):108–118. [PMC free article] [PubMed]
  • Aris JP, Blobel G. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):931–935. [PubMed]
  • Berger J, Aepinus C, Dobrovnik M, Fleckenstein B, Hauber J, Böhnlein E. Mutational analysis of functional domains in the HIV-1 Rev trans-regulatory protein. Virology. 1991 Aug;183(2):630–635. [PubMed]
  • Block T, Jordan R. Herpes simplex virus type 1 alpha gene containing plasmids can inhibit expression regulated from an alpha promoter in CV-1 but not HeLa cells. Virus Res. 1988 Nov;11(4):269–279. [PubMed]
  • Booher RN, Alfa CE, Hyams JS, Beach DH. The fission yeast cdc2/cdc13/suc1 protein kinase: regulation of catalytic activity and nuclear localization. Cell. 1989 Aug 11;58(3):485–497. [PubMed]
  • Buvoli M, Biamonti G, Tsoulfas P, Bassi MT, Ghetti A, Riva S, Morandi C. cDNA cloning of human hnRNP protein A1 reveals the existence of multiple mRNA isoforms. Nucleic Acids Res. 1988 May 11;16(9):3751–3770. [PMC free article] [PubMed]
  • Calnan BJ, Biancalana S, Hudson D, Frankel AD. Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition. Genes Dev. 1991 Feb;5(2):201–210. [PubMed]
  • Chapman CJ, Harris JD, Hardwicke MA, Sandri-Goldin RM, Collins MK, Latchman DS. Promoter-independent activation of heterologous virus gene expression by the herpes simplex virus immediate-early protein ICP27. Virology. 1992 Feb;186(2):573–578. [PubMed]
  • Cochrane AW, Perkins A, Rosen CA. Identification of sequences important in the nucleolar localization of human immunodeficiency virus Rev: relevance of nucleolar localization to function. J Virol. 1990 Feb;64(2):881–885. [PMC free article] [PubMed]
  • Dingwall C, Laskey RA. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. [PubMed]
  • Dworetzky SI, Lanford RE, Feldherr CM. The effects of variations in the number and sequence of targeting signals on nuclear uptake. J Cell Biol. 1988 Oct;107(4):1279–1287. [PMC free article] [PubMed]
  • Everett RD. The products of herpes simplex virus type 1 (HSV-1) immediate early genes 1, 2 and 3 can activate HSV-1 gene expression in trans. J Gen Virol. 1986 Nov;67(Pt 11):2507–2513. [PubMed]
  • Frost E, Williams J. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology. 1978 Nov;91(1):39–50. [PubMed]
  • Gao M, Knipe DM. Distal protein sequences can affect the function of a nuclear localization signal. Mol Cell Biol. 1992 Mar;12(3):1330–1339. [PMC free article] [PubMed]
  • Garcia-Bustos J, Heitman J, Hall MN. Nuclear protein localization. Biochim Biophys Acta. 1991 Mar 7;1071(1):83–101. [PubMed]
  • Ghisolfi L, Joseph G, Amalric F, Erard M. The glycine-rich domain of nucleolin has an unusual supersecondary structure responsible for its RNA-helix-destabilizing properties. J Biol Chem. 1992 Feb 15;267(5):2955–2959. [PubMed]
  • Girard JP, Lehtonen H, Caizergues-Ferrer M, Amalric F, Tollervey D, Lapeyre B. GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast. EMBO J. 1992 Feb;11(2):673–682. [PubMed]
  • Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. [PubMed]
  • Hall MN, Craik C, Hiraoka Y. Homeodomain of yeast repressor alpha 2 contains a nuclear localization signal. Proc Natl Acad Sci U S A. 1990 Sep;87(18):6954–6958. [PubMed]
  • Jong AY, Clark MW, Gilbert M, Oehm A, Campbell JL. Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins. Mol Cell Biol. 1987 Aug;7(8):2947–2955. [PMC free article] [PubMed]
  • Kalderon D, Richardson WD, Markham AF, Smith AE. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature. 1984 Sep 6;311(5981):33–38. [PubMed]
  • Kalderon D, Roberts BL, Richardson WD, Smith AE. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. [PubMed]
  • Kiledjian M, Dreyfuss G. Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box. EMBO J. 1992 Jul;11(7):2655–2664. [PubMed]
  • Knipe DM, Senechek D, Rice SA, Smith JL. Stages in the nuclear association of the herpes simplex virus transcriptional activator protein ICP4. J Virol. 1987 Feb;61(2):276–284. [PMC free article] [PubMed]
  • Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem. 1991 Oct 25;266(30):19867–19870. [PubMed]
  • Lanford RE, Butel JS. Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell. 1984 Jul;37(3):801–813. [PubMed]
  • Maeda Y, Hisatake K, Kondo T, Hanada K, Song CZ, Nishimura T, Muramatsu M. Mouse rRNA gene transcription factor mUBF requires both HMG-box1 and an acidic tail for nucleolar accumulation: molecular analysis of the nucleolar targeting mechanism. EMBO J. 1992 Oct;11(10):3695–3704. [PubMed]
  • Malim MH, Cullen BR. HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency. Cell. 1991 Apr 19;65(2):241–248. [PubMed]
  • Mattaj IW. RNA recognition: a family matter? Cell. 1993 Jun 4;73(5):837–840. [PubMed]
  • McCarthy AM, McMahan L, Schaffer PA. Herpes simplex virus type 1 ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient. J Virol. 1989 Jan;63(1):18–27. [PMC free article] [PubMed]
  • McGeoch DJ, Cunningham C, McIntyre G, Dolan A. Comparative sequence analysis of the long repeat regions and adjoining parts of the long unique regions in the genomes of herpes simplex viruses types 1 and 2. J Gen Virol. 1991 Dec;72(Pt 12):3057–3075. [PubMed]
  • McGeoch DJ, Dalrymple MA, Davison AJ, Dolan A, Frame MC, McNab D, Perry LJ, Scott JE, Taylor P. The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J Gen Virol. 1988 Jul;69(Pt 7):1531–1574. [PubMed]
  • McLauchlan J, Phelan A, Loney C, Sandri-Goldin RM, Clements JB. Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing. J Virol. 1992 Dec;66(12):6939–6945. [PMC free article] [PubMed]
  • Newmeyer DD. The nuclear pore complex and nucleocytoplasmic transport. Curr Opin Cell Biol. 1993 Jun;5(3):395–407. [PubMed]
  • O'Hare P, Hayward GS. Expression of recombinant genes containing herpes simplex virus delayed-early and immediate-early regulatory regions and trans activation by herpesvirus infection. J Virol. 1984 Nov;52(2):522–531. [PMC free article] [PubMed]
  • Phelan A, Carmo-Fonseca M, McLaughlan J, Lamond AI, Clements JB. A herpes simplex virus type 1 immediate-early gene product, IE63, regulates small nuclear ribonucleoprotein distribution. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9056–9060. [PubMed]
  • Post LE, Mackem S, Roizman B. Regulation of alpha genes of herpes simplex virus: expression of chimeric genes produced by fusion of thymidine kinase with alpha gene promoters. Cell. 1981 May;24(2):555–565. [PubMed]
  • Quinlan MP, Chen LB, Knipe DM. The intranuclear location of a herpes simplex virus DNA-binding protein is determined by the status of viral DNA replication. Cell. 1984 Apr;36(4):857–868. [PubMed]
  • Rice SA, Knipe DM. Gene-specific transactivation by herpes simplex virus type 1 alpha protein ICP27. J Virol. 1988 Oct;62(10):3814–3823. [PMC free article] [PubMed]
  • Rice SA, Knipe DM. Genetic evidence for two distinct transactivation functions of the herpes simplex virus alpha protein ICP27. J Virol. 1990 Apr;64(4):1704–1715. [PMC free article] [PubMed]
  • Rice SA, Lam V. Amino acid substitution mutations in the herpes simplex virus ICP27 protein define an essential gene regulation function. J Virol. 1994 Feb;68(2):823–833. [PMC free article] [PubMed]
  • Rice SA, Lam V, Knipe DM. The acidic amino-terminal region of herpes simplex virus type 1 alpha protein ICP27 is required for an essential lytic function. J Virol. 1993 Apr;67(4):1778–1787. [PMC free article] [PubMed]
  • Rice SA, Su LS, Knipe DM. Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities. J Virol. 1989 Aug;63(8):3399–3407. [PMC free article] [PubMed]
  • Richardson WD, Roberts BL, Smith AE. Nuclear location signals in polyoma virus large-T. Cell. 1986 Jan 17;44(1):77–85. [PubMed]
  • Robbins J, Dilworth SM, Laskey RA, Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. [PubMed]
  • Roberts BL, Richardson WD, Smith AE. The effect of protein context on nuclear location signal function. Cell. 1987 Jul 31;50(3):465–475. [PubMed]
  • Rokeach LA, Haselby JA, Hoch SO. Molecular cloning of a cDNA encoding the human Sm-D autoantigen. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4832–4836. [PubMed]
  • Sacks WR, Greene CC, Aschman DP, Schaffer PA. Herpes simplex virus type 1 ICP27 is an essential regulatory protein. J Virol. 1985 Sep;55(3):796–805. [PMC free article] [PubMed]
  • Sandri-Goldin RM, Mendoza GE. A herpesvirus regulatory protein appears to act post-transcriptionally by affecting mRNA processing. Genes Dev. 1992 May;6(5):848–863. [PubMed]
  • Schmidt-Zachmann MS, Nigg EA. Protein localization to the nucleolus: a search for targeting domains in nucleolin. J Cell Sci. 1993 Jul;105(Pt 3):799–806. [PubMed]
  • Sekulovich RE, Leary K, Sandri-Goldin RM. The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0. J Virol. 1988 Dec;62(12):4510–4522. [PMC free article] [PubMed]
  • Silver PA. How proteins enter the nucleus. Cell. 1991 Feb 8;64(3):489–497. [PubMed]
  • Siomi H, Shida H, Nam SH, Nosaka T, Maki M, Hatanaka M. Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing. Cell. 1988 Oct 21;55(2):197–209. [PubMed]
  • Siomi H, Siomi MC, Nussbaum RL, Dreyfuss G. The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein. Cell. 1993 Jul 30;74(2):291–298. [PubMed]
  • Smith IL, Hardwicke MA, Sandri-Goldin RM. Evidence that the herpes simplex virus immediate early protein ICP27 acts post-transcriptionally during infection to regulate gene expression. Virology. 1992 Jan;186(1):74–86. [PubMed]
  • Smith MR, Greene WC. Characterization of a novel nuclear localization signal in the HTLV-I tax transactivator protein. Virology. 1992 Mar;187(1):316–320. [PubMed]
  • Southern PJ, Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed]
  • Srivastava M, Fleming PJ, Pollard HB, Burns AL. Cloning and sequencing of the human nucleolin cDNA. FEBS Lett. 1989 Jun 19;250(1):99–105. [PubMed]
  • Su L, Knipe DM. Herpes simplex virus alpha protein ICP27 can inhibit or augment viral gene transactivation. Virology. 1989 Jun;170(2):496–504. [PubMed]
  • Underwood MR, Fried HM. Characterization of nuclear localizing sequences derived from yeast ribosomal protein L29. EMBO J. 1990 Jan;9(1):91–99. [PubMed]
  • Upton C, Schiff L, Rice SA, Dowdeswell T, Yang X, McFadden G. A poxvirus protein with a RING finger motif binds zinc and localizes in virus factories. J Virol. 1994 Jul;68(7):4186–4195. [PMC free article] [PubMed]
  • Weeks KM, Ampe C, Schultz SC, Steitz TA, Crothers DM. Fragments of the HIV-1 Tat protein specifically bind TAR RNA. Science. 1990 Sep 14;249(4974):1281–1285. [PubMed]
  • Yan C, Mélèse T. Multiple regions of NSR1 are sufficient for accumulation of a fusion protein within the nucleolus. J Cell Biol. 1993 Dec;123(5):1081–1091. [PMC free article] [PubMed]
  • Zhao LJ, Padmanabhan R. Nuclear transport of adenovirus DNA polymerase is facilitated by interaction with preterminal protein. Cell. 1988 Dec 23;55(6):1005–1015. [PubMed]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)