Transcriptional Activation by the Parvoviral Nonstructural Protein NS-1 Is Mediated via a Direct Interaction with Sp1

Abstract

The nonstructural protein NS-1, encoded by the parvovirus minute virus of mice, is a potent regulator of viral gene expression. NS-1 does not bind DNA in a sequence-specific manner, and the mechanism by which it modulates viral promoter function is unclear. We have used Gal4-NS-1 fusion protein constructs to identify and characterize an activating domain encoded within the C-terminal 88 amino acids of NS-1 which competes effectively with the acidic activator domain of the herpes simplex virus VP16 protein. DNA affinity chromatography and immunoprecipitation experiments demonstrate that protein-protein interactions between the transcription factor Sp1 and NS-1 are required to bind NS-1 to promoter DNA in vitro. Cotransfection of Gal4-NS-1 and Sp1-VP16 acidic activator constructs into Drosophila melanogaster Schneider cells, which lack endogenous Sp1, stimulates transcription from a minimal promoter containing five Gal4 binding sites, while single-construct transfections do not. Cotransfection of Schneider cells with wild-type NS-1 and Sp1 constructs activates transcription from a simian virus 40 promoter 10-to 30-fold over that of either construct alone. Thus, Sp1-NS-1 interactions in vivo can stimulate transcription from a heterologous promoter containing Sp1 binding sites.NS-1, an 83-kDa nuclear phosphoprotein, is the major nonstructural protein encoded by the parvovirus minute virus of mice (MVM). In addition to prominent roles in viral replication (32, 45) and the cytopathic effects associated with parvoviral infection (3, 5), NS-1 is an important regulator of viral gene expression. The MVM genome contains two transcription units; a promoter at map unit 4, termed P4, drives expression of the nonstructural proteins, including NS-1, while the downstream promoter, P38, controls expression of the structural capsid proteins (9). Regulatory effects on transcription from both promoters have been ascribed to NS-1. NS-1 transactivates the expression of viral structural proteins from the downstream P38 promoter (12, 31), enhancing expression approximately 1,000-fold over the low basal level (2). Effects of NS-1 on expression from its own promoter (P4) have been shown to range from activation to inhibition, presumably in a concentration-dependent manner (1,11,17,33). NS-1 also exhibits a trans-inhibitory effect on expression from a number of heterologous promoters (21, 33), most notably the Rous sarcoma virus long terminal repeat promoter.The mechanism by which NS-1 exerts this transcriptional regulation is currently not understood. Cellular and viral transcriptional regulatory proteins have generally been shown to contain two functionally separate domains (4, 30, 43). A binding domain, responsible for linking the protein to the appropriate promoter, allows an activation domain to interact with the transcriptional machinery to stimulate transcription. Activation domains tend to be acidic in nature although domains rich in glutamine or proline residues have also been described (26). Binding domains can interact either di...

Sp1 Binds Two Sites in the CD11c Promoter In Vivo Specifically in Myeloid Cells and Cooperates with AP1 to Activate Transcription

“…However, in the presence of all three expression plasmids, CD11c promoter activity increased 12-fold. Sp1 has been shown to cooperate through direct physical interactions with a number of proteins, including the erythroid transcription factor GATA-1 (12,31), the parvoviral nonstructural protein NS-1 (27), and the TATA box-associated factor dTAF II 110 (14). We have shown by in vitro DNase I footprinting analysis that the observed partial protection of the AP1 sites in the presence of a suboptimal amount of purified c-jun protein could be enhanced in the presence of increasing amounts of purified Sp1 protein.…”

“…That Sp1 binding can be influenced by other transcription factors has been documented in other gene systems. For example, the transcription factor Egr-1 (10), the erythroid-specific factors GATA (12,31) and NF-E1 (51), the parvoviral nonstructural protein NS-1 (27), and the pituitaryspecific factor Pit-1 (47) either act synergistically with Sp1 for gene activation or promote the displacement of bound Sp1 from overlapping sites within promoters.…”

Molecular Pathways: Rodent Parvoviruses—Mechanisms of Oncolysis and Prospects for Clinical Cancer Treatment

“…2) reveals a striking interplay between PVs and cellular components of various metabolic pathways related to gene expression or DNA replication and repair. Some of these components are direct binding partners of PV proteins, as exemplified by the formation of complexes between NS1 and cellular transcription factors (TBP, TFIIA, and SP1/3) or replication factors [replicator protein A (RPA) (12)(13)(14)]. Others are known effectors and/or targets of the PV life cycle that are not found in complexes with PV proteins.…”

Novel PKCηIs Required To Activate Replicative Functions of the Major Nonstructural Protein NS1 of Minute Virus of Mice

“…NS1 is endowed with numerous biochemical activities, such as ATP binding and hydrolysis (12,62), helicase (44,62), site-specific binding to the cognate recognition motif [ACCA] 2-3 which is scattered throughout the viral genome (13,19), and site-and strand-specific endonuclease (11,18,44). Furthermore, NS1 takes part in protein-protein interactions to form homo-oligomers (40,52) or complexes with cellular partner proteins like the transcription factor SP1 (35), the cochaperone SGT (20,58), or heterogeneous nuclear ribonucleoproteins (28). NS1 plays a key role in many processes necessary for progeny virus production.…”