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Cytodifferentiating Agents Affect the Replication of Herpes Simplex Virus Type 1 in the Absence of Functional VP16
Abstract
The herpes simplex virus type 1 (HSV-1) mutant in1814 encodes an altered form of the virion protein VP16 that is unable to transactivate immediate-early (IE) transcription. As a consequence of the mutation, in1814 initiates productive replication inefficiently after infection of tissue culture cells. Previous studies showed that this defect could be overcome by the inclusion in the culture medium of hexamethylene bisacetamide (HMBA), a compound that promotes the differentiation of murine erythroleukemia cells (MELCs). The effects of additional agents known to induce differentiation of MELCs were investigated. N'-Methylnicotinamide, at concentrations optimal for the induction of MELCs, complemented the replication of in1814 and stimulated IE gene expression. Suberoyl bishydroxamic acid and suberoylanilide hydroxamic acid, which induce differentiation of MELCs at micromolar concentrations, did not complement in1814 but specifically blocked the action of HMBA. The histone deacetylase inhibitor trichostatin A, which also induces differentiation of MELCs, antagonized the effect of HMBA in a manner similar to that of suberoyl bishydroxamic acid and suberoylanilide hydroxamic acid. The results demonstrate that the requirement for VP16 activity is dependent on the metabolic state of the host cell and that the pathways leading to complementation of in1814 and differentiation of MELCs are overlapping but not identical.
... The restoration of serine at position 375 and the absence of any adventitious mutations in the VP16 gene were confirmed by DNA sequencing (not shown). Because mutants lacking the transactivation function of VP16 enter the lytic cycle inefficiently at low multiplicity, the standard plaque assay (a low multiplicity assay which is used to quantify infectious viral particles) will underestimate the amount of virus present (Ace, McKee et al. 1989;Smiley and Duncan 1997;Preston and McFarlane 1998). The addition of the cell differentiating agent hexamethylbisacetamide (HMBA) increases the plaquing efficiency of in1814 and other VP16 transactivation deficient mutants with minimal effect on wild type virus (Preston and McFarlane 1998). ...
... Because mutants lacking the transactivation function of VP16 enter the lytic cycle inefficiently at low multiplicity, the standard plaque assay (a low multiplicity assay which is used to quantify infectious viral particles) will underestimate the amount of virus present (Ace, McKee et al. 1989;Smiley and Duncan 1997;Preston and McFarlane 1998). The addition of the cell differentiating agent hexamethylbisacetamide (HMBA) increases the plaquing efficiency of in1814 and other VP16 transactivation deficient mutants with minimal effect on wild type virus (Preston and McFarlane 1998). A comparison of the plaquing efficiency of SJO2, SJO2R, and parental strain KOS in rabbit skin cells (RSC) demonstrated that as reported for in1814 (McFarlane, Daksis et al. 1992;Smiley and Duncan 1997;Preston and McFarlane 1998), treatment with 5mm HMBA resulted in a ∼10 fold increase in plaques of SJO2 (for example, a typical SJO2 stock yields 1.×10 7 pfu/ml in the absence of HMBA and 1.7×10 8 pfu/ml in the presence of HMBA). ...
... The addition of the cell differentiating agent hexamethylbisacetamide (HMBA) increases the plaquing efficiency of in1814 and other VP16 transactivation deficient mutants with minimal effect on wild type virus (Preston and McFarlane 1998). A comparison of the plaquing efficiency of SJO2, SJO2R, and parental strain KOS in rabbit skin cells (RSC) demonstrated that as reported for in1814 (McFarlane, Daksis et al. 1992;Smiley and Duncan 1997;Preston and McFarlane 1998), treatment with 5mm HMBA resulted in a ∼10 fold increase in plaques of SJO2 (for example, a typical SJO2 stock yields 1.×10 7 pfu/ml in the absence of HMBA and 1.7×10 8 pfu/ml in the presence of HMBA). In contrast, SJO2R and parental strain KOS exhibited < 1.8 fold differential, confirming that by this criterion, the rescued virus was restored to wild type. ...
Development of novel prevention and treatment strategies for herpes simplex virus (HSV) mediated diseases is dependent upon an accurate understanding of the central molecular events underlying the regulation of latency and reactivation. We have recently shown that the transactivation function of the virion protein VP16 is a critical determinant in the exit from latency in vivo. HSV-1 strain SJO2 carries a single serine to alanine substitution at position 375 in VP16 which disrupts its interaction with its essential co-activator Oct-1. Here we report that SJO2 is severely impaired in its ability to exit latency in vivo. This result reinforces our prior observations with VP16 transactivation mutant, in1814, in which VP16 interaction with Oct-1 is also disrupted and solidifies the importance of the VP16-Oct-1 interaction in the early steps in HSV-1 reactivation.
... These results are in general agreement with previous reports [16,30]. Interpretation of this result is complicated by the fact that at low moi (such as a plaque assay), mutants lacking the transactivation function of VP16 enter the lytic cycle inefficiently, leading to an underestimate the amount of virus present [40,41,74]. Several strategies have been utilized to overcome this problem, including VP16 expressing cell lines, and superinfection with a replication impaired virus [16,30]. ...
... Several strategies have been utilized to overcome this problem, including VP16 expressing cell lines, and superinfection with a replication impaired virus [16,30]. The addition of the cell differentiating agent, hexamethylene bisacetamide (HMBA), to cell cultures increases the plaquing efficiency of in1814 [74]. As shown inFigure 3C and 3D, the addition of HMBA to the culture medium revealed the presence of 100 and 500 fold more virus in in1814 and 17VP16D422 eye homogenates (day 2 pi), respectively. ...
... The generation and characterization of the VP16 transactivation deficient mutant in1814 and its genomically restored counterpart, 1814R, have been described [16,40]. Where indicated, the plaquing efficiency of in1814 was enhanced by the addition of 3 mM hexamethylene bisacetamide (HMBA, Sigma) as described [74,103]. The mutant DTfi and its genomically restored counterpart, DTfiR were a kind gift of David Leib, Washington University, and have been described in detail [29,104]. ...
The mechanism controlling the exit from herpes simplex virus latency (HSV) is of central importance to recurrent disease and transmission of infection, yet interactions between host and viral functions that govern this process remain unclear. The cascade of HSV gene transcription is initiated by the multifunctional virion protein VP16, which is expressed late in the viral replication cycle. Currently, it is widely accepted that VP16 transactivating function is not involved in the exit from latency. Utilizing the mouse ocular model of HSV pathogenesis together with genetically engineered viral mutants and assays to quantify latency and the exit from latency at the single neuron level, we show that in vivo (i) the VP16 promoter confers distinct regulation critical for viral replication in the trigeminal ganglion (TG) during the acute phase of infection and (ii) the transactivation function of VP16 (VP16TF) is uniquely required for the exit from latency. TG neurons latently infected with the VP16TF mutant in1814 do not express detectable viral proteins following stress, whereas viruses with mutations in the other major viral transcription regulators ICP0 and ICP4 do exit the latent state. Analysis of a VP16 promoter/reporter mutant in the background of in1814 demonstrates that the VP16 promoter is activated in latently infected neurons following stress in the absence of other viral proteins. These findings support the novel hypothesis that de novo expression of VP16 regulates entry into the lytic program in neurons at all phases of the viral life cycle. HSV reactivation from latency conforms to a model in which stochastic derepression of the VP16 promoter and expression of VP16 initiates entry into the lytic cycle.
... This mutant contains a 12 bp insertion at aa379 in VP16 that retains the protein's essential contribution to virion structure but selectively disrupts the interaction of VP16 with Oct-1, thus preventing the formation of the VIC (Ace et al., 1988;Ace et al., 1989;Campbell et al., 1984;Wysocka and Herr, 2003). This and other VP16TF mutants are severely deficient in replication at low moi (McFarlane et al., 1992;Preston and McFarlane, 1998;Smiley and Duncan, 1997 from latently-infected ganglia explanted into culture (Steiner et al., 1990). ...
We are at an interesting time in the understanding of alpha herpesvirus latency and reactivation and their implications to human disease. Conceptual advances have come from both animal and neuronal culture models. This review focuses on the concept that the tegument protein and viral transactivator VP16 plays a major role in the transition from latency to the lytic cycle. During acute infection, regulation of VP16 transactivation balances spread in the nervous system, establishment of latent infections and virulence. Reactivation is dependent on this transactivator to drive entry into the lytic cycle. In vivo de novo expression of VP16 protein is mediated by sequences conferring pre-immediate early transcription embedded in the normally leaky late promoter. In vitro, alternate mechanisms regulating VP16 expression in the context of latency have come from the SCG neuron culture model and include the concepts that (i) generalized transcriptional derepression of the viral genome and sequestration of VP16 in the cytoplasm for ~48 hours (Phase I) precedes and is required for VP16-dependent reactivation (Phase II); and (ii) a histone methyl/phospho switch during Phase I is required for Phase II reactivation. The challenge to the field is reconciling these data into a unified model of virus reactivation. The task of compiling this review was uncomfortably humbling, as if cataloging the stars in the universe. While not completely dark, our night sky is missing a multitude of studies which are among the many points of light contributing to our field. This article is a focused review in which we discuss from the vantage point of our expertise, just a handful of concepts that have or are emerging. A lookback at some of the pioneering work that grounds our field is also included.
... Viral IE mRNAs are synthesized within the first few minutes of infection, even in the total absence of new protein synthesis. They are usually overexpressed when the infection is carried out in the presence of protein synthesis inhibitors such as cycloheximide (CHX) or anysomycin (Preston and McFarlane, 1998). This suggests that only host factors are required for IE expression, which can be activated by the virion VP16 protein or regulated for their own genes, like ICP4 that autoregulates its own gene expression (Fields et al., 2007;Spector et al., 1991). ...
... The mechanism has yet to be defined. One peculiar feature of its purported mode of action on virus gene expression kinetics is that the effect is transient and requires a short exposure (1.5 to 5 h) to the agent early after infection using a VP16-null mutant [55,57]. Various signaling events, UV light, or chemicals such as HMBA cause transiently release of P-TEFb from its inhibitory apparatus, resulting in host gene transcription [58,59]. ...
The human HSV-1 and -2 are common pathogens of human diseases. Both host and viral factors are involved in HSV lytic infection, although detailed mechanisms remain elusive. By screening a chemical library of epigenetic regulation, we identified bromodomain-containing protein 4 (BRD4) as a critical player in HSV infection. We show that treatment with pan BD domain inhibitor enhanced both HSV infection. Using JQ1 as a probe, we found that JQ1, a defined BD1 inhibitor, acts through BRD4 protein since knockdown of BRD4 expression ablated JQ1 effect on HSV infection. BRD4 regulates HSV replication through complex formation involving CDK9 and RNAP II; whereas, JQ1 promotes HSV-1 infection by allocating the complex to HSV gene promoters. Therefore, suppression of BRD4 expression or inhibition of CDK9 activity impeded HSV infection. Our data support a model that JQ1 enhances HSV infection by switching BRD4 to transcription regulation of viral gene expression from chromatin targeting since transient expression of BRD4 BD1 or BD1/2 domain had similar effect to that by JQ1 treatment. In addition to the identification that BRD4 is a modulator for JQ1 action on HSV infection, this study demonstrates BRD4 has an essential role in HSV infection.
... VP16 is an essential viral tegument protein required for virion morphogenesis [12]. In infected cell cultures VP16 also provides an important role in initiating the viral lytic cycle at low multiplicity of infection (moi), increasing plaquing efficiency 10-1000 fold in a cell type dependent manner [13][14][15]. VP16 has a potent carboxy-terminal acidic activation domain and an upstream core domain region that interacts with host cell proteins HCF and Oct-1 to form a VP16 induced complex (VIC) [16][17][18] (for review see [19]). These host cell partners are required for binding of the VIC to the TAATGARAT motifs unique to 5 viral immediate early (IE) gene promoters [20][21][22][23][24][25]. ...
The life long relationship between herpes simplex virus and its host hinges on the ability of the virus to aggressively replicate in epithelial cells at the site of infection and transport into the nervous system through axons innervating the infection site. Interaction between the virus and the sensory neuron represents a pivot point where largely unknown mechanisms lead to a latent or a lytic infection in the neuron. Regulation at this pivot point is critical for balancing two objectives, efficient widespread seeding of the nervous system and host survival. By combining genetic and in vivo in approaches, our studies reveal that the balance between latent and lytic programs is a process occurring early in the trigeminal ganglion. Unexpectedly, activation of the latent program precedes entry into the lytic program by 12 -14hrs. Importantly, at the individual neuronal level, the lytic program begins as a transition out of this acute stage latent program and this escape from the default latent program is regulated by de novo VP16 expression. Our findings support a model in which regulated de novo VP16 expression in the neuron mediates entry into the lytic cycle during the earliest stages of virus infection in vivo. These findings support the hypothesis that the loose association of VP16 with the viral tegument combined with sensory axon length and transport mechanisms serve to limit arrival of virion associated VP16 into neuronal nuclei favoring latency. Further, our findings point to specialized features of the VP16 promoter that control the de novo expression of VP16 in neurons and this regulation is a key component in setting the balance between lytic and latent infections in the nervous system.
... The ICP0 promoter was significantly induced by the etoposide treatment ( fig 2d) and there was around a two-fold induction by camptothecin treatment (fig 2c). Of note, camptothecin treatment of infected fibroblast cells showed significant improvement in viral titer, although, the ICP0 promoter was induced by only two folds as observed here (Preston and McFarlane, 1998). Neither drug treatments significantly affected RR and VP16 promoter activity under this experimental condition. ...
Although the induction of the cellular DNA damage response by herpes simplex virus-1 (HSV-1) infection of epithelial cells in tissue culture promotes productive infection, there has been no experimental observation of the effect of the cellular DNA damage response on HSV-1 infection in vivo or in neuronal derived cell lines in tissue culture. Thus, it has been speculated that the lack of cellular DNA damage induction during infection of neurons may promote latency in these cells. This work examines the profile of HSV-1 promoter induction and protein expression, in the absence or presence of infection; using cellular DNA damage inducing topoisomerase inhibitors (Camptothecin and Etoposide) on a neuroblastoma cell line (C1300) in which HSV-1 infection fails to induce the DNA damage response. In the absence of infection, a plasmid expressing the immediate early ICP0 promoter was the most induced by the DNA damage drug treatments compared to the early (RR) and late (VP16) gene promoters. Similarly, drug treatment of C1300 cells infected with HSV-1 virus showed enhanced protein expression for ICP0, but not ICP4 and VP16 proteins. However, when the cells were infected with a HSV-1 virus defective in the immediate early gene trans-activator VP16 (in814) and treated with the DNA damaging drugs, there was enhanced expression of immediate early and late HSV-1 proteins. Although, viral infection of the neuroblastoma cell alone did not induce DNA damage, cellular DNA damage induced by drug treatments facilitated viral promoter induction and viral protein expression. This implicates a mechanism by which HSV-1 viral genes in a quiescent or latent state may become induced by cellular DNA damage in neuronal cells to facilitate productive infection.
... HMBA induces the differentiation of murine erythroleukemia cells (MELC) and other transformed cells to a less transformed phenotype [73]. Comparison of the effects of various other agents known to promote the differentiation of MELCs demonstrated that some of these substances complement the growth of VP16-deficient HSV-1 whereas others antagonize the effect of HMBA [74]. HMBA-treatment also increases the replication of VP16-positive HSV-1 and HSV-2 in epidermal and neuronal cells [75], and the oncolytic activity of a gamma1-34.5negative ...
Following productive, lytic infection in epithelia, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons that is interrupted by episodes of reactivation. In order to better understand what triggers this lytic/latent decision in neurons, we set up an organotypic model based on chicken embryonic trigeminal ganglia explants (TGEs) in a double chamber system. Adding HSV-1 to the ganglion compartment (GC) resulted in a productive infection in the explants. By contrast, selective application of the virus to distal axons led to a largely nonproductive infection that was characterized by the poor expression of lytic genes and the presence of high levels of the 2.0-kb major latency-associated transcript (LAT) RNA. Treatment of the explants with the immediate-early (IE) gene transcriptional inducer hexamethylene bisacetamide, and simultaneous co-infection of the GC with HSV-1, herpes simplex virus type 2 (HSV-2) or pseudorabies virus (PrV) helper virus significantly enhanced the ability of HSV-1 to productively infect sensory neurons upon axonal entry. Helper-virus-induced transactivation of HSV-1 IE gene expression in axonally-infected TGEs in the absence of de novo protein synthesis was dependent on the presence of functional tegument protein VP16 in HSV-1 helper virus particles. After the establishment of a LAT-positive silent infection in TGEs, HSV-1 was refractory to transactivation by superinfection of the GC with HSV-1 but not with HSV-2 and PrV helper virus. In conclusion, the site of entry appears to be a critical determinant in the lytic/latent decision in sensory neurons. HSV-1 entry into distal axons results in an insufficient transactivation of IE gene expression and favors the establishment of a nonproductive, silent infection in trigeminal neurons.
... TSA and sodium butyrate have been shown to reactivate lytic HSV1 infection in other systems. [18][19][20] Application of 1μM TSA to latently infected cultures at DIV 9 yields viral reactivation in 61.7% of wells by DIV15 (or the tenth day after application of TSA) (Fig. 6A). Higher doses of TSA result in cell death which does not allow for viral reactivation. ...
Vestibular neuritis is a common cause of both acute and chronic vestibular dysfunction. Multiple pathologies have been hypothesized to be the causative agent of vestibular neuritis; however, whether herpes simplex type I (HSV1) reactivation occurs within the vestibular ganglion has not been demonstrated previously by experimental evidence. We developed an in vitro system to study HSV1 infection of vestibular ganglion neurons (VGNs) using a cell culture model system.
basic science study.
Lytic infection of cultured rat VGNs was observed following low viral multiplicity of infection (MOI). Inclusion of acyclovir suppressed lytic replication and allowed latency to be established. Upon removal of acyclovir, latent infection was confirmed with reverse-transcription polymerase chain reaction and by RNA fluorescent in situ hybridization for the latency-associated transcript (LAT). A total of 29% cells in latently infected cultures were LAT positive. The lytic ICP27 transcript was not detected by reverse-transcription polymerase chain reaction (RT-PCR). Reactivation of HSV1 occurred at a high frequency in latently infected cultures following treatment with trichostatin A (TSA), a histone deactylase inhibitor.
VGNs can be both lytically and latently infected with HSV1. Furthermore, latently infected VGNs can be induced to reactivate using TSA. This demonstrates that reactivation of latent HSV1 infection in the vestibular ganglion can occur in a cell culture model, and suggests that reactivation of HSV1 infection a plausible etiologic mechanism of vestibular neuritis.
... Treatment with chemical HDAC inhibitors mimics the functional consequences of pp71 and IE1 expression, and PML-NB protein knockdown [2,45,51]. The effects of HDAC inhibitors on HSV-1 gene expression from either wild-type, VP16 defective or ICP0-null genomes appear to show a cell type dependence, failing to stimulate HSV-1 replication in certain cell types [48,52] while enhancing replication or reactivation in others, particularly neuronal cells53545556. Thus, while ICP0 is thought to associate with and modify the function of cellular chromatin remodeling complexes that contain HDACs57585960, it is evident that ICP0 may also possess additional mechanisms to alter viral gene expression. ...
The promyelocytic leukemia (PML) protein gathers other cellular proteins, such as Daxx and Sp100, to form subnuclear structures termed PML-nuclear bodies (PML-NBs) or ND10 domains. Many infecting viral genomes localize to PML-NBs, leading to speculation that these structures may represent the most efficient subnuclear location for viral replication. Conversely, many viral proteins modify or disrupt PML-NBs, suggesting that viral replication may be more efficient in the absence of these structures. Thus, a debate remains as to whether PML-NBs inhibit or enhance viral replication. Here we review and discuss recent data indicating that for herpesviruses, PML-NB proteins inhibit viral replication in cell types where productive, lytic replication occurs, while at the same time may enhance the establishment of lifelong latent infections in other cell types.
... The OBP Ϫ mutant virus hr94 and complementing cell line 2B.11 were kindly provided by Sandra Weller (University of Connecticut Health Sciences Center, Farmington), and hr94 was propagated on 2B.11 cells as previously described (31). The VP16 Ϫ mutant virus RP5 (54), kindly provided by Rath Pichyangkura and Steve Triezenberg (Michigan State University, East Lansing), was propagated on ICP4-complementing E5 cells in the presence of 5 mM hexamethylenebisacetamide (HMBA), a compound that complements the replication of ICP0 Ϫ and VP16 Ϫ mutants in vitro (33,41). The parental adenovirus vector H5.010CMVEGFP (5), hereafter referred to as Ad.C-GFP, was obtained from the Institute of Human Gene Therapy at the University of Pennsylvania School of Medicine, Philadelphia. ...
In a previous study, we demonstrated that infected-cell polypeptide 0 (ICP0) is necessary for the efficient reactivation of herpes simplex virus type 1 (HSV-1) in primary cultures of latently infected trigeminal ganglion (TG) cells (W. P. Halford and P. A. Schaffer, J. Virol. 75:3240-3249, 2001). The present study was undertaken to determine whether ICP0 is sufficient to trigger HSV-1 reactivation in latently infected TG cells. To test this hypothesis, replication-defective adenovirus vectors that express wild-type and mutant forms of ICP0 under the control of a tetracycline response element (TRE) promoter were constructed. Similar adenovirus vectors encoding wild-type ICP4, wild-type and mutant forms of the HSV-1 origin-binding protein (OBP), and wild-type and mutant forms of VP16 were also constructed. The TRE promoter was induced by coinfection of Vero cells with the test vector and an adenovirus vector that expresses the reverse tetracycline-regulated transactivator in the presence of doxycycline. Northern blot analysis demonstrated that transcription of the OBP gene in the adenovirus expression vector increased as a function of doxycycline concentration over a range of 0.1 to 10 microM. Likewise, Western blot analysis demonstrated that addition of 3 microM doxycycline to adenovirus vector-infected Vero cells resulted in a 100-fold increase in OBP expression. Wild-type forms of ICP0, ICP4, OBP, and VP16 expressed from adenovirus vectors were functional based on their ability to complement plaque formation in Vero cells by replication-defective HSV-1 strains with mutations in these genes. Adenovirus vectors that express wild-type forms of ICP0, ICP4, or VP16 induced reactivation of HSV-1 in 86% +/- 5%, 86% +/- 5%, and 97% +/- 5% of TG cell cultures, respectively (means +/- standard deviations). In contrast, vectors that express wild-type OBP or mutant forms of ICP0, OBP, or VP16 induced reactivation in 5% +/- 5%, 8% +/- 0%, 0% +/- 0%, and 13% +/- 6% of TG cell cultures, respectively. In control infections, an adenovirus vector expressed green fluorescent protein efficiently in TG neurons but did not induce HSV-1 reactivation. Therefore, expression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation in latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular and viral proteins are sufficient to induce HSV-1 reactivation from neuronal latency.
... The sequences encoding the fusion protein were cloned as an AgeI-NotI fragment into pCP1802 to yield pMJ129, in which the YFP-pp71 protein was controlled by the HCMV major IE promoter and embedded in the HSV-1 TK coding region Viruses. HSV-1 (strain 17) mutants in1312, in1324, in1372, in1382, in1383 and in1388 have been described previously Homer et al., 1999 ;Rinaldi et al., 1999 ;Preston & McFarlane, 1998 ;Marshall et al., 2000). Mutant in1357 was constructed by cotransfecting ScaI-cleaved pCP7991 with in1312 DNA. ...
The human cytomegalovirus (HCMV) tegument phosphoprotein pp71 activates viral immediate early (IE) transcription and thus has a role in initiating lytic infection. Protein pp71 stimulates expression from a range of promoters in a sequence-independent manner, and in this respect behaves similarly to the herpes simplex virus type 1 (HSV-1) IE protein ICP0. The intracellular localization of pp71 was investigated after its expression from transfected plasmids or from HSV-1 mutants constructed to produce pp71 transiently. The protein colocalized with the cell promyelocytic leukaemia (PML) protein at nuclear domain 10 (ND10) structures but, unlike ICP0, pp71 did not induce disruption of ND10. The activity of pp71 in mouse sensory neurons in vivo was investigated after co-inoculation of animals with pairs of HSV-1 mutants, one expressing pp71 and the second containing the E. coli lacZ gene controlled by various promoters. In this system, pp71 stimulated beta-galactosidase expression from a range of viral IE promoters when mice were analysed at 4 days postinoculation. At later times, expression of pp71 resulted in a reduction in numbers of neurons containing beta-galactosidase, indicating cytotoxicity or promoter shutoff. The HSV-1 latency-active promoter was not responsive to pp71, demonstrating specificity in the activity of the protein. Pp71 was as active in mice lacking both copies of the PML gene (PML-/-) as in control animals, and in PML-/- fibroblasts pp71 stimulated gene expression as effectively as in other cell types. Therefore, neither the PML protein nor the normal ND10 structure is necessary for pp71 to stimulate gene expression.
... AD169 per cell and determination of the percentage of GFP-positive cells at 3 days post-infection (p.i.). The HSV-1 mutants in1324, in1372 and in1382 have been described previously (Homer et al., 1999;Preston & McFarlane, 1998;Rinaldi et al., 1999). They are derived from mutant in1312, which contains a 12 bp insertion in the VP16-coding region, a deletion of the RING domain of ICP0 and the ICP4 temperaturesensitive mutation of HSV-1, tsK (Davison et al., 1984;. ...
Human cytomegalovirus (HCMV) immediate-early (IE) transcription is stimulated by virion phosphoprotein pp71, the product of gene UL82. It has previously been shown that pp71 interacts with the cellular protein hDaxx and, in the studies presented here, the significance of this interaction was investigated for HCMV IE gene expression. In co-transfection experiments, the presence of hDaxx increased the transcriptional response of the HCMV major IE promoter (MIEP) to pp71, but it was not possible to determine whether the effect was due to an interaction between the two proteins or to stimulation of hDaxx synthesis by pp71. The use of small interfering RNA (siRNA) in long- and short-term transfection approaches reduced intracellular hDaxx levels to no more than 3 % of normal. Infection of hDaxx-depleted cells with herpes simplex virus recombinants containing the HCMV MIEP revealed significantly greater promoter activity when hDaxx levels were minimal. Similarly, reducing intracellular hDaxx amounts resulted in greater IE gene expression during infection with an HCMV mutant lacking pp71, but had no effect on IE transcription during infection with wild-type HCMV. The results suggest that hDaxx is not important as a positive-acting factor for the stimulation of HCMV IE transcription by pp71. Instead, it appears that hDaxx acts as a repressor of IE gene expression, and it is proposed here that the interaction of pp71 with hDaxx is important to relieve repression and permit efficient initiation of productive replication.
... At the time of infection in some experiments, cells were subjected to one of the following six treatments: (i) control medium alone; (ii) retinoic acid (RA) in medium; (ii) hexamethylene bisacetamide (HMBA) in medium; (iv) RA and cortisol in medium; (v) HMBA and cortisol in medium; or (vi) RA, HMBA, and cortisol in medium. The RA concentration was 0.01 mM, the HMBA concentration was 5 mM (29,45), and cortisol levels were 1 M. In initial experiments, stock solutions of RA and cortisol were dissolved in dimethyl sulfoxide, with a final concentration of 0.3% dimethyl sulfoxide. ...
In the course of examining the trafficking pathways of varicella-zoster virus (VZV) glycoproteins gE, gI, gH, and gB, we discovered that all four are synthesized within 4 to 6 h postinfection (hpi) in cultured cells. Thereafter, they travel via the trans-Golgi network to the outer cell membrane. When we carried out a similar analysis on VZV gC, we observed little gC biosynthesis in the first 72 hpi. Further examination disclosed that gC was present in the inocula of infected cells, but no new gC biosynthesis occurred during the first 24 to 48 h thereafter, during which time new synthesis of gE, gH, and major capsid protein was easily detectable. Similarly, delayed gC biosynthesis was confirmed with three different VZV strains and two different cell lines. Bioinformatics analyses disclosed the presence of PBX/HOX consensus binding domains in the promoter/enhancer regions of the genes for VZV gC and ORF4 protein (whose orthologs transactivate gC in other herpesviruses). Bioinformatics analysis also identified two HOXA9 activation regions on ORF4 protein. Treatment of infected cultures with chemicals known to induce the production of PBX/HOX transcription proteins, namely, hexamethylene bisacetamide (HMBA) and retinoic acid, led to more rapid gC biosynthesis. Immunoblotting demonstrated a fivefold increase in the HOXA9 protein after HMBA treatment. In summary, these results documented that gC was not produced during early VZV replication cycles, presumably related to a deficiency in the PBX/HOX transcription factors. Furthermore, these results explain the apparent spontaneous loss of VZV gC in some passaged viruses, as well as other anomalous gC results.
We report toxic effects of a photoactivatable platinum(IV) complex conjugated with suberoyl-bis-hydroxamic acid in tumor cells. The conjugate exerts, after photoactivation, two functions: activity as both a platinum(II) anticancer drug and histone deacetylase (HDAC) inhibitor in cancer cells. This approach relies on the use of a Pt(IV) pro-drug, acting by two independent mechanisms of biological action in a cooperative manner, which can be selectively photoactivated to a cytotoxic species in and around a tumor, thereby increasing selectivity towards cancer cells. These results suggest that this strategy is a valuable route to design new platinum agents with higher efficacy for photodynamic anticancer chemotherapy.
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Because of its very low titer, varicella-zoster virus (VZV) infectivity is usually transferred by passage of trypsin dispersed infected cells. Previously, we observed that gC biosynthesis was markedly delayed in monolayers inoculated with cell free virus. In this report, we investigated the kinetics of gC expression in more detail and included studies of monolayers inoculated with trypsin dispersed infected cells, the more traditional method of VZV infection. Extensive imaging analyses disclosed that gC was detectable in some inoculum cells, but little gC biosynthesis occurred during the first 48 hpi in the newly infected underlying monolayer. In contrast, during the first 24-48 hpi, expression of VZV gE and gB was easily detectable. Using real-time RT-PCR, we found a delay in accumulation of VZV gC transcripts that paralleled the delay in expression of VZV gC protein. Treatment with hexamethylene bisacetamide (HMBA) increased expression of both gC protein and gC mRNA. HMBA treatment also increased virus titer by 4-fold, but paradoxically reduced plaque size in the titration assay. Finally, we examined skin vesicles from cases of chickenpox and zoster in humans and observed abundant amounts of gC expression. In short, this report documents an unexpected delay in both gC mRNA and protein production under all conditions of VZV infection of cultured cells.
The activation of gene expression by the human cytomegalovirus (HCMV) particle was investigated. The HCMV major immediate-early (IE) promoter was cloned upstream of the Escherichia coli lacZ coding sequences, and the resulting cassette was introduced into the genome of a herpes simplex virus type 1 (HSV-1) mutant lacking functional VP16. Upon infection with the HSV-1 recombinant in the presence of cycloheximide, to block de novo protein synthesis, expression of lacZ-specific transcripts was increased by fivefold when HCMV was included in the inoculum. Accumulation of HSV-1 IE RNAs was also stimulated by coinfection with HCMV, as was expression of the adenovirus 5 VAI transcript when the VAI gene was cloned into the HSV-1 genome. Coinfection with HCMV did not alter mRNA stability or uncoating of the HSV-1 genome. The coding sequences for the HCMV phosphoprotein pp71, controlled by the HCMV IE promoter, were cloned into an HSV-1 recombinant impaired for the production of the three major transactivators (VP16, ICP0, and ICP4) to yield a recombinant (in1324) which expressed pp71 but did not cause significant cytotoxicity. Infection with in1324 resulted in stimulation of HCMV IE, HSV-1 IE, and VAI expression, demonstrating that pp71 is responsible for the effects we observed when using the entire HCMV particle. Therefore, HCMV pp71 exhibits novel properties in its ability to stimulate gene expression from a range of promoters present in a herpesvirus genome.
The effect of hexamethylane bisacetamide (HMBA), a hybrid polar compound, on gene expression and replication of human cytomegalovirus (HCMV) was studied. When HCMV-infected human thyroid papillary carcinoma (TPC-1) and human embryonic lung (HEL) fibroblast cells were maintained with medium containing 2.5 and 5 mM HMBA for 10 days, there was a greater than 2- to 3-log reduction in virus yield compared to that in untreated cells. Infection of TPC-1 cells with HCMV resulted in an establishment of persistent infection and the cells continuously produced virus with titer of over 10(5) PFU/ml, whereas HMBA prevented the infected cells from entering into the persistent infection. Moreover, treatment of the persistently infected cultures with HMBA reduced production of infectious HCMV more efficiently than did ganciclovir, and eventually ceased HCMV production. Western blotting analysis revealed that HMBA blocks accumulation of the immediate early 2 (IE2) protein in TPC-1 cells and delays synthesis of this protein in HEL cells, but has little effect on the level of the IE1 protein during the early times after infection. Synthesis of the viral early and late proteins in both cells was also substantially blocked by HMBA. The results indicate that the inhibition or the delay of the critical IE2 protein synthesis in the presence of HMBA would actually be a process that fails to proceed beyond the IE stages in HCMV replication cycle.
(R)-Trichostatin A (TSA) is a Streptomyces product which causes the induction of Friend cell differentiation and specific inhibition of the cell cycle of normal rat fibroblasts in the G1 and G2 phases at the very low concentrations. We found that TSA caused an accumulation of acetylated histone species in a variety of mammalian cell lines. Pulse-labeling experiments indicated that TSA markedly prolonged the in vivo half-life of the labile acetyl groups on histones in mouse mammary gland tumor cells, FM3A. The partially purified histone deacetylase from wild-type FM3A cells was effectively inhibited by TSA in a noncompetitive manner with Ki = 3.4 nM. A newly isolated mutant cell line of FM3A resistant to TSA did not show the accumulation of the acetylated histones in the presence of a higher concentration of TSA. The histone deacetylase preparation from the mutant showed decreased sensitivity to TSA (Ki = 31 nM, noncompetitive). These results clearly indicate that TSA is a potent and specific inhibitor of the histone deacetylase and that the in vivo effect of TSA on cell proliferation and differentiation can be attributed to the inhibition of the enzyme.
Hexamethylene bisacetamide (HMBA) and DMSO are known to induce differentiation of cultured erythroleukaemic cells and to enhance the reactivation of latent herpes simplex virus (HSV) after explantation of ganglia. We report that the presence of these compounds in cell culture medium overcomes the replication defect of in1814, an HSV-1 mutant with an insertion mutation that inactivates the virion trans-inducing factor, Vmw65 (VP16). The effect of HMBA was not cell type-specific and was attained even by a short exposure (1.5 to 5 h) to the agent early after infection. The presence of HMBA resulted in an increase in immediate early (IE) RNA accumulation after infection of cells in the presence of cycloheximide, such that RNA levels in in1814-infected cells approached the values observed in wild-type HSV-1-infected cells in the absence of HMBA. Transport of viral DNA to the cell nucleus was not affected by HMBA. The results suggest that HMBA- and DMSO-mediated enhancement of reactivation from latency is due to an increase in IE RNA production. In addition, these studies demonstrate a primary effect of HMBA on gene regulation which may be a paradigm for initial events during erythroleukaemic cell differentiation.
In a mouse model for herpes simplex virus type 1 (HSV-1) latency in which the virus was inoculated via the eye after corneal scarification, HSV-1 replicated in corneal epithelial cells and infected the nerve cell endings. HSV-1 reached the trigeminal ganglia by fast axonal transport between 2 and 10 days postinfection (p.i.) and established a latent infection in neuronal cells or replicated and spread to nonneuronal cells. By using in situ hybridization, we showed that cellular transcription factors are stimulated by HSV-1 infection in trigeminal ganglia. This stimulation is biphasic, peaking at 1 and 3 to 4 days p.i. The first peak involves c-jun and oct-1 expression in neurons, and the second involves c-jun, c-fos, and oct-1 expression in neurons and nonneuronal cells. Corneal scarification, alone or followed by infection with UV-inactivated HSV-1, induced monophasic c-jun and oct-1 expression in some neurons of the trigeminal ganglia, with a peak at 1 day p.i. Corneal infection without prior scarification induced c-jun, c-fos, and oct-1 expression in some neuronal and nonneuronal cells of the trigeminal ganglia 2 to 9 days p.i. Explanation of ganglia from latently infected animals resulted in reactivation of the latent virus. Independently of the presence of latent HSV-1 in explanted ganglia, expression of c-fos, c-jun, and oct-1 was induced first in nonneuronal cells, peaking 6 to 10 h postexplantation, and then in neuronal cells, with a peak at 24 h after explantation when expression of viral replicative genes was first detectable. Since ocular HSV-1 infection, corneal scarification, and explantation of trigeminal ganglia all resulted in induction of expression of cellular transcription factors in ganglia, these factors may play a critical role in the permissiveness of cells for HSV-1 replication during acute infection, latency, and reactivation.
Exposure of mammalian cells to DNA-damaging agents leads to activation of a genetic response known as the UV response. Because
several previously identified UV-inducible genes contain AP-1 binding sites within their promoters, we investigated the induction
of AP-1 activity by DNA-damaging agents. We found that expression of both c-jun and c-fos, which encode proteins that participate
in formation of the AP-1 complex, is rapidly induced by two different DNA-damaging agents: UV and H2O2. Interestingly, the
c-jun gene is far more responsive to UV than any other immediate-early gene that was examined, including c-fos. Other jun
and fos genes were only marginally affected by UV or H2O2. Furthermore, UV is a much more efficient inducer of c-jun than
phorbol esters, the standard inducers of c-jun expression. This preferential response of the c-jun gene is mediated by its
5' control region and requires the TPA response element, suggesting that this element also serves as an early target for the
signal transduction pathway elicited by DNA damage. Both UV and H2O2 lead to a long-lasting increase in AP-1 binding activity,
suggesting that AP-1 may mediate the induction of other damage-inducible genes such as human collagenase.
(R)-Trichostatin A (TSA) is a Streptomyces product which causes the induction of Friend cell differentiation and specific inhibition of the cell cycle of normal rat fibroblasts in the G1 and G2 phases at the very low concentrations. We found that TSA caused an accumulation of acetylated histone species in a variety of mammalian cell lines. Pulse-labeling experiments indicated that TSA markedly prolonged the in vivo half-life of the labile acetyl groups on histones in mouse mammary gland tumor cells, FM3A. The partially purified histone deacetylase from wild-type FM3A cells was effectively inhibited by TSA in a noncompetitive manner with Ki = 3.4 nM. A newly isolated mutant cell line of FM3A resistant to TSA did not show the accumulation of the acetylated histones in the presence of a higher concentration of TSA. The histone deacetylase preparation from the mutant showed decreased sensitivity to TSA (Ki = 31 nM, noncompetitive). These results clearly indicate that TSA is a potent and specific inhibitor of the histone deacetylase and that the in vivo effect of TSA on cell proliferation and differentiation can be attributed to the inhibition of the enzyme.
Using nonsense and deletion mutants of herpes simplex virus type 1, we investigated the roles of three immediate-early proteins (ICP4, ICP27 and ICP0) in the establishment and reactivation of ganglionic latency in a mouse ocular model. DNA hybridization, superinfection-rescue, and cocultivation techniques provided quantitative data that distinguished between the failure of a virus to establish latency in the ganglion and its failure to reactivate. Null mutants with lesions in the genes for ICP4 and ICP27 did not replicate in the eye or in ganglia and failed to establish reactivatable latent infections. Three ICP0 deletion mutants which could replicate in the eye and ganglia varied in their ability to establish and reactivate from the latent state, demonstrating that ICP0 plays a role both in the establishment and the reactivation of latency. The use of viral mutants and a variety of stage-specific assays allowed us to better define the stages in the establishment and reactivation of herpes simplex virus type 1 latency.
A herpes simplex virus mutant, in1814, possessing a 12-base-pair insertion in the gene encoding the transinducing factor Vmw65 has been constructed. The insertion abolished the ability of Vmw65 to transinduce immediate-early (IE) gene expression and to form a protein-DNA complex with cell proteins and the IE-specific regulatory element TAATGAGAT. Accumulation of IE RNA 1 and 2 was reduced four- to fivefold in in1814-infected cells, but the level of IE RNA 4 was reduced only by twofold, and IE RNA 3 was unaffected. Mutant in1814 had a high particle/PFU ratio, but many of the particles, although unable to form plaques, were capable of normal participation in the early stages of infection at high multiplicity of infection. The defect of in1814 was overcome partially by transfection of a plasmid encoding the IE protein Vmw110 into cells prior to titration and by prior infection with ultraviolet light-inactivated herpes simplex virus. Mutant in1814 was essentially avirulent when injected into mice. The results demonstrate that transinduction of IE transcription by Vmw65 is important at low multiplicity of infection and in vivo but that at high multiplicity of infection the function is redundant.
Transcription from the early and late classes of the herpes simplex virus type 1 (HSV-1) promoters requires prior immediate early (IE) gene expression. Although the product of IE gene 1, Vmw110, is not absolutely essential for virus growth in tissue culture, transfection experiments have demonstrated that Vmw110 can activate gene expression both by itself and in a synergistic manner with the product of IE gene 3, Vmw175. This paper describes the construction of 10 mutant HSV-1 viruses with deletion and insertion mutations in Vmw110. The mutant viruses were then studied in single-step growth curve experiments, by assaying for plaques in a variety of cell types and by analysis of viral polypeptide synthesis during productive infection at high and low multiplicities. The results show that mutations in Vmw110 reduce the efficiency of plaque formation by HSV-1; the extent of this reduction depends on cell type and the position of the mutation in the polypeptide. In particular, a potential zinc finger domain is crucial for Vmw110 function. The patterns and amounts of viral polypeptide synthesis during high multiplicity infections with mutant and wild-type viruses were similar in all cell types. At low multiplicity, mutations in Vmw110 reduced viral gene expression in the least permissive cell type. The data suggest that the role of Vmw110 during virus infection in tissue culture is at a very early stage of low multiplicity infections; its inactivity leads to the failure to express viral genes so that the virus does not enter the lytic cycle.
Reactivation of latent herpes simplex virus type 2 (HSV-2) by the immediate-early protein Vmw110 was studied by using an in vitro latency system. Adenovirus recombinants that express Vmw110 reactivated latent HSV-2. An HSV-1 mutant possessing a deletion in a carboxy-terminal region of Vmw110 reactivated latent HSV-2, whereas mutant FXE, which has a deletion in the second exon, did not. Therefore, Vmw110 alone is required to reactivate latent HSV-2 in vitro, and the region of Vmw110 defined by the deletion in FXE is important for this process.
Hexamethylenebisacetamide (HMBA)-induced differentiation of murine erythroleukemia cells (MELC) is a multistep process involving an early latent period during which a number of metabolic changes have been detected, but the cells are not yet committed irreversibly to differentiate. Commitment is defined as the capacity of MELC to go on to express the program of terminal cell division and gene expression (such as the accumulation of globin mRNA) upon removal of the HMBA from the culture. In the presence of HMBA, a small proportion of MELC are committed by 10-12 hr and greater than 90% by 48-60 hr. The present study shows that, during the initial 4 hr of culture, HMBA causes a marked decrease in c-myb and c-myc and an increase in c-fos mRNA levels. With continued culture, the decrease in c-myb and the increase in c-fos mRNA persists, while c-myc mRNA returns to control levels before the time that MELC begin to show irreversible differentiation. Dexamethasone, which blocks expression of HMBA-induced MELC differentiation, does not alter the early pattern of changes in protooncogene mRNA nor the sustained elevation of c-fos, but it does inhibit the continued suppression of c-myb allowing c-myb to return toward control levels. Hemin, which induces MELC to accumulate globins but does not initiate commitment to terminal cell division, does not alter these protooncogene mRNA levels. These studies suggest that, although the early decrease in c-myb and c-myc and increase in c-fos mRNAs may be involved in the multistep events leading to differentiation, the continued suppression of c-myb is critical for HMBA-induced MELC commitment to terminal cell division.
Chromatin proteins are covalently modified by at least five different processes; in no case has the precise physiological function been established. One of these post-synthetic, covalent modifications is effected by the enzyme poly(ADP-ribose) polymerase, which uses the coenzyme NAD+ to ADP-ribosylate chromatin proteins. The modification consists largely of mono(ADP-ribose), but long, homopolymer chains of (ADP-ribose) are also present. Various physiological functions have been suggested for (ADP-ribose)n. Here we demonstrate that one function of (ADP-ribose)n is to participate in the cellular recovery from DNA damage. Specific inhibitors of poly(ADP-ribose) polymerase prevent rejoining of DNA strand breaks caused by dimethyl sulphate and cytotoxicity is enhanced thereby. The rejoining of strand breaks is prevented also by nutritionally depleting the cells of NAD.
M-TAT is a cytokine-dependent cell line with the potential to differentiate along the erythroid and megakaryocytic lineages. We cultured M-TAT cells long term (> 1 year) in the continuous presence of erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), or stem cell factor (SCF). These long term cultures are referred to as M-TAT/EPO, M-TAT/GM-CSF, and M-TAT/SCF cells, respectively. Hemoglobin concentration and gamma-globin and erythroid delta-aminolevulinate synthase mRNA levels were significantly higher in M-TAT/EPO cells than in M-TAT/GM-CSF cells. When the supplemented cytokine was switched from GM-CSF to EPO, hemoglobin synthesis in M-TAT/GM-CSF cells increased rapidly (within 5 h), and the level of GATA-1 mRNA increased. In contrast, the addition of GM-CSF to the M-TAT/EPO cell culture decreased the amount of hemoglobin, even in the presence of EPO, indicating that the EPO signal for erythroid differentiation is suppressed by GM-CSF. Thus, erythroid development of M-TAT cells is promoted by EPO and suppressed by GM-CSF. These results support the hypothesis that EPO actively influences the programming of gene expression required for erythroid progenitor cell differentiation.
We have examined the cis- and trans-acting factors involved in constitutive transcription of the promoter for the IE110k protein of herpes simplex virus type 1. Our results indicate that while the IE110k gene is activated by Vmw65, it also exhibits very efficient constitutive expression approximating that from the simian virus 40 early enhancer-promoter region. We show that despite the presence of multiple copies of the octamer consensus site which mediate Oct-1 binding and subsequent Vmw65 activation, these upstream sequences have a minor effect on constitutive transcription. By progressive exonuclease digestion and subsequent site-directed mutagenesis of the promoter, we have identified a 15-bp region (termed the EC region), from position -89 to -74, which is required for efficient constitutive expression from the IE110k promoter. We demonstrate that two cellular proteins interact with this region and, by competition and methylation interference analyses, show they have distinct but overlapping sequence requirements for binding. One of these proteins is identified as NF-Y, a CCAAT box-binding factor, which binds an inverted CCAAT box located between positions -71 and -75. The second cellular factor, F2, appears to be novel and binds a region with the sequence CGCGCGGC CAT which overlaps the 3' end of the CCAAT box. The terminal AT of the recognition site for F2 represents, on the opposite strand, the terminal AT of the CCAAT box, and these and adjacent bases are critically required for the binding of both factors. These results together with further competition analysis indicate that these factors bind in a mutually exclusive manner to the EC region. The implications of these results for regulation of expression of the IE110k gene are discussed.
Hexamethylenebisacetamide (HMBA), a potent inducer of differentiation of transformed cells such as murine erythroleukemia cells, causes a prolongation of the G1 phase of the cell cycle during which commitment to terminal differentiation is first detected. Removal of HMBA prior to the G1 phase aborts commitment. To further define the relationship between the G1 phase and commitment to differentiation, we used two inhibitors of cell cycle progression: aphidicolin, which blocks cells at the G1/S interphase, and deferoxamine, which blocks cells at an earlier stage during G1. HMBA-induced prolongation of G1 is associated with the accumulation of underphosphorylated retinoblastoma protein, decrease in cyclin A protein levels, and commitment to differentiation. G1 arrest of murine erythroleukemia cells induced by aphidicolin or deferoxamine is not associated with accumulation of under-phosphorylated retinoblastoma protein, suppression of cyclin A protein, or commitment of cells to terminal differentiation. Neither of the cell cycle inhibitors alters the effect of HMBA in inducing the G1-associated changes or commitment to differentiation. Taken together, the present findings indicate that the site of action of HMBA which leads to commitment is in a stage of the G1 phase prior to the point of cell cycle block caused by deferoxamine or aphidicolin. HMBA appears to cause cell differentiation with suppression of cell cycle progression by an action that affects events required for cell progression through G1, including accumulation of underphosphorylated retinoblastoma protein and changes in regulation of cyclin levels.
Hybrid polar compounds, of which hexamethylenebisacetamide (HMBA) is the prototype, are potent inducers of differentiation of murine erythroleukemia (MEL) cells and a wide variety of other transformed cells. HMBA has been shown to induce differentiation of neoplastic cells in patients, but is not an adequate therapeutic agent because of dose-limiting toxicity. We report on a group of three potent second generation hybrid polar compounds, diethyl bis-(pentamethylene-N,N-dimethylcarboxamide) malonate (EMBA), suberoylanilide hydroxamic acid (SAHA), and m-carboxycinnamic acid bis-hydroxamide (CBHA) with optimal concentrations for inducing MEL cells of 0.4 mM, 2 microM, and 4 microM, respectively, compared to 5 mM for HMBA. All three agents induce accumulation of underphosphorylated pRB; increased levels of p2l protein, a prolongation of the initial G1 phase of the cell cycle; and accumulation of hemoglobin. However, based upon their effective concentrations, the cross-resistance or sensitivity of an HMBA-resistant MEL cell variant, and differences in c-myb expression during induction, these differentiation-inducing hybrid polar compounds can be grouped into two subsets, HMBA/EMBA and SAHA/CBHA. This classification may prove of value in selecting and planning prospective preclinical and clinical studies toward the treatment of cancer by differentiation therapy.
Herpes simplex virus (HSV) replicates in peripheral tissues and forms latent infections in neurons of the peripheral nervous system. It can be reactivated from latency by various stimuli to cause recurrent disease. During lytic infection in tissue culture cells, there is a well-described temporal pattern of (i) immediate-early, (ii) early, and (iii) late gene expression. However, latency is characterized by little if any expression of genes of the lytic cycle of infection. During reactivation, the pattern of gene expression is presumed to be similar to that during the lytic cycle in tissue culture, though recent work of W. P. Halford et al. (J. Virol. 70:5051-5060, 1996) and P. F. Nichol et al. (J. Virol. 70:5476-5486, 1996) suggests that it is modified in neuronal cell cultures. We have used the mouse trigeminal ganglion explant model and reverse transcription-PCR to determine the pattern of viral and cellular gene expression during reactivation. Surprisingly, the pattern of viral gene expression during lytic infection of cell cultures is not seen during reactivation. During reactivation, early viral transcripts were detected before immediate-early transcripts. The possibility that a cellular factor upregulates early genes during the initial reactivation stimulus is discussed.
Herpes simplex virus type 1 (HSV-1) transcription can be arrested at the immediate early (IE) stage by continuous treatment of cells with inhibitors of protein synthesis, usually cycloheximide, from the time of infection. We have analysed the effect of cycloheximide on IE gene expression with HSV-1 mutants deficient in the production of functional levels of the three major transactivators, the virion protein (VP16) and two IE proteins (ICP0 and ICP4). Expression from the HSV-1 IE promoters that control synthesis of ICP0 and ICP27 was, unexpectedly, stimulated by inhibition of protein synthesis. The effect was observed for the ICP0 promoter in its normal genome location and also when cloned upstream of the Escherichia coli lacZ coding sequences and inserted into the viral thymidine kinase locus. Expression from the human cytomegalovirus major IE promoter, when cloned into the genome of HSV-1 mutants, was also increased by inhibition of protein synthesis. Cycloheximide did not affect the intracellular stability of lacZ-specific RNA, suggesting that the response represented an increase in mRNA production. Activation of the ICP0 promoter was observed when protein synthesis was blocked by alternative agents. Since inhibitors of protein synthesis are known to activate cellular signal transduction pathways, our findings demonstrate new mechanisms for the regulation of HSV-1 IE gene expression which may be important during latency and reactivation. The results also highlight previously unrecognized difficulties in analysing the intrinsic activities of promoters when cloned into the HSV-1 genome.
A herpes simplex virus mutant, in1814, possessing a 12-base-pair insertion in the gene encoding the transinducing factor Vmw65 has been constructed. The insertion abolished the ability of Vmw65 to transinduce immediate-early (IE) gene expression and to form a protein-DNA complex with cell proteins and the IE-specific regulatory element TAATGAGAT. Accumulation of IE RNA 1 and 2 was reduced four- to fivefold in in1814-infected cells, but the level of IE RNA 4 was reduced only by twofold, and IE RNA 3 was unaffected. Mutant in1814 had a high particle/PFU ratio, but many of the particles, although unable to form plaques, were capable of normal participation in the early stages of infection at high multiplicity of infection. The defect of in1814 was overcome partially by transfection of a plasmid encoding the IE protein Vmw110 into cells prior to titration and by prior infection with ultraviolet light-inactivated herpes simplex virus. Mutant in1814 was essentially avirulent when injected into mice. The results demonstrate that transinduction of IE transcription by Vmw65 is important at low multiplicity of infection and in vivo but that at high multiplicity of infection the function is redundant.
— In HeLa cells transcription of the c-jun gene is activated strongly and rapidly by ultraviolet (UV) irradiation and, to a somewhat lesser extent, by treatment with phorbol ester tumor promoters. In the same cells UV and phorbol esters only marginally enhance the abundance of RNA transcribed from the jun D gene and from the gene coding for the serum response factor (which in turn acts on the UV and phorbol ester response element of the c-fos gene). In contrast to c-jun, jun B transcription is induced more efficiently by phorbol ester than by UV irradiation, suggesting that the members of the jun family are differently regulated. The promoter of c-jun carries two enhancer elements resembling AP-1 binding sites: the junl UV response element (URE-71 TGACATCA-64) and the jun2 URE (-190 TTACCTCA-183). These elements act independently in the UV induced expression of c-jun. In the context of the complete c-jun promoter they seem not to be required for c-jun induction by phorbol esters. When fused to the Herpes simplex thymidine kinase promoter, however, the isolated elements mediate induction by both UV and phorbol esters. UV and phorbol ester treatment of cells increases the binding of transcription factors to both elements. Both elements bind factors different in modification or/and constitution from AP-1, the heterodimeric transcription factor composed of c-Fos and c-Jun that controls the activity of the UV and phorbol ester response element (-72 TGAGTCA-66) of the human collagenase gene.
Vmw65 (VP16) is a structural component of herpes simplex virus which is essential for proper replication and morphogenesis. This protein also performs an unusual additional role, in that it contains one of the most powerful transcriptional activation domains yet described. By an intricate pathway utilising host cell regulatory factors, Vmw65 selectively induces viral immediate early gene expression as one of the first steps in the viral replicative pathway. Studies of this function of Vmw65 have been the subject of considerable interest, and have provided insights both into the selective assembly of regulatory factors into multicomponent transcriptional complexes and into the transcriptional activation process itself. The fact that viral immediate early genes are positively regulated opens up the prospect that this step could represent a pivotal control point in determining the outcome of virus infection.
Summary Effects of DNA hypomethylating agents on reactivation of herpes simplex virus from latently infected mouse ganglia in vitro were examined. L-ethionine and 5-azacytidine increased the incidence of reactivation. Dimethylsulphoxide and 5-azacytidine allowed earlier detection of virus.
n-Butyrate (butyrate) has been shown to amplify transgene expression in cells infected with E1-defective adenoviruses. The present studies were undertaken in order to better define the actions of butyrate in the context of adenovirus gene expression, and to attempt to elucidate the mechanism by which butyrate mediates the transgene amplification. It was found that butyrate amplified viral transgene expression over a concentration range of 0.5–5 mM,and that the amplification required an exposure of 12–24 hr for maximal effect. Western blot analysis of representative viral proteins showed that butyrate treatment amplified DNA-binding protein, but not fiber protein. A transient adenoviral replication system suggested that butyrate had a modest inhibitory effect on replication of the E1-defective adenovirus. Use of a specific inhibitor of histone deacetylase, trichostatin A (TSA), reproduced the amplification of the viral transgene product achieved with the butyrate. In contrast, adenoviral transgene expression could not be amplified by TSA treatment in a cell line known to have a TSA-resistant histone deacetylase. Butyrate amplified steady-state gene expression of the viral transgene, but had no detectable effects on either DNA-binding protein or fiber steady-state gene expression. Nuclear run-off experiments showed that both butyrate and TSA caused an increase in the viral transgene transcription. It was concluded that inhibitors of histone deacetylase amplify adenoviral transgene expression at the transcriptional level.
The immediate-early (IE) proteins of herpes simplex virus (HSV) function on input genomes and affect many aspects of host cell metabolism to ensure the efficient expression and regulation of the remainder of the genome and, subsequently, the production of progeny virions. Due to the many and varied effects of IE proteins on host cell metabolism, their expression is not conducive to normal cell function and viability. This presents a major impediment to the use of HSV as a vector system. In this study, we describe a series of ICP4 mutants that are defective in different subsets of the remaining IE genes. One mutant, d109, does not express any of the IE proteins and carries a green fluorescent protein (GFP) transgene under the control of the human cytomegalovirus IE promoter (HCMVIEp). d109 was nontoxic to Vero and human embryonic lung (HEL) cells at all multiplicities of infection tested and was capable of establishing persistent infections in both of these cell types. Paradoxically, the genetic manipulations that were required to eliminate toxicity and allow the genome to persist in cells for long periods of time also dramatically lowered the level of transgene expression. Efficient expression of the HCMVIEp-GFP transgene in the absence of ICP4 was dependent on the ICP0 protein. In d109-infected cells, the level of transgene expression was very low in most cells but abundant in a small subpopulation of cells. However, expression of the transgene could be induced in cells containing quiescent d109 genomes weeks after the initial infection, demonstrating the functionality of the persisting genomes.
Hybrid polar compounds (HPCs) have been synthesized that induce terminal differentiation and/or apoptosis in various transformed cells. We have previously reported on the development of the second-generation HPCs suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bishydroxamide (CBHA) that are 2,000-fold more potent inducers on a molar basis than the prototype HPC hexamethylene bisacetamide (HMBA). Herein we report that CBHA and SAHA inhibit histone deacetylase 1 (HDAC1) and histone deacetylase 3 (HDAC3) activity in vitro. Treatment of cells in culture with SAHA results in a marked hyperacetylation of histone H4, but culture with HMBA does not. Murine erythroleukemia cells developed for resistance to SAHA are cross-resistant to trichostatin A, a known deacetylase inhibitor and differentiation inducer, but are not cross-resistant to HMBA. These studies show that the second-generation HPCs, unlike HMBA, are potent inhibitors of HDAC activity. In this sense, HMBA and the second-generation HPCs appear to induce differentiation by different pathways.
Nicotinamide and its analogues were evaluated for their activity as inducers of differentiation of murine erythroleukemia cells in culture. N'-Methylnicotinamide was the most effective of the compounds tested; at its optimal concentration it was more effective than dimethyl sulfoxide. With 8-10 mM N'-methylnicotinamide, almost all the cells contained hemoglobin (benzidine-reactive) after a 60-hr culture. Commitment to differentiate, assayed by transfer of the cells to semisolid medium without inducers, occurred much earlier and was more extensive with N'-methylnicotinamide than that with dimethyl sulfoxide or nicotinamide. Increase in globin mRNA was greater in the cells cultured with N'-methylnicotinamide than in cells cultured with dimethyl sulfoxide or nicotinamide. The relationship between the inducing activities of nicotinamide analogues and their effect on poly(ADP-ribose) polymerase in vitro was studied. All the compounds studied that had strong inhibitory effects on poly(ADP-ribose) polymerase in vitro induced differentiation of erythroleukemia cells in culture. This property is not a prerequisite of inducers; N'-methylnicotinamide did not inhibit the enzyme in vitro.
N'-Methylnicotinamide and nicotinamide, which decreased in vitro ADP-ribosylation of nuclear proteins and/or cellular NAD+ content, selectively increased the basal expression of the rat growth hormone (GH) gene promoter and its response to triiodothyronine (T3). This increase was not found when the thyroid hormone response element (TRE) was deleted from the promoter. Transfection with an expression vector for the T3 receptor inhibited basal activity of the TRE-containing promoter and repressed the stimulatory effect of N'-methylnicotinamide. The addition of hormone relieved this inhibition and enhanced transcription above levels found in the absence of the transfected receptors. These results suggest a modulatory role of ADP-ribosylation in hormonal regulation of gene expression.
The requirement for the herpes simplex virus type 1 (HSV-1) protein Vmw65 (VP16) for activation of immediate early (IE) gene expression was examined in synchronized HeLa cells. Analyses of IE RNA levels were conducted during infection with a viral Vmw65 mutant, in1814. The results revealed an increased requirement for Vmw65 when cultures reached G2 phase of the cell cycle. The levels of IE RNAs 1, 2, and 4 were reduced 5-10 times more in G2 than G1/S for in1814-infected cells when compared to cells infected with wild-type virus or 1814R (a rescued virus), and similar but smaller effects were observed on IE RNA 3 levels. The relative decrease at G2 was reversed by resynchronization of cells to G1/S. Mutant in1814 formed plaques less efficiently on cells at G2 than on cells synchronized at G1/S. The results show that, in the absence of functional Vmw65, HSV-1 IE gene expression and replication vary during the cell cycle.
The herpes simplex virus type 1 (HSV-1) mutant in1814 possesses an insertion mutation that abolishes trans-activation of immediate early (IE) transcription by the virion protein Vmw65. Interactions between in1814 and the host cell were examined by use of an in vitro latency system which relies on infection of human foetal lung (HFL) cells at 42 degrees C to prevent lytic growth of virus. Mutant in1814 was retained in HFL cells after infection at low m.o.i. and incubation at 42 degrees C, and was reactivated by superinfection of monolayers with viruses that express the HSV-1 IE protein Vmw110. Moreover, latency was established by in1814 in an analogous manner at 37 degrees C. The low cytotoxicity of in1814 enabled an investigation of latency after infection at high m.o.i. (five particles per cell) to be undertaken. At 42 degrees C, or at 37 degrees C in the presence of an inhibitor of DNA synthesis, in1814 DNA was maintained at low abundance (one to eight copies per infected cell) in a non-linear configuration. The absence of trans-activation by Vmw65 therefore predisposes HSV to latency, as opposed to lytic growth, in HFL cells, resulting in the retention of the genome in a form resembling that found in vivo.
The herpes simplex virus type 1 genome contains four open reading frames (ORFs) which are predicted to encode hydrophobic proteins with the potential to cross a membrane several times. The products of these genes (genes UL10, UL20, UL43 and UL53) have not previously been identified. To investigate the role of these proteins in the virus life cycle, we attempted to inactivate the genes individually by inserting the lacZ gene from Escherichia coli within the ORFs. Using this approach we have isolated insertion mutants for UL10 and UL43, as well as a deletion mutant lacking the majority of the UL43 ORF. The growth of the UL10-lacZ virus was slightly impaired in tissue culture compared to that of the wild-type virus parent, whereas the growth of the UL43 mutants was indistinguishable from that of wild-type virus. Furthermore, deletion of the majority of the UL43 ORF did not impair the ability of the virus to replicate in vivo at the periphery, or to spread to and replicate within the nervous system, in a mouse ear model. Repeated attempts to isolate lacZ insertion mutants for UL20 and UL53 were unsuccessful, suggesting that these genes may be essential for virus growth, at least in tissue culture. Using antipeptide sera, the products of genes UL10 and UL20 have been detected.
Bishydroxamic acids are effective inducers of differentiation in murine erythroleukemia cells. Flexible analogs of suberic acid bisdimethylamide are approximately 100 times as active as the parent compound or hexamethylenebisacetamide. They also induce differentiation of human promyelocytic leukemia cells (HL-60) and a subclone of human colon carcinoma cells (HT-29-U4). Some rigid bishydroxamic acids with benzene rings in the spacers are even more active toward murine erythroleukemia cells but show curious biological differences. In contrast to the flexible molecules, those with benzene spacers show poor activity toward HL-60 cells; they also have different geometric requirements, and they are not additive with hexamethylenebisacetamide in their effect. It is likely that rigid bishydroxamic acids, with a benzene ring spacer, induce differentiation by a different mechanism in spite of their chemical resemblance to the flexible bisamide and bishydroxamic acid inducers.
The fungistatic antibiotics trichostatins (TS) A and C were isolated from culture broth of Streptomyces platensis No. 145 and were found to be potent inducers of differentiation in murine erythroleukemia (Friend and RV133) cells at concentrations of 1.5 X 10(-8) M for TSA and 5 X 10(-7) M for TSC. Differentiation induced by TS was cooperatively enhanced by UV irradiation but not by treatment with dimethyl sulfoxide. This enhanced activity was completely inhibited by adding cycloheximide to the culture medium 2 h after exposure to TS, suggesting that TS are dimethyl sulfoxide-type inducers of erythroid differentiation. No inhibitory effect of TS was observed on macromolecular synthesis in cultured cells.
We evaluated the effect of the demethylating agent hexamethylenebisacetamide on reactivation of latent herpes simplex virus type 2 (HSV-2) from guinea pig neural and extraneural tissues. Four explant cultures from the dorsal root ganglia of 42 latently infected guinea pigs and vaginal and cervical explant cultures from 33 animals were divided so that half received 5 mM of hexamethylenebisacetamide supplemented media and half media alone. HSV-2 was recovered earlier and from a greater percentage of treated cultures than controls. For example, seven days after explant, HSV-2 was recovered from 35 of 84 (42%) treated dorsal root ganglia cultures compared to seven of 84 control cultures (p less than 0.0001). Likewise, HSV-2 was recovered seven days after explant from 11 of 66 (17%) treated external genital skin cultures and 2 of 66 control cultures (p less than 0.009), Hexamethylenebisacetamide had no effect on productive HSV-2 infection in guinea pig dorsal root ganglia cultures. This study provides evidence for a role of demethylation in the reactivation of latent HSV from neural as well as peripheral tissues and suggests that latent virus exists at these sites in a similar state. Hexamethylenebisacetamide should be useful in studies of herpes virus latency because it decreases the time necessary to recover virus from latently infected tissues and enhances the recovery of virus.
DNA is a highly reactive chemical species and therefore is the target of numerous physical and chemical agents. Heat causes deamination of bases, and base loss by gycosylic hydrolysis; UV radiation produces pyrimidine dimers and 6-4 photoproducts; ionizing radiation results in ring opening, base fragmentation, and single- and double-strand breaks. Chemical agents that modify DNA range from activated oxygen species generated during oxidative metabolism to such common metabolites as glucose to diverse inorganic and organic electrophiles including metals, alkylating agents, and polycyclic aromatic hydrocarbons. It is therefore quite remarkable that DNA is functionally more stable than the two other cellular macromolecules, RNA and protein. [A 3.4-kilobase fragment containing Alu sequences was recently cloned from a 2400-year-old mummy]. This stability can be attributed to the double-helical structure, which carries the information in duplicate, and to the fact that the primary structure of DNA is all that is needed for transfer of information. Equally important for functional stability of DNA are the various repair mechanisms. There are molecular mechanisms of different complexities to undo the DNA damage (modification) and maintain cellular survival as well as genetic integrity. In direct DNA repair the chemical change in DNA is simply reversed. DNA photolyase, which breaks the cyclobutane ring of a pyrimidine dimer, and m-O6Gua DNA methyltransferase, which removes methyl groups from DNA, are examples of this class of enzymes. Other repair pathways remove modified (incorrect) nucleotides and rely on the redundant information in the complementary strand to restore the duplex. In base-excision repair the base is first removed by a glycosylase, then the abasic sugar is removed by apurinic/apyrimidinic (AP) endonucleases and DNA polymerase. Abasic sugars (AP sites) are also generated spontaneously, by a facilitated mechanism or by direct action of damaging agents. In nucleotide-excision repair, modified bases are removed in the form of an oligonucleotide. The single-strand gaps so generated are filled in by polymerases. In recombinational repair, the gaps that are generated during replication of damaged duplexes are filled in by strand transfer from an intact duplex. In the past five years, mostly through the use of recombinant DNA technology, the enzymes involved in these various repair processes have been overproduced, purified, and characterized, and the regulation of the cellular responses to DNA damage has been studied in detail. The most dramatic progress has been in studies of DNA repair in Escherichia coli, and therefore this organism will be emphasized. However, some eukaryotic repair enzymes have been purified extensively, and DNA repair genes from yeast and humans have also been cloned, and therefore eukaryotic DNA repair will also be briefly reviewed. Accounts of older literature have appeared in this series and in a comprehensive book on DNA repair. The highly specialized cellular response to alkylation damage (adaptation) is reviewed elsewhere in this volume. In this review we follow the traditional classification of repair mechanisms and do not discuss 'mismatch repair', which is essentially an error-correcting mechanism and has been reviewed recently.
The expression of viral genes during infection of tissue culture cells by herpesviruses can be divided into three broad classes termed Immediate-Early (IE), early and late. The IE polypeptides include trans-acting regulators of gene expression; prior expression of IE gene products is essential for the activation of transcription from early gene promoters. During the past five years, the use of modern gene manipulation techniques, short-term transfection assays and in vitro methods has resulted in a significant increase in our understanding of the IE polypeptides. This review concentrates on the genetic, physical and functional properties of herpesvirus IE gene products with particular emphasis on those of herpes simplex virus type 1.
Based on evidence that 50% of herpes simplex 1 DNA is transcribed in HEp 2 cells in the absence of protein synthesis the order and rates of synthesis of viral polypeptides in infected cells after reversal of cycloheximide or puromycin mediated inhibition of protein synthesis were examined. These experiments showed that viral polypeptides formed three sequentially synthesized, coordinately regulated groups designated α, β, and γ. Specifically: the α group, containing one minor structural and several nonstructural polypeptides, was synthesized at highest rates from 3 to 4 hr postinfection in untreated cells and at diminishing rates thereafter. The β group, also containing minor strucutural and nonstructural polypeptides, was synthesized at highest rates from 5 to 7 hr and at decreasing rates thereafter. The γ group containing major structural polypeptides was synthesized at increasing rates until at least 12 hr postinfection.
This paper describes the activation of cellular genes after infection of chick embryo fibroblasts (CEF) with temperature-sensitive (ts) mutants of herpes simplex virus type 1. One mutant, tsK, has a mutation in the gene sequences which encode the immediate-early (IE) polypeptide Vmw175. After infection of CEF with tsK at the nonpermissive temperature (38.5°), IE polypeptides are overproduced but early and late viral gene products cannot be detected. In addition, the synthesis of three cellular polypeptides is stimulated in a manner which closely resembles the “stress” or “heat-shock” response. Peptide mapping confirmed that the tsK-induced polypeptides are stress proteins. No induction was observed when viral gene expression was prevented, leading to the conclusion that one or more IE polypeptides is responsible for the stress response. Two other mutants, tsD and tsT, which have mutations in different regions of the Vmw175 coding sequences, also induce stress proteins at 38.5°.
The murine erythroleukemia cell line is a virus transformed system whose expression of erythroid differentiation appears to be blocked at an early stage of erythroid development. The ability to alter the transcriptional program by treatment with chemical compounds of defined structure has proved to be of great value for studying controls of macromolecular biosynthesis. Understanding the mechanism whereby these chemical inducers relieve the virus-imposed block in the erythroid program should provide insight into the regulation of the coordinated expression of the differentiation program. Examination of temporal and cell cycle relationships to the expression of erythroid characteristics in differentiating and in resistant variants as well as in inhibitor-treated cells should also be useful in elucidating control mechanisms. The ability to reverse the block in normal differentiation and thereby reverse the malignant phenotype may eventually prove to be of clinical interest.
We have developed a practical synthesis of N-hydroxy-N'-phenyloctanediamide from the methyl ester of suberanilic acid. It provides the product in high yield and purity with a simple purification process. We have found that at 10(-5) M it has a dramatic effect on T/5 AXC/SSh rat prostate cancer cells in vitro. It is a potent inhibitor of cell proliferation and it changes the cell morphology to resemble nonmalignant cells.
Explanation into culture of dorsal root ganglia (DRG) latently infected with herpes simplex virus type 1 (HSV-1) causes reactivation of the virus. Previous studies have suggested that either latency-associated transcripts (LATs) were removed as an early consequence of reactivation or, alternatively, there was a population of latently infected cells which did not contain LATs. We have now attempted to detect this population of neurons by inserting a reporter gene (Escherichia coli lacZ gene), under the control of promoters other than LAT, into the HSV-1 strain 17 mutant in 1814, which was used in the earlier studies. One of these promoters, the human cytomegalovirus enhancer, resulted in weak expression of beta-galactosidase in DRG neurons for at least 5 months. The pattern of staining was predominantly homogeneous in neurons at 3 or 5 days post-infection or at 3 days post-explanation, and was predominantly speckled in latently infected neurons (1 to 5 months post-infection). About 30% of the beta-galactosidase-positive neurons did not contain LATs by in situ hybridization. However, the detergents used to enable penetration of the substrate for beta-galactosidase had also reduced the levels of the LATs; in neurons which originally had only small numbers of LATs this may have reduced levels to below those detectable by the methods used. There was, therefore, no unequivocal evidence for a population of latently HSV-1-infected cells which did not express LATs.
The development and utilization of a tissue culture system for the analysis of quiescent, nonreplicating herpes simplex virus type 1 (HSV-1) genomes is described. It was demonstrated previously that the HSV-1 Vmw65 mutant in1814, which is impaired for immediate early (IE) transcription, was retained for many days in human fetal lung (HFL) fibroblasts in a quiescent 'latent' state. Molecular analysis of the viral genome was not possible, however, due to residual expression of IE proteins and consequent cytotoxicity at high m.o.i. In the study reported here, IE transcription was reduced further by pretreatment of cells with interferon-alpha (IFN-alpha) and by the use of mutant in1820, a derivative of in1814 in which the Vmw110 promoter was replaced by the Moloney murine leukaemia virus (Momulv) enhancer. The Momulv enhancer was not expressed under IE conditions; thus in1820 was more impaired for replication than in1814 and behaved as if deficient for both Vmw65 and Vmw110. In cells pretreated with IFN-alpha and subsequently infected with in1820 cytotoxicity was overcome, enabling a tissue culture system to be developed in which all cells stably retained at least one quiescent viral genome. To assist the analysis of gene expression, in1820 was further modified by insertion of the Escherichia coli lacZ gene controlled by the human cytomegalovirus enhancer (mutant in1883) or the HSV-1 immediate early Vmw110 promoter (in1884). Expression of beta-galactosidase was not detected after infection of IFN-alpha-pretreated cells with in1883 or in1884 but could be induced in almost all cells containing a viral genome, by superinfection of cultures. In1820-derived viruses were retained for at least 9 days and were not reactivated by subculture of cells. A regular arrangement of nucleosomes, as found in cellular chromatin, was not detected on the viral genome at the thymidine kinase locus. The non-linear genome was a template for reactivation with no requirement for prior conversion to a linear form. A small number of remaining linear genomes resulted from incomplete uncoating of input virus.
Infected cell protein 0 (ICP0), a major immediate-early regulatory protein of herpes simplex virus type 1 (HSV-1), activates expression of all classes of HSV genes as well as a variety of heterologous viral and cellular genes. Previous studies have shown that a cellular activity expressed maximally in Vero cells 8 h after release from growth arrest in the G0/G1 phase of the cell cycle can enhance plaque formation and gene expression of a mutant virus (7134) lacking both copies of the gene encoding ICP0 (W. Cai and P. Schaffer, J. Virol. 65:4078-4090, 1991). This observation suggests that the cellular activity can substitute for ICP0 to activate viral gene expression. To further characterize this cellular activity, Vero and NB41A3 (mouse neuroblastoma) cells were transfected at various times after release from growth arrest with promoter-chloramphenicol acetyltransferase (CAT) constructs containing promoters representing the major kinetic classes of HSV genes, and CAT activity was measured from 2 to 24 h postrelease. The results of these tests demonstrate that CAT expression from immediate-early promoter-CAT plasmids was enhanced 10- and 3-fold when Vero and NB41A3 cells were transfected at 6 and 2 h postrelease, respectively. In contrast, only low levels of immediate-early promoter-driven CAT activity were apparent when cells were transfected at later times postrelease. No significant stimulation of CAT activity was observed from promoter-CAT plasmids containing representative early or late HSV promoters or a heterologous viral (simian virus 40 early) promoter. Differences in the efficiency of uptake of plasmid DNA by cells at various times postrelease did not account for the observed differences in CAT expression. Unlike Vero cells, in which cell division resumed after release from growth arrest, division of NB41A3 cells did not resume. Rather, these cells displayed morphological features suggestive of a differentiated phenotype. Collectively, these findings demonstrate that a cellular activity expressed in Vero and NB41A3 cells after release from growth arrest can activate HSV gene expression by enhancing immediate-early gene expression.
Transformed cells do not necessarily lose their capacity to differentiate. Various agents can induce many types of neoplastic cells to terminal differentiation. Among such inducers, a particularly potent group consists of hybrid polar compounds; hexamethylene bisacetamide (HMBA) is the prototype of this group. With virus-transformed murine erythroleukemia cells as a model, HMBA was shown to cause these cells to arrest in G1 phase and express globin genes. This review focuses on HMBA-induced modulation of factors regulating G1-to-S phase progression, including a decrease in the G1 cyclin-dependent kinase cdk4, associated with inhibition of phosphorylation of the retinoblastoma protein pRB and possibly other related proteins that, in turn, sequester factors required for initiation of DNA synthesis; this provides a possible mechanism for HMBA-induced terminal cell division. Evidence that hybrid polar compounds have therapeutic potential for cancer treatment will also be reviewed.
A highly conserved, cysteine-rich region plays a crucial role in the function of a family of regulatory proteins encoded by alpha herpes viruses. The so-called C3HC4 motif spans approximately 60 residues and has been predicted to bind zinc. This motif occurs in a number of other viral and cellular proteins, many of which appear to be involved in some aspect of the regulation of gene expression. We have cloned and expressed in bacteria a portion of immediate-early protein Vmw110 of herpes simplex virus type 1 that encompasses the C3HC4 motif, and the equivalent regions from the homologous proteins of varicella zoster virus and equine herpes virus type 1 (EHV-1). All three polypeptides were purified and found to bind zinc stably. None of the three interacted significantly with either DNA or RNA under our assay conditions. The EHV-1 domain yielded interpretable proton nuclear magnetic resonance spectra. Assignment of resonances and analysis of nuclear Overhauser effects revealed its secondary structure. Starting from the N terminus, this consists of an ordered but irregular loop, the first two strands of a triple-stranded antiparallel beta-sheet, two turns of an alpha-helix, a second irregular loop, and the third strand of the beta-sheet. It appears that, taking the cysteine and histidine residues in turn, cysteine residues I, II, IV and V co-ordinate one zinc atom while the histidine residue and cysteine residues III, VI and VII co-ordinate a second zinc atom. This arrangement of secondary structure differs from that found in other characterized zinc-containing proteins.
Retroviral and adeno-associated viral sequences can dramatically silence transgene expression in mice. We now report that this repression also occurs in stably infected HeLa cells when the cells are grown without selection. Expression of a transduced lacZ gene (rAAV/CMVlacZ) is silenced in greater than 90% of cells after 60 days in culture. Surprisingly, high-level expression can be reactivated by treating the cells with sodium butyrate or trichostatin A but not with 5-azacytidine. When cell clones with integrated copies of rAAV/CMVlacZ were isolated, lacZ expression was silenced in 80% of the clones; however, lacZ expression was reactivated in all of the silenced clones by treatment with butyrate or trichostatin A. The two drugs also reactivated a silenced globin gene construct (rAAV/HS2alphabetaAS3) in stably infected K562 cells. Trichostatin A is a specific inhibitor of histone deacetylase; therefore, we propose that hyperacetylation of histones after drug treatment changes the structure of chromatin on integrated viral sequences and relieves repression of transduced genes. The reactivation of silenced, transduced genes has implications for gene therapy. Efficient viral gene transfer followed by drug treatment to relieve suppression may provide a powerful combination for treatment of various genetic and infectious diseases.
Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.
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