Herpes simplex virus type-1 (HSV-1) establishes latency in peripheral neurons, creating a permanent source of recurrent infections. The latent genome is assembled into chromatin and lytic cycle genes are silenced. Processes that orchestrate reentry into productive replication (reactivation) remain poorly understood. We have used latently infected cultures of primary superior cervical ganglion (SCG) sympathetic neurons to profile viral gene expression following a defined reactivation stimulus. Lytic genes are transcribed in two distinct phases, differing in their reliance on protein synthesis, viral DNA replication and the essential initiator protein VP16. The first phase does not require viral proteins and has the appearance of a transient, widespread de-repression of the previously silent lytic genes. This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron. During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters. The transactivation function supplied by VP16 promotes increased viral lytic gene transcription leading to the onset of genome amplification and the production of infectious viral particles. Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons.

p53 stimulates the transcription of a number of genes, such as MDM2, Waf1, and GADD45. We and others have shown previously that this activity of p53 can be inhibited by adenovirus type 2 or 12 large E1B proteins. Here we show that the adenovirus E1A proteins also can repress the stimulation of transcription by p53, both in transient transfections and in stably transfected cell lines. The inhibition by E1A occurs without a significant effect on the DNA-binding capacity of p53. Furthermore, the activity of a fusion protein containing the N-terminal part of p53 linked to the GAL4 DNA-binding domain can be suppressed by E1A. This indicates that E1A affects the transcription activation domain of p53, although tryptic phosphopeptide mapping revealed that the level of phosphorylation of this domain does not change significantly in E1A-expressing cell lines. Gel filtration studies, however, showed p53 to be present in complexes of increased molecular weight as a result of E1A expression. Apparently, E1A can cause increased homo-or hetero-oligomerization of p53, which might result in the inactivation of the transcription activation domain of p53. Additionally, we found that transfectants stably expressing E1A have lost the ability to arrest in G 1 after DNA damage, indicating that E1A can abolish the normal biological function of p53.Cell proliferation is a tightly regulated process in which the p53 tumor suppressor protein plays an important role. A possible mechanism by which p53 can, at least partially, fulfill its tumor suppressor function is by regulating the expression of a set of target genes. p53 is known to be able to down-regulate the expression of a number of genes (15,33,46,53), probably by interacting with the basal transcription machinery (1,41,48). On the other hand, p53 has also been found to activate the transcription of genes containing a p53-responsive element (12,43,57). The list of p53-inducible genes at the moment features genes like Waf1 (11), MDM2 (2, 64), GADD45 (19), the cyclin G gene (38, 67), EGFR (7), and Bax (36, 47), but probably many more p53-responsive genes will be identified in the near future. Most naturally occurring p53 mutants have lost their normal transcription-regulatory functions (20). In addition, a gain of function has been shown for certain p53 mutants (9, 24).We have used the adenovirus transformation system to obtain more insight into the mechanism of regulation of transcription by p53 and to answer the question whether structural changes of p53 play a role in these modulations. It has been shown that the two major adenovirus E1B proteins have different effects on the transcription-regulatory functions of p53. The small E1B protein can inhibit only the transcription-inhibitory function of p53 (44,49,51), whereas the large E1B protein can inhibit the stimulation (66) as well as the repression of transcription by p53 (51). We have previously shown that in adenovirus type 12 (Ad12)-transformed cells, p53 is present in complexes of increased molecular weight compared with those...

Inactivation of p53 and p73 is known to promote thyroid cancer progression. We now describe p63 expression and function in human thyroid cancer. TAp63a is expressed in most thyroid cancer specimens and cell lines, but not in normal thyrocytes. However, in thyroid cancer cells TAp63a fails to induce the target genes (p21Cip1, Bax, MDM2) and, as a consequence, cell cycle arrest and apoptosis occur. Moreover, TAp63a antagonizes the effect of p53 on target genes, cell viability and foci formation, and p63 gene silencing by small interfering (si) RNA results in improved p53 activity. This unusual effect of TAp63a depends on the protein C-terminus, since TAp63b and TAp63g isoforms, which have a different arrangement of their C-terminus, are still able to induce the target genes and to exert tumour-restraining effects in thyroid cancer cells. Our data outline the existence of a complex network among p53 family members, where TAp63a may promote thyroid tumour progression by inactivating the tumour suppressor activity of p53.

A vaccine consisting of rhesus cytomegalovirus (RhCMV) pp65-2, gB and IE1 expressed via modiWed vaccinia Ankara (MVA) was evaluated in rhesus macaques with or without prior priming with expression plasmids for the same antigens. Following two MVA treatments, comparable levels of anti-gB, pp65-2 and neutralizing antibody responses, and pp65-2-and IE1-speciWc cellular immune responses were detected in both vaccinated groups. Similar reductions in plasma peak viral loads were observed in these groups compared to untreated controls. This study demonstrates the immunogenicity and protective eYcacy of rMVA-based RhCMV subunit vaccines in a primate host and warrants further investigation to improve the eYcacy of subunit vaccines against CMV.

A therapeutic CMV vaccine incorporating an antigenic repertoire capable of eliciting a cellular immune response has yet to be successfully implemented for patients who already have acquired an infection. To address this problem, we have developed a vaccine candidate derived from modified vaccinia Ankara (MVA) that expresses three immunodominant antigens (pp65, IE1, IE2) from CMV. The novelty of this vaccine is the fusion of two adjacent exons from the immediate-early region of CMV, their successful expression in MVA, and robust immunogenicity in both primary and memory response models. Evaluation of the immunogenicity of the viral vaccine in mouse models shows that it can stimulate primary immunity against all three antigens in both the CD4(+) and CD8(+) T cell subsets. Evaluation of human PBMC from healthy CMV-positive donors or patients within 6 months of receiving hematopoietic cell transplant shows robust stimulation of existing CMV-specific CD4(+) and CD8(+) T cell subsets.