Herpes simplex virus 1 ICP22 inhibits the transcription of viral gene promoters by binding to and blocking the recruitment of P-TEFb.

ICP22 is a multifunctional herpes simplex virus 1 (HSV-1) immediate early protein that functions as a general repressor of a subset of cellular and viral promoters in transient expression systems. Although the exact mechanism of repression remains unclear, this protein induces a decrease in RNA polymerase II Serine 2 (RNAPII Ser-2) phosphorylation, which is critical for transcription elongation. To characterize the mechanism of transcriptional repression by ICP22, we established an in vivo transient expression reporter system. We found that ICP22 inhibits transcription of the HSV-1 α, ß and γ gene promoters. The viral tegument protein VP16, which plays vital roles in initiation of viral gene expression and viral proliferation, can overcome the inhibitory effect of ICP22 on α-gene transcription. Further immunoprecipitation studies indicated that both ICP22 and VP16 bind to positive transcription elongation factor b (P-TEFb) and form a complex with it in vivo. We extended this to show that P-TEFb regulates transcription of the viral α-gene promoters and affects transcriptional regulation of ICP22 and VP16 on the α-genes. Additionally, ChIP assays demonstrated that ICP22 blocks the recruitment of P-TEFb to the viral promoters, while VP16 reverses this blocking effect by recruiting P-TEFb to the viral α-gene promoters through recognition of the TAATGARAT motif. Taken together, our results suggest that ICP22 interacts with and blocks the recruitment of P-TEFb to viral promoter regions, which inhibits transcription of the viral gene promoters. The transactivator VP16 binds to and induces the recruitment of P-TEFb to viral α-gene promoters, which counteracts the transcriptional repression of ICP22 on α-genes by recruiting p-TEFb to the promoter region.

Transient reversal of episome silencing precedes VP16-dependent transcription during reactivation of latent HSV-1 in neurons.

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.

Herpes simplex virus VP16, but not ICP0, is required to reduce histone occupancy and enhance histone acetylation on viral genomes in U2OS osteosarcoma cells.

The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure.

The major determinant for addition of tegument protein pUL48 (VP16) to capsids in herpes simplex virus type 1 is the presence of the major tegument protein pUL36 (VP1/2).

In this study a number of herpes simplex virus type 1 (HSV-1) proteins were screened, using a yeast-two-hybrid assay, for interaction with the tegument protein pUL48 (VP16). This approach identified interactions between pUL48 and the capsid proteins pUL19 (VP5), pUL38 (VP19C), and pUL35 (VP26). In addition, the previously identified interaction of pUL48 with the major tegument protein pUL36 (VP1/2) was confirmed. All of these interactions, except that of pUL35 and pUL48, could be confirmed using an in vitro pulldown assay. A subsequent pulldown assay with intact in vitro-assembled capsids, consisting of pUL19, pUL38, and pUL18 (VP23) with or without pUL35, showed no binding of pUL48, suggesting that the capsid/pUL48 interactions initially identified were more then likely not biologically relevant. This was confirmed by using a series of HSV-1 mutants lacking the gene encoding either pUL35, pUL36, or pUL37. For each HSV-1 mutant, analysis of purified deenveloped C capsids indicated that only in the absence of pUL36 was there a complete loss of capsid-bound pUL48, as well as pUL37. Collectively, this study shows for the first time that pUL36 is a major factor for addition of both pUL48 and pUL37, likely through a direct interaction of both with nonoverlapping sites in pUL36, to unenveloped C capsids during assembly of HSV-1.

Multiple hTAF(II)31-binding motifs in the intrinsically unfolded transcriptional activation domain of VP16.

Transcriptional activation domain (TAD) in virion protein 16 (VP16) of herpes simplex virus does not have any globular structure, yet exhibits a potent transcriptional activity. In order to probe the structural basis for the transcriptional activity of VP16 TAD, we have used NMR spectroscopy to investigate its detailed structural features. Results show that an unbound VP16 TAD is not merely "unstructured" but contains four short motifs (residues 424-433, 442-446, 465-467 and 472-479) with transient structural order. Pre-structured motifs in other intrinsically unfolded proteins (IUPs) were shown to be critically involved in target protein binding. The 472-479 motif was previously shown to bind to hTAF(II)31, whereas the hTAF(II)31-binding ability of other motifs found in this study has not been addressed. The VP16 TAD represents another IUP whose prestructured motifs mediate promiscuous binding to various target proteins.

Knockin mice expressing a chimeric p53 protein reveal mechanistic differences in how p53 triggers apoptosis and senescence.

The contribution of transcriptional activation to the p53 effector functions critical for tumor suppression, apoptosis and cellular senescence, remains unclear because of p53's ability to regulate diverse cellular processes in a transactivation-independent manner. Dissociating the importance of transactivation from other p53 functions, including regulating transcriptional repression, DNA replication, homologous recombination, centrosome duplication, and mitochondrial function, has been difficult because of overlapping motifs for these functions in the amino terminus. To determine the relative contribution of these activities and transactivation to p53 function, we generated knockin mice expressing a p53 mutant lacking domains involved in these transactivation-independent functions, while remaining competent for transactivation through fusion to the Herpes Simplex Virus VP16 transactivation domain. This chimeric mutant, termed p53(VP16), robustly activates the transcription of a range of p53 targets involved in both apoptosis and senescence. Intriguingly, despite being transactivation-competent, this chimeric protein shows selectivity in p53 effector function in mouse fibroblasts, with a capacity to trigger senescence but not apoptosis under a variety of conditions. Our study highlights the central role of p53 transactivation for senescence while suggesting that transactivation is insufficient for apoptosis, and provides insight into the mechanisms by which p53 serves as a tumor suppressor.

Doubling the size of the glucocorticoid receptor ligand binding pocket by deacylcortivazol.

A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 A(3), effectively doubling the size of the GR dexamethasone-binding pocket of 540 A(3) and yet leaving the structure of the coactivator binding site intact. DAC occupies only approximately 50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.

Incorporation of the herpes simplex virus type 1 tegument protein VP22 into the virus particle is independent of interaction with VP16.

Herpes simplex virus type 1 (HSV-1) virions contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The mechanisms underlying tegumentation remain largely undefined for all herpesviruses. Using glutathione S-transferase (GST) pulldowns and coimmunoprecipitation studies, we have identified a domain of the tegument protein VP22 that facilitates interaction with VP16. This region of VP22 (residues 165-225) overlaps the glycoprotein E (gE) binding domain of VP22 (residues 165-270), which is sufficient to mediate VP22 packaging into assembling virus particles. To ascertain the contribution of the VP16 and gE binding activities of VP22 to its virion incorporation, a transfection/infection based virion incorporation assay, using point mutants that discern between the two binding activities, was utilized. Our results suggest that interaction with VP16 is not required for incorporation of VP22 into virus particles and that binding to the cytoplasmic tail of gE is sufficient to facilitate packaging.

Replication-competent herpes simplex virus 1 isolates selected from cells transfected with a bacterial artificial chromosome DNA lacking only the UL49 gene vary with respect to the defect in the UL41 gene encoding host shutoff RNase.

To generate a null U(L)49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the U(L)49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-DeltaU(L)49 DNA yielded foci of degenerated cells that could not be expanded and a few replication-competent clones. The replication-competent viral clones derived from independent transfections yielded viruses that expressed genes with some delay, produced smaller plaques, and gave lower yields than wild-type virus. A key finding is that the independently derived replication-competent viruses lacked the virion host shutoff (vhs) activity expressed by the RNase encoded by the U(L)41 gene. One mutant virus expressed no vhs protein, whereas two others, derived from independent transfections, produced truncated vhs proteins consistent with the spontaneous in-frame deletion. In contrast, cells infected with the virus recovered upon transfection of the BAC-U(L)49R DNA (R-U(L)49) accumulated a full-length vhs protein, indicating that in the parental BAC-DeltaU(L)49 DNA, the U(L)41 gene was intact. We conclude that expression of the vhs protein in the absence of U(L)49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cells vhs requires both VP16 and VP22, the product of U(L)49, to be neutralized.

Liver stem cell-derived beta-cell surrogates for treatment of type 1 diabetes.

Consistent with the common embryonic origin of liver and pancreas as well the similar glucose-sensing systems in hepatocytes and pancreatic beta-cells, it should not be surprising that liver stem cells/hepatocytes can transdifferentiate into insulin-producing cells under high-glucose culture conditions or by genetic reprogramming. Persistent expression of the pancreatic duodenal homeobox-1 (Pdx1) transcription factor or its super-active form Pdx1-VP16 fusion protein in hepatic cells reprograms these cells into pancreatic beta-cell precursors. In vitro culture at elevated glucose concentrations or in vivo exposure to a hyperglycemia are required for further differentiation and maturation of liver-derived pancreatic beta-cell precursor into functional insulin-producing pancreatic beta-like cells. Under appropriate conditions, multiple pancreatic transcription factors can work in concert to reprogram liver stem/adult liver cells into functional insulin-producing cells. If such autologous liver-derived insulin-producing cells can be made to escape the type 1 diabetes-associated autoimmunity, they may serve as a valuable cell source for future cell replacement therapy without the need for life-long immunosuppression.

Role of Pax4 in Pdx1-VP16-mediated liver-to-endocrine pancreas transdifferentiation.

Although Pdx1-VP16 expression induces hepatic cell transdifferentiation into pancreatic precursor cells (WB-1), these incompletely reprogrammed cells fail to become glucose-sensitive insulin-producing cells in the absence of the activation of late-stage pancreatic transcription factors. As Pax4 promotes late-stage beta-cell differentiation and maturation, we generated lentiviral vector (LV) containing mouse Pax4 gene and developed two hepatic cell lines expressing Pax4 in the absence (WB-2 cells) or presence (WB-1A cells) of Pdx1-VP16, via LV-mediated gene transfer. Functional Pax4 protein expression in WB-2 and WB-1A cells was confirmed by electrophoretic mobility shift assay and Pdx1-VP16 protein expression in WB-1 and WB-1A cells was confirmed by Western blotting. Activation of Pax4 resulted in the expression of the late-stage transcription factors, including Pax6, Isl-1, and MafA, and generated a gene expression profile for WB-1A cells similar to that of functional rat insulinoma INS-1 cells. Insulin abundance in WB-1A cells was demonstrated by immunostaining. WB-1A cells exhibited glucose-responsive insulin release in vitro, and caused a rapid reversal of hyperglycemia following cell transplantation into streptozotocin-induced diabetic mice. Intraperitoneal glucose tolerance test showed a normal glucose response in WB-1, and WB-1A transplanted mice similar to that of normal mice. Removal of transplanted WB-1A cells resulted in a return of hyperglycemia, confirming that they were responsible for the observed normoglycemia. The explanted WB-1A cells exhibited strong insulin staining comparable to native islet beta-cells. These studies indicate that activation of Pax4 in Pdx1-VP16-expressing cells reprograms pancreatic precursor-like WB-1 cells into glucose-responsive, more mature insulin-producing cells.

ICP0 is not required for efficient stress-induced reactivation of herpes simplex virus type 1 from cultured quiescently infected neuronal cells.

Viral genes sufficient and required for herpes simplex virus type 1 (HSV-1) reactivation were identified using neuronally differentiated PC12 cells (ND-PC12 cells) in which quiescent infections with wild-type and recombinant strains were established. In this model, the expression of ICP0, VP16, and ICP4 from adenovirus vectors was sufficient to reactivate strains 17+ and KOS. The transactivators induced similar levels of reactivation with KOS; however, 17+ responded more efficiently to ICP0. To identify viral transactivators required for reactivation, we examined quiescently infected PC12 cell cultures (QIF-PC12 cell cultures) established with HSV-1 deletion mutants R7910 (deltaICP0), KD6 (deltaICP4), and in1814, a virus containing an insertion mutation in VP16. Although growth of these mutant viruses was impaired in ND-PC12 cells, R7910 and in1814 reactivated at levels equivalent to or better than their respective parental controls following stress (i.e., heat or forskolin) treatment. After treatment with trichostatin A, in1814 and 17+ reactivated efficiently, whereas the F strain and R7910 reactivated inefficiently. In contrast, KD6 failed to reactivate. In experiments with the recombinant KM100, which contains the in1814 mutation in VP16 and the n212 mutation in ICP0, spontaneous and stress-induced reactivation was observed. However, two strains, V422 and KM110, which lack the acidic activation domain of VP16, did not reactivate above low spontaneous levels after stress. These results demonstrate that in QIF-PC12 cells ICP0 is not required for efficient reactivation of HSV-1, the acidic activation domain of VP16 is essential for stress-induced HSV-1 reactivation, and HSV-1 reactivation is modulated uniquely by different treatment constraints and phenotypes.

Molecular dissection of Meis1 reveals 2 domains required for leukemia induction and a key role for Hoxa gene activation.

The Hoxa9 and Meis1 genes represent important oncogenic collaborators activated in a significant proportion of human leukemias with genetic alterations in the MLL gene. In this study, we show that the transforming property of Meis1 is modulated by 3 conserved domains, namely the Pbx interaction motif (PIM), the homeodomain, and the C-terminal region recently described to possess transactivating properties. Meis1 and Pbx1 interaction domain-swapping mutants are dysfunctional separately, but restore the full oncogenic activity of Meis1 when cotransduced in primary cells engineered to overexpress Hoxa9, thus implying a modular nature for PIM in Meis1-accelerated transformation. Moreover, we show that the transactivating domain of VP16 can restore, and even enhance, the oncogenic potential of the Meis1 mutant lacking the C-terminal 49 amino acids. In contrast to Meis1, the fusion VP16-Meis1 is spontaneously oncogenic, and all leukemias harbor genetic activation of endogenous Hoxa9 and/or Hoxa7, suggesting that Hoxa gene activation represents a key event required for the oncogenic activity of VP16-Meis1.

Tat gets the "green" light on transcription initiation.

Human immunodeficiency virus type 1 (HIV-1) Tat transactivation is an essential step in the viral life cycle. Over the past several years, it has become widely accepted that Tat exerts its transcriptional effect by binding the transactivation-responsive region (TAR) and enhancing transcriptional elongation. Consistent with this hypothesis, it has been shown that Tat promotes the binding of P-TEFb, a transcription elongation factor composed of cyclin T1 and cdk9, and the interaction of Tat with P-TEFb and TAR leads to hyperphosphorylation of the C-terminal domain (CTD) of RNA Pol II and increased processivity of RNA Pol II. A recent report, however, has generated renewed interest that Tat may also play a critical role in transcription complex (TC) assembly at the preinitiation step. Using in vivo chromatin immunoprecipitation assays, the authors reported that the HIV TC contains TBP but not TBP-associated factors. The stimulatory effect involved the direct interaction of Tat and P-TEFb and was evident at the earliest step of TC assembly, the TBP-TATA box interaction. In this article, we will review this data in context of earlier data which also support Tat's involvement in transcriptional complex assembly. Specifically, we will discuss experiments which demonstrated that Tat interacted with TBP and increased transcription initiation complex stability in cell free assays. We will also discuss studies which demonstrated that over expression of TBP alone was sufficient to obtain Tat activated transcription in vitro and in vivo. Finally, studies using self-cleaving ribozymes which suggested that Tat transactivation was not compatible with pausing of the RNA Pol II at the TAR site will be discussed.

Gene expression profiling of facilitated L-LTP in VP16-CREB mice reveals that BDNF is critical for the maintenance of LTP and its synaptic capture.

Expression of VP16-CREB, a constitutively active form of CREB, in hippocampal neurons of the CA1 region lowers the threshold for eliciting the late, persistent phase of long-term potentiation (L-LTP) in the Schaffer collateral pathway. This VP16-CREB-mediated L-LTP differs from the conventional late phase of LTP in not being dependent on new transcription. This finding suggests that in the transgenic mice the mRNA transcript(s) encoding the protein(s) necessary for this form of L-LTP might already be present in CA1 neurons in the basal condition. We used high-density oligonucleotide arrays to identify the mRNAs differentially expressed in the hippocampus of transgenic and wild-type mice. We then explored the contribution of the most prominent candidate genes revealed by our screening, namely prodynorphin, BDNF, and MHC class I molecules, to the facilitated LTP of VP16-CREB mice. We found that the overexpression of brain-derived neurotrophic factor accounts for an important component of this phenotype.

Purification and characterization of the Caenorhabditis elegans HCF protein and domains of human HCF.

The human cellular factor (HCF) is a multidomain protein that is implicated in processes of cell cycle progression, and it is recruited into a multicomponent assembly that triggers the expression of the herpes simplex virus genome. The amino-terminal domain of HCF has been proposed to form a "kelch" type beta-propeller fold, and the carboxy-terminal domain contains a repeat of a fibronectin-like motif. We describe the expression, purification, and characterization of the domains from the human HCF and of the full-length HCF from Caenorhabditis elegans. The purified recombinant C. elegans HCF can substitute for the human HCF in efficiently forming a multiprotein complex on a herpes simplex virus promoter element. As noted in earlier studies, a segment of human HCF encompassing the human kelch domain forms a stable complex on a viral promoter element. The purified fibronectin domain can also be recruited into this complex, but not into the stable complex formed with the minimal kelch domain. These results suggest that the fibronectin domain can interact with HCF in the transcriptional activating complex and that the association requires a region outside the putative beta-propeller.

Identification in vivo of different rate-limiting steps associated with transcriptional activators in the presence and absence of a GAGA element.

We analyzed the impact of a GAGA element on a transgenic promoter in Drosophila melanogaster that was activated by proteins composed of the Tet(on) DNA binding domain and either the heat shock factor (HSF) activation domain or a potent subdomain of VP16. Permanganate footprinting was used to monitor polymerase II (Pol II) on the transgenic promoters in vivo. Activation by Tet(on)-HSF but not by Tet(on)-VP16(A2) required the GAGA element; this correlated with the ability of the GAGA element to establish a paused Pol II. Although the GAGA element was not required for activation by Tet(on)-VP16(A2), the GAGA element greatly accelerated the rate of activation. The permanganate data also provided evidence that Pol II encountered different rate-limiting steps, following initiation in the presence of Tet(on)-HSF and Tet(on)-VP16(A2). The rate-limiting step in the presence of Tet(on)-HSF was release of Pol II paused about 20 to 40 nucleotides downstream from the start site. The rate-limiting step in the presence of Tet(on)-VP16(A2) occurred much closer to the transcription start site. Several biochemical studies have provided evidence for a structural transition shortly after Pol II initiates transcription. The behavior of Pol II in the presence of Tet(on)-VP16(A2) provides the first evidence that this transition occurs in vivo.

Gal4-VP16 directs ATP-independent chromatin reorganization in a yeast chromatin assembly system.

Major insights into the regulation of chromatin organization have stemmed from biochemical studies using Gal4-VP16, a chimeric transcriptional activator in which the DNA binding domain of Gal4p is fused to the activation domain of viral protein VP16. Unexpectedly, given previous intensive efforts to understand how Gal4-VP16 functions in the context of chromatin, we have uncovered a new mode of chromatin reorganization that is dependent on Gal4-VP16. This reorganization is performed by an activity in a crude DEAE (CD) fraction from budding yeast which also supports ATP-dependent assembly of physiologically spaced nucleosome arrays. Biochemical analysis reveals that the activity tightly associates with chromatin and reorganizes nucleosome arrays by a mechanism which is insensitive to ATP depletion after nucleosome assembly. It generates a chromatin organization in which a nucleosome is stably positioned immediately adjacent to Gal4p binding sites in the template DNA. Individual deletion of genes previously implicated in chromatin assembly and remodeling, namely, the histone chaperones NAP1, ASF1, and CAC1 and the SNF2-like DEAD/H ATPases SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20, does not significantly perturb reorganization. Therefore, Gal4-VP16-directed chromatin reorganization in yeast can occur by an ATP-independent mechanism that does not require SAGA, SWI/SNF, Isw1, or Isw2 chromatin remodeling complexes.

PDX-1/VP16 fusion protein, together with NeuroD or Ngn3, markedly induces insulin gene transcription and ameliorates glucose tolerance.

Diabetes is the most prevalent and serious metabolic disease, and the number of diabetic patients worldwide is increasing. The reduction of insulin biosynthesis in pancreatic beta-cells is closely associated with the onset and progression of diabetes, and thus it is important to search for ways to induce insulin-producing cells in non-beta-cells. In this study, we showed that a modified form of the pancreatic and duodenal homeobox factor 1 (PDX-1) carrying the VP16 transcriptional activation domain (PDX-1/VP16) markedly increases insulin biosynthesis and induces various pancreas-related factors in the liver, especially in the presence of NeuroD or neurogenin 3 (Ngn3). Furthermore, in streptozotocin-induced diabetic mice, PDX-1/VP16 overexpression, together with NeuroD or Ngn3, drastically ameliorated glucose tolerance. Thus PDX-1/VP16 expression, together with NeuroD or Ngn3, markedly induces insulin gene transcription and ameliorates glucose tolerance. This approach warrants further investigation and may have utility in the treatment of diabetes.

Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells.

Green fluorescent protein (GFP) has gained widespread use as a tool to visualize spatial and temporal patterns of gene expression in vivo. However, it is not generally accepted that GFP can also be used as a quantitative reporter of gene expression. We report that GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured by flow cytometry. Two pieces of evidence support this conclusion: GFP fluorescence increases in direct proportion to the GFP gene copy number delivered to cells by a replication-defective adenovirus vector, Ad.CMV-GFP, and the intensity of GFP fluorescence is directly proportional to GFP mRNA abundance in cells. This conclusion is further supported by the fact that the induction of GFP gene expression from two inducible promoters (i.e., the TRE and ICP0 promoters) is readily detected by flow cytometric measurement of GFP fluorescent intensity. Collectively, the results presented herein indicate that GFP fluorescence is a reliable and quantitative reporter of underlying differences in gene expression.