Recruitment of TRRAP required for oncogenic transformation by E1A.

TRRAP links Myc with histone acetylases and appears to be an important mediator of its oncogenic function. Here we show that interaction with TRRAP is required for cellular transformation not only by Myc, but also by the adenovirus E1A protein. Substitution of the 262 N-terminal residues of Myc with a small domain of E1A (residues 12-54) restores Myc transforming function. E1A(12-54) contains a TRRAP-interaction domain, that recruits TRRAP to either E1A-Myc chimeras, or the native 12S E1A protein. Overexpression of a competing TRRAP fragment in vivo blocks interaction of cellular TRRAP with either E1A-Myc or E1A, and suppresses cellular transformation by both oncoproteins. Moreover, E1A(Delta26-35) that fails to bind TRRAP but is capable of binding the Retinoblastoma (Rb)-family and p300/CBP proteins is defective in cellular immortalization, transformation and cell cycle deregulation. Thus in addition to disrupting Rb and p300/CBP functions, E1A must recruit TRRAP to transform cells.

Induction of S phase and apoptosis by the human papillomavirus type 16 E7 protein are separable events in immortalized rodent fibroblasts.

The HPV16 E7 oncoprotein neutralizes several cell cycle checkpoints, favouring the entry of quiescent cells into S phase. This activity is mediated in part by association of E7 with the pocket proteins and consequent activation of E2F transcription factors. In addition, HPV16 E7 protein is able to promote apoptosis. In this study we demonstrate that the ability to induce apoptosis is a common property of E7s belonging to both benign and malignant HPV types. The E7-induced apoptosis is mediated by inactivation of pRb, whilst neutralization of the other two pRB-related proteins, p107 and 130, is not sufficient to trigger apoptosis. Moreover, we show that certain point mutations in the conserved region 1 (CR1) of HPV16 E7 abolish the induction of apoptosis without altering the ability to stimulate S phase. Thus, these two E7-mediated cellular events, apoptosis and S phase entry, can be separated in immortalized rodent fibroblasts. Our findings demonstrate that the E7-mediated pRb destabilization is not required for its ability to drive quiescent cells into S phase and to induce apoptosis. Finally, expression of E7 proteins in NIH3T3, which lack a functional p19ARF, does not lead to p53 accumulation, indicating that the E7 impacts upon additional cellular pathways to promote apoptosis.

Activation of promoter P4 of the autonomous parvovirus minute virus of mice at early S phase is required for productive infection.

Autonomous parvoviruses are tightly dependent on host cell factors for various steps of their life cycle. In particular, DNA replication and gene expression of the prototype strain of the minute virus of mice (MVMp) are closely linked to the onset of host cell DNA replication, pointing to the involvement of an S-phase-specific cellular factor(s) in parvovirus multiplication. The viral nonstructural protein NS-1 is absolutely required for parvovirus DNA replication and is able to transcriptionally regulate parvoviral and heterologous promoters. We previously showed that the promoter P4, which directs the transcription unit encoding the NS proteins, is activated at the onset of S phase. This activation is dependent on an E2F motif in the proximal region of promoter P4. An infectious MVM DNA clone was mutated in the E2F motif of P4. The wild type and the E2F mutant derivative were tested for their ability to produce progeny viruses after transfection of permissive cells. In the context of the whole MVMp genome, the E2F mutation abolished P4 induction in S phase and inactivated the infectious molecular clone, which failed to become amplified and generate progeny particles. The virus could be rescued when NS proteins were supplied in trans, showing that P4 hyperactivity in S is needed to reach a level of NS-1 expression that is sufficient to drive the viral replication cycle. These data show that E2F-mediated P4 activation at the early S phase is a limiting factor for parvovirus production. The primary barrier to parvovirus gene expression in G1 is thought to be promoter formation rather than activation, due to the poor conversion of the parental single-strand genome to a duplex form. The S dependence of P4 activation may therefore be a sign of the virus adaptation to life in the S-phase host cell. If the conversion block in G1 were to be leaky, the S induction of promoter P4 could be envisioned as a safeguard against the production of toxic NS proteins until cells reach the S phase and provide the full machinery for parvovirus replication.

Opposite transcriptional effects of cyclic AMP-responsive elements in confluent or p27KIP-overexpressing cells versus serum-starved or growing cells.

The minute virus of mice, an autonomous parvovirus, requires entry of host cells into the S phase of the cell cycle for its DNA to be amplified and its genes expressed. This work focuses on the P4 promoter of this parvovirus, which directs expression of the transcription unit encoding the parvoviral nonstructural polypeptides. These notably include protein NS1, necessary for the S-phase-dependent burst of parvoviral DNA amplification and gene expression. The activity of the P4 promoter is shown to be regulated in a cell cycle-dependent manner. At the G1/S-phase transition, the promoter is activated via a cis-acting DNA element which interacts with phase-specific complexes containing the cellular transcription factor E2F. It is inhibited, on the other hand, in cells arrested in G1 due to contact inhibition. This inhibitory effect is not observed in serum-starved cells. It is mediated in cis by cyclic AMP response elements (CREs). Unlike serum-starved cells, confluent cells accumulate the cyclin-dependent kinase inhibitor p27, suggesting that the switch from CRE-mediated activation to CRE-mediated repression involves the p27 protein. Accordingly, plasmid-driven overexpression of p27 causes down-modulation of promoter P4 in growing cells, depending on the presence of at least two functional CREs. No such effect is observed with two other cyclin-dependent kinase inhibitors, p16 and p21. Given the importance of P4-driven synthesis of protein NS1 in parvoviral DNA amplification and gene expression, the stringent S-phase dependency of promoter P4 is likely a major determinant of the absolute requirement of the minute virus of mice for host cell proliferation.

Inhibition of parvovirus minute virus of mice replication by a peptide involved in the oligomerization of nonstructural protein NS1.

The large nonstructural protein NS1 of the minute virus of mice and other parvoviruses is involved in essential steps of the viral life cycle, such as DNA replication and transcriptional regulation, and is a major contributor to the toxic effect on host cells. Various biochemical functions, such as ATP binding, ATPase, site-specific DNA binding and nicking, and helicase activities, have been assigned to NS1. Homo-oligomerization is a prerequisite for a number of proteins to be fully functional. In particular, helicases generally act as homo-oligomers. Indirect evidence of NS1 self-association has been recently obtained by a nuclear cotransport assay (J. P. Nüesch and P. Tattersall, Virology 196:637-651, 1993). In order to demonstrate the oligomerizing property of NS1 in a direct way and localize the protein region(s) involved, the yeast two-hybrid system was used in combination with deletion mutagenesis across the whole NS1 molecule, followed by high-resolution mapping of the homo-oligomerization domain by a peptide enzyme-linked immunosorbent assay method. This study led to the identification of a distinct NS1 peptide that contains a bipartite domain involved in NS1 oligomerization. Furthermore, this isolated peptide was found to act as a specific competitive inhibitor and suppress NS1 helicase activity in vitro and parvovirus DNA replication in vivo, arguing for the involvement of NS1 oligomerization in these processes. Our results point to drug targeting of oligomerization motifs of viral regulatory proteins as a potentially useful antiviral strategy.

The minute virus of mice (MVM) nonstructural protein NS1 induces nicking of MVM DNA at a unique site of the right-end telomere in both hairpin and duplex conformations in vitro.

The right-end telomere of replicative form (RF) DNA of the autonomous parvovirus minute virus of mice (MVM) consists of a sequence that is self-complementary except for a three nucleotide loop around the axis of symmetry and an interior bulge of three unpaired nucleotides on one strand (designated the right-end 'bubble'). This right-end inverted repeat can exist in the form of a folded-back strand (hairpin conformation) or in an extended form, base-paired to a copy strand (duplex conformation). We recently reported that the right-end telomere is processed in an A9 cell extract supplemented with the MVM nonstructural protein NS1. This processing is shown here to result from the NS1-dependent nicking of the complementary strand at a unique position 21 nt inboard of the folded-back genomic 5' end. DNA species terminating in duplex or hairpin configurations, or in a mutated structure that has lost the right-end bulge, are all cleaved in the presence of NS1, indicating that features distinguishing these structures are not prerequisites for nicking under the in vitro conditions tested. Cleavage of the hairpin structure is followed by strand-displacement synthesis, generating the right-end duplex conformation, while processing of the duplex structure leads to the release of free right-end telomeres. In the majority of molecules, displacement synthesis at the right terminus stops a few nucleotides before reaching the end of the template strand, possibly due to NS1 which is covalently bound to this end. A fraction of the right-end duplex product undergoes melting and re-folding into hairpin structures (formation of a 'rabbit-ear' structure).

ras oncogene-dependent activation of the P4 promoter of minute virus of mice through a proximal P4 element interacting with the Ets family of transcription factors.

The P4 promoter of parvovirus minute virus of mice (MVMp) directs transcription of the genes coding for nonstructural proteins. The activity of promoter P4 is regulated by several cis-acting DNA elements. Among these, a promoter-proximal GC box was shown to be essential for P4 activity (J.K. Ahn, B.J. Gavin, G. Kumar, and D.C. Ward, J. Virol. 63:5425-5439, 1989). In this study, a motif homologous to an Ets transcription factor-binding site (EBS), located immediately upstream from the GC box, was found to be required for the full activity of promoter P4 in the ras-transformed rat fibroblast cell line FREJ4. In normal parental FR3T3 cells, the transcriptional function of P4 EBS was insignificant but could be restored by transient cell transfection with the c-Ha-ras oncogene. P4 EBS may thus contribute to the stimulation of promoter P4 in ras-transformed cells. Electrophoretic mobility shift assays using crude extracts from FREJ4 cells revealed the binding of a member(s) of the Ets family of transcription factors to the P4 EBS, as well as the interaction of two members of the Sp1 family, Sp1 and Sp3, with the adjacent GC box. When produced in Drosophila melanogaster SL2 cells, Ets-1 and Sp1 proteins acted synergistically to transactivate promoter P4 through their respective cognate sites.

Upstream CREs participate in the basal activity of minute virus of mice promoter P4 and in its stimulation in ras-transformed cells.

The activity of the P4 promoter of the parvovirus minute virus of mice (prototype strain MVMp) is stimulated in ras-transformed FREJ4 cells compared with the parental FR3T3 line. This activation may participate in the oncolytic effect of parvoviruses, given that P4 drives a transcriptional unit encoding cytotoxic nonstructural proteins. Our results suggest that the higher transcriptional activity of promoter P4 in FREJ4 cells is mediated at least in part by upstream CRE elements. Accordingly, mutations in the CRE motifs impair P4 function more strongly in the FREJ4 derivative than in its FR3T3 parent. Further evidence that these elements contribute to hyperactivity of the P4 promoter in the ras transformant is the fact that they form distinct complexes with proteins from FREJ4 and FR3T3 cell extracts. This difference can be abolished by treating the FREJ4 cell extracts with cyclic AMP-dependent protein kinase (PKA) or treating original cultures with a PKA activator. These findings can be linked with two previously reported features of ras-transformed cells: the activation of a PKA-inhibited protein kinase cascade and the reduction of PKA-induced protein phosphorylation. In keeping with these facts, P4-directed gene expression can be up- or downmodulated in vivo by exposing cells to known inhibitors or activators of PKA, respectively.

Mapping of upstream regulatory elements in the P4 promoter of parvovirus minute virus of mice.

The early promoter (P4) of the autonomous parvovirus minute virus of mice (prototype strain) directs the expression of the transcription unit coding for the nonstructural proteins NS1 and NS2. Although proximal promoter elements (GC and TATA boxes) are essential for P4 activity in vivo, additional upstream sequences appear to be required for optimal transcription. Therefore, associations of proteins with the upstream regulatory region of promoter P4 were studied in the rat fibroblast cell line FREJ4 by gel retardation and in vitro as well as in vivo footprinting assays. This led to the identification of at least four distinct upstream elements that interacted with cellular proteins. The functionality of these elements was supported by the reduced level of gene expression driven by corresponding linker-substitutive mutants of promoter P4.