Angela Pearson

BINDING OF BASAL TRANSCRIPTION FACTOR TFIIH TO THE ACIDIC ACTIVATION DOMAINS OF VP16 AND P53

Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIIH and its Saccharomyces cerevisiae counterpart, factor b. The VP16- and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.

Modular organization of the E2F1 activation domain and its interaction with general transcription factors TBP and TFIIH

The transcriptional activator E2F1 regulates the expression of genes at the G1/S boundary. We have characterized interactions of the E2F1 activation domain with two general transcription factors, the TATA-box binding protein (TBP) and TFIIH. Two distinct binding sites on E2F1 were identified for TBP (amino acids 386 – 417 and 415 – 437) each of which supported activation in mammalian cells when expressed as a fusion to a heterologous DNA-binding domain. Neither of these minimal activation domains independently bound TFIIH; rather, the TFIIH binding site of E2F1 overlaps both domains. Loss of TFIIH-binding by E2F1 resulted in a 60 – 65% reduction in transactivation, suggesting that the E2F1/TFIIH interaction is important, but not essential, for transactivation. The retinoblastoma protein (Rb) binds directly to E2F1 and represses E2F1-mediated transactivation. We have demonstrated that recombinant Rb can compete with TBP and the p62 subunit of TFIIH for binding to immobilized E2F1. A tumorigenic form of Rb deficient in repressing E2F1-mediated transactivation is likewise deficient in displacing TBP from E2F1. We propose that competition between Rb and both TBP and TFIIH for binding to E2F1 is a mechanism by which Rb inhibits transactivation by E2F1.

Activation of the Murine Dihydrofolate Reductase Promoter by E2F1 A REQUIREMENT FOR CBP RECRUITMENT

The E2F family of heterodimeric transcription factors plays an important role in the regulation of gene expression at the G1/S phase transition of the mammalian cell cycle. Previously, we have demonstrated that cell cycle regulation of murine dihydrofolate reductase (dhfr) expression requires E2F-mediated activation of the dhfr promoter in S phase. To investigate the mechanism by which E2F activates an authentic E2F-regulated promoter, we precisely replaced the E2F binding site in the dhfr promoter with a Gal4 binding site. Using Gal4-E2F1 derivatives, we found that E2F1 amino acids 409–437 contain a potent core transactivation domain. Functional analysis of the E2F1 core domain demonstrated that replacement of phenylalanine residues 413, 425, and 429 with alanine reduces both transcriptional activation of the dhfr promoter and protein-protein interactions with CBP, transcription factor (TF) IIH, and TATA-binding protein (TBP). However, additional amino acid substitutions for phenylalanine 429 demonstrated a strong correlation between activation of thedhfr promoter and binding of CBP, but not TFIIH or TBP. Finally, transactivator bypass experiments indicated that direct recruitment of CBP is sufficient for activation of the dhfrpromoter. Therefore, we suggest that recruitment of CBP is one mechanism by which E2F activates the dhfr promoter.

Identification, Localization, and Regulation of Expression of the UL24 Protein of Herpes Simplex Virus Type 1

ABSTRACT The UL24 gene of herpes simplex virus type 1 is conserved across many herpesviruses, but its protein product has not been identified. We expressed the UL24 gene in insect cells from a recombinant baculovirus and used the resulting protein to raise a rat antiserum. In immunoblotting experiments, this antiserum recognized a 30-kDa protein in lysates from infected cells. The identity of this species as UL24 was confirmed by using a virus encoding a truncated form of UL24. On the basis of biochemical fractionation of infected cells, UL24 appeared to be predominantly nucleus associated, especially at later times in infection. Although certain UL24 transcripts exhibit early kinetics, UL24 protein accumulated at later times in infection and levels were decreased sixfold in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid; thus, UL24 was expressed with leaky-late kinetics. The entire UL24 open reading frame is encoded by mRNAs with two different 5′ ends. A mutation that eliminates the more abundant transcripts that originate at the first transcription start site resulted in a 10-fold reduction in the level of UL24 expressed but did not eliminate expression. Thus, the less abundant transcripts originating at the second transcription start site can evidently be translated, although transcripts originating at the first start site appear to be the major contributors to the expression of UL24. We conclude that UL24 is a bona fide herpes simplex virus type 1 protein that associates primarily with nuclei and whose expression is regulated at multiple levels.

The conserved N-terminal domain of herpes simplex virus 1 UL24 protein is sufficient to induce the spatial redistribution of nucleolin

UL24 is widely conserved among herpesviruses but its function during infection is poorly understood. Previously, we discovered a genetic link between UL24 and the herpes simplex virus 1-induced dispersal of the nucleolar protein nucleolin. Here, we report that in the absence of viral infection, transiently expressed UL24 accumulated in both the nucleus and the Golgi apparatus. In the majority of transfected cells, nuclear staining for UL24 was diffuse, but a minor staining pattern, whereby UL24 was present in nuclear foci corresponding to nucleoli, was also observed. Expression of UL24 correlated with the dispersal of nucleolin. This dispersal did not appear to be a consequence of a general disaggregation of nucleoli, as foci of fibrillarin staining persisted in cells expressing UL24. The conserved N-terminal region of UL24 was sufficient to cause this change in subcellular distribution of nucleolin. Interestingly, a bipartite nuclear localization signal predicted within the C terminus of UL24 was dispensable for nuclear localization. None of the five individual UL24 homology domains was required for nuclear or Golgi localization, but deletion of these domains resulted in the loss of nucleolin-dispersal activity. We determined that a nucleolar-targeting signal was contained within the first 60 aa of UL24. Our results show that the conserved N-terminal domain of UL24 is sufficient to specifically induce dispersal of nucleolin in the absence of other viral proteins or virus-induced cellular modifications. These results suggest that UL24 directly targets cellular factors that affect the composition of nucleoli.

Failure of Thymidine Kinase-Negative Herpes Simplex Virus To Reactivate from Latency following Efficient Establishment

ABSTRACT Thymidine kinase-negative mutants of herpes simplex virus did not reactivate from latency in mouse trigeminal ganglia, even when their latent viral loads were comparable to those that permitted reactivation by wild-type virus. Thus, reduced establishment of latency does not suffice to account for the failure to reactivate.

Involvement of the UL24 protein in herpes simplex virus 1-induced dispersal of B23 and in nuclear egress

UL24 of herpes simplex virus 1 (HSV-1) is widely conserved within the Herpesviridae family. Herein, we tested the hypothesis that UL24, which we have previously shown to induce the redistribution of nucleolin, also affects the localization of the nucleolar protein B23. We found that HSV-1-induced dispersal of B23 was dependent on UL24. The conserved N-terminal portion of UL24 was sufficient to induce the redistribution of B23 in transient transfection assays. Mutational analysis revealed that the endonuclease motif of UL24 was important for B23 dispersal in both transfected and infected cells. Nucleolar protein relocalization during HSV-1 infection was also observed in non-immortalized cells. Analysis of infected cells by electron microscopy revealed a decrease in the ratio of cytoplasmic versus nuclear viral particles in cells infected with a UL24-deficient strain compared to KOS-infected cells. Our results suggest that UL24 promotes nuclear egress of nucleocapsids during HSV-1 infection, possibly though effects on nucleoli.

Conserved Residues in the UL24 Protein of Herpes Simplex Virus 1 Are Important for Dispersal of the Nucleolar Protein Nucleolin

ABSTRACT The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39°C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.

ICP27 selectively regulates the cytoplasmic localization of a subset of viral transcripts in herpes simplex virus type 1-infected cells.

ABSTRACT Evidence suggests that the herpes simplex virus regulatory protein ICP27 mediates the nuclear export of viral transcripts; however, the extent of this activity during infection is unclear. ICP27 is required for efficient expression of the long, leaky-late UL24 transcripts, but not for that of the short, early UL24 transcripts. We found that infection by an ICP27-null mutant resulted in undetectable UL24 protein expression, which represented at least a 70-fold decrease relative to that of wild-type virus. Because lack of ICP27 had a greater effect on levels of UL24 protein than on transcripts, we examined its effect on subcellular localization of UL24 transcripts. In wild-type-infected cells, both short and long UL24 transcripts fractionated predominantly with the cytoplasm. However, in the absence of ICP27, greater than 50% of long UL24 transcripts were nuclear, while the percentage of short UL24 transcripts that were cytoplasmic was not reduced. These results also imply that the short UL24 transcripts are translated poorly. The effect of ICP27 on cytoplasmic localization of the long UL24 transcripts did not extend to other transcripts with which it shared a common 3′ end or to other transcripts tested, including gC and UL42, whose overall expression is highly dependent on ICP27. Thus, the dual effects of ICP27 on mRNA accumulation and cytoplasmic localization are not always linked. These results identify viral transcripts that are dependent on ICP27 for efficient cytoplasmic localization during infection, but they also indicate the existence of ICP27-independent nuclear export pathways that are accessible to many viral transcripts during infection.

The Ribonucleotide Reductase R1 Subunits of Herpes Simplex Virus 1 and 2 Protect Cells against Poly(I · C)-Induced Apoptosis

ABSTRACT We recently provided evidence that the ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 (HSV-1 and -2) protect cells against tumor necrosis factor alpha- and Fas ligand-induced apoptosis by interacting with caspase 8. Double-stranded RNA (dsRNA) is a viral intermediate known to initiate innate antiviral responses. Poly(I · C), a synthetic analogue of viral dsRNA, rapidly triggers caspase 8 activation and apoptosis in HeLa cells. Here, we report that HeLa cells after HSV-1 and HSV-2 infection were quickly protected from apoptosis caused by either extracellular poly(I · C) combined with cycloheximide or transfected poly(I · C). Cells infected with the HSV-1 R1 deletion mutant ICP6Δ were killed by poly(I · C), indicating that HSV-1 R1 plays a key role in antiapoptotic responses to poly(I · C). Individually expressed HSV R1s counteracted caspase 8 activation by poly(I · C). In addition to their binding to caspase 8, HSV R1s also interacted constitutively with receptor-interacting protein 1 (RIP1) when expressed either individually or with other viral proteins during HSV infection. R1(1-834)-green fluorescent protein (GFP), an HSV-2 R1 deletion mutant protein devoid of antiapoptotic activity, did not interact with caspase 8 and RIP1, suggesting that these interactions are required for protection against poly(I · C). HSV-2 R1 inhibited the interaction between the Toll/interleukin-1 receptor domain-containing adaptor-inducing beta interferon (IFN-β) (TRIF) and RIP1, an interaction that is essential for apoptosis triggered by extracellular poly(I · C) plus cycloheximide or TRIF overexpression. TRIF silencing reduced poly(I · C)-triggered caspase 8 activation in mock- and ICP6Δ-infected cells, confirming that TRIF is involved in poly(I · C)-induced apoptosis. Thus, by interacting with caspase 8 and RIP1, HSV R1s impair the apoptotic host defense mechanism prompted by dsRNA.