Biomedical subjects

M A Hardwicke

Publications and source records attributed to M A Hardwicke.

10 recordsLinked to original sources

PubMed1997

Differential effects of nerve growth factor and dexamethasone on herpes simplex virus type 1 oriL- and oriS-dependent DNA replication in PC12 cells.

The herpes simplex virus type 1 (HSV-1) genome contains three origins of DNA replication, one copy of oriL and two copies of oriS. Although oriL and oriS are structurally different, they have extensive nucleotide sequence similarity and can substitute for each other to initiate viral DNA replication. A fundamental question that remains to be answered is why the HSV-1 genome contains two types of origin. We have recently identified a novel glucocorticoid response element (GRE) within oriL that is not present in oriS and have shown by gel mobility shift assays that purified glucocorticoid receptor (GR), as well as GR present in cellular extracts, can bind to the GRE in oriL. To determine whether glucocorticoids and the GRE affect the efficiency of oriL-dependent DNA replication, we performed transient DNA replication assays in the presence and absence of dexamethasone (DEX). Because HSV-1 is a neurotropic virus and establishes latency in cells of neural origin, these tests were conducted in PC12 cells, which assume the properties of sympathetic neurons when differentiated with nerve growth factor (NGF). In NGF-differentiated PC12 cells, oriL-dependent DNA replication was enhanced 5-fold by DEX, whereas in undifferentiated cells, DEX enhanced replication approximately 2-fold. Notably, the enhancement of oriL function by DEX was abolished when the GRE was mutated. NGF-induced differentiation alone had no effect. In contrast to oriL, oriS-dependent DNA replication was reduced approximately 5-fold in NGF-differentiated PC12 cells and an additional 4-fold in differentiated cells treated with DEX. In undifferentiated PC12 cells, DEX had only a minor inhibitory effect (approximately 2-fold) on oriS function. Although the cis-acting elements that mediate the NGF- and DEX-specific repression of oriS-dependent DNA replication are unknown, a functional GRE is critical for the DEX-induced enhancement of oriL function in NGF-differentiated PC12 cells. The enhancement of oriL-dependent DNA replication by DEX in differentiated PC12 cells suggests the possibility that glucocorticoids, agents long recognized to enhance reactivation of latent herpesvirus infections, act through the GRE in oriL to stimulate viral DNA replication and reactivation in terminally differentiated neurons in vivo.

Animals↗

PubMed1996

Properties of the novel herpes simplex virus type 1 origin binding protein, OBPC.

We have recently identified a novel 53-kDa herpes simplex virus type 1 (HSV-1) protein encoded by, and in frame with, the 3' half of the UL9 open reading frame, designated OBPC (K. Baradaran, C. Dabrowski and P. A. Schaffer, J. Virol. 68:4251-4261, 1994). Here we show that OBPC is a nuclear protein synthesized at both early and late times postinfection. In gel-shift assays in vitro-synthesized OBPC bound to oriS site I DNA to form a complex identical in mobility to complex A, generated with infected cell extracts and site I DNA. OBPC inhibited both plaque formation and viral DNA replication in transient assays, consistent with its ability to bind to site I DNA and its limited ability to interact with other essential DNA replication proteins. These properties suggest that OBPC may play a role in the initiation, elongation, or packaging of viral DNA.

DNA-Binding Proteins↗

PubMed1995

The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing.

Herpes simplex virus type 1 infection results in a reorganization of antigens associated with the small nuclear ribonucleoprotein particles (snRNPs), resulting in the formation of prominent clusters near the nuclear periphery. In this study, we show that the immediate-early protein ICP27, which is involved in the impairment of host cell splicing and in the changes in the distribution of snRNPs, is also required for reassorting the SR domain splicing factor SC35. Other viral processes, such as adsorption and penetration, shutoff of host protein synthesis, early and late gene expression, and DNA replication, do not appear to play a role in changing the staining pattern of splicing antigens. Furthermore, the C-terminal repressor region of ICP27, which is required for the inhibitory effects on splicing, also is involved in redistributing the snRNPs and SC35. During infection or transfection with five different repressor mutants, the speckled staining pattern characteristic of uninfected cells was seen and the level of a spliced target mRNA was not reduced. Infections in the presence of activator mutants showed a redistributed snRNP pattern and a decreased accumulation of spliced target mRNA. Moreover, two arginine-rich regions in the N-terminal half of ICP27 were not required for the redistribution of snRNPs or SC35. Substitution of these regions with a lysine-rich sequence from simian virus 40 large-T antigen resulted in a redistribution of splicing antigens. Unexpectedly, a repressor mutant with a ts phenotype showed a redistributed staining pattern like that seen with wild-type infected cells. During infections with this ts mutant, splicing was not inhibited, as shown in this and previous studies, confirming its repressor phenotype. Furthermore, both the mutant and the wild-type protein colocalized with snRNPs. Therefore, the redistribution of snRNPs and SC35 correlates with ICP27-mediated impairment of host cell splicing, but these alterations are not sufficient to fully inhibit splicing. This indicates that active splicing complexes are still present even after dramatic changes in the organization of the snRNPs.

Animals↗

PubMed1995

Cloning and characterization of herpes simplex virus type 1 oriL: comparison of replication and protein-DNA complex formation by oriL and oriS.

The herpes simplex virus type 1 genome contains three origins of DNA replication: two copies of oriS and one copy of oriL. Although oriS has been characterized extensively, characterization of oriL has been severely limited by the inability to amplify oriL sequences in an undeleted form in Escherichia coli. We report the successful cloning of intact oriL sequences in an E. coli strain, SURE, which contains mutations in a series of genes involved in independent DNA repair pathways shown to be important in the rearrangement and deletion of DNA containing irregular structures such as palindromes. The oriL-containing clones propagated in SURE cells contained no deletions, as determined by Southern blot hybridization and DNA sequence analysis, and were replication competent in transient DNA replication assays. Deletion of 400 bp of flanking sequences decreased the replication efficiency of oriL twofold in transient assays, demonstrating a role for flanking sequences in enhancing replication efficiency. Comparison of the replication efficiencies of an 822-bp oriS-containing plasmid and an 833-bp oriL-containing plasmid demonstrated that the kinetics of replication of the two plasmids were similar but that the oriL-containing plasmid replicated 60 to 70% as efficiently as the oriS-containing plasmid at both early and late times after infection with herpes simplex virus type 1. The virus-specified origin-binding protein (OBP) and a cellular factor(s) (OF-1) have been shown in gel mobility shift experiments to bind specific sequences in oriS (C.E. Dabrowski, P. Carmillo, and P.A. Schaffer, Mol. Cell. Biol. 14:2545-2555, 1994; C.E. Dabrowski and P.A. Schaffer, J. Virol. 65:3140-3150, 1991). Although the nucleotides required for the binding of OBP to OBP binding site I in oriL and oriS are the same, a single nucleotide difference distinguishes OBP binding site III in the two origins. The nucleotides adjacent to oriS sites I and III have been shown to be important for the binding of OF-1 to oriS site I. Several nucleotide differences exist in these sequences in oriL and oriS. Despite these minor nucleotide differences, the protein-DNA complexes that formed with oriL and oriS sites I and III were indistinguishable when extracts of infected and uninfected cells were used as the source of protein. Furthermore, the results of competition analysis suggest that the proteins involved in protein-DNA complex formation with sites I and III of the two origins are likely the same.

Animals↗

PubMed1994

The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection.

We have previously shown that the herpes simplex virus immediate-early regulatory protein ICP27 acts posttranscriptionally to affect mRNA processing (R. M. Sandri-Goldin and G. E. Mendoza, Genes Dev. 6:848-863, 1992). Specifically, in the presence of ICP27, spliced target mRNAs were decreased 5- to 10-fold in transfections with target genes containing a 5' or 3' intron. Here, we have investigated the effect of ICP27 during herpes simplex virus type 1 (HSV-1) infection on accumulation of spliced cellular mRNAs. ICP27 viral mutants have been shown to be defective in host shutoff (W. R. Sacks, C. C. Greene, D. P. Aschman, and P. A. Schaffer, J. Virol. 55:796-805, 1985). Therefore, we examined whether ICP27 could contribute to this complex process by decreasing cellular mRNA levels through its effects on host cell splicing. It was found that in infections with viral mutants defective in ICP27, the accumulated levels of three spliced host mRNAs were higher than those seen with wild-type HSV-1. The differences occurred posttranscriptionally as shown by nuclear runoff transcription assays. The stabilities of the spliced products during infection with wild-type or ICP27 mutant viruses were similar, and unspliced precursor mRNA for a viral spliced gene was detected in infections with wild-type HSV-1 but not in infections in which ICP27 was not expressed. These results suggest that the reduction in cellular mRNA levels and the accumulation of pre-mRNA are related and may be caused by an impairment in host cell splicing. These data further show that ICP27 is required for these effects to occur.

Actins↗

PubMed1992

Promoter-independent activation of heterologous virus gene expression by the herpes simplex virus immediate-early protein ICP27.

The herpes simplex virus (HSV) immediate-early protein ICP27 has been postulated to play an auxillary role in HSV. gene expression, augmenting or inhibiting the activation of different viral promoters by the other immediate-early proteins ICPO and ICP4. Here we show that ICP27 alone can up-regulate gene expression of a retroviral vector containing Moloney murine leukemia virus (MoMuLV) regulatory sequences. This is the first report of an effect of ICP27 on gene expression driven by heterologous virus regulatory sequences. The effect does not involve the region of ICP27 which inhibits gene activation of HSV early gene promoters by ICPO and ICP4, but rather is dependent on the same region of ICP27 as is required to augment the activation of HSV late gene promoters by ICPO and ICP4. This indicates that the two activation effects are likely to operate via the same mechanism. Activation by ICP27 is dependent on the 3' LTR and adjacent region of MoMuLV but is independent of the promoter in the 5' LTR which can be replaced by a heterologous promoter such as that of SV40 without affecting activation by ICP27. The significance of this effect for an understanding of the mechanism of action of ICP27 and its role in regulating the gene expression of HSV and potentially of other viruses is discussed.

3T3 Cells↗

PubMed1992

Evidence that the herpes simplex virus immediate early protein ICP27 acts post-transcriptionally during infection to regulate gene expression.

The herpes simplex virus (HSV-1) immediate early protein ICP27 is a regulatory protein which is essential for virus replication. The phenotype of temperature-sensitive and deletion mutants in ICP27 includes overexpression of some immediate early and early gene products and greatly reduced levels of late gene products. To determine whether regulation by ICP27 occurs primarily at the transcriptional level, we have studied the expression of two immediate early products (ICP4 and ICP27) and two late gene products (glycoprotein B and glycoprotein C) at the level of transcription initiation, accumulation of steady state mRNA, and protein synthesis in an ICP27 temperature-sensitive mutant tsLG4, compared to wild-type HSV-1. At the nonpermissive temperature in tsLG4-infected cells, the two immediate early gene products ICP4 and ICP27 were overexpressed both at the mRNA and protein level although synthesis of these transcripts as measured by nuclear runoff assays was reduced relative to the wild-type HSV-1 infections. The transcription of late gene products glycoprotein B (gB) and glycoprotein C (gC) was lower in runoff assays from tsLG4 infections suggesting that the reduction in the level of late products occurred at the transcriptional level. However, temperature shift experiments in which tsLG4-infected cells were shifted to the nonpermissive temperature at various times after infection showed that the synthesis of late transcripts was not altered 2 hr after the shift whereas both the accumulation of leaky late and late mRNA and the incorporation of [35S]methionine into newly synthesized gB and gC was reduced by 2 hr after the shift to nonpermissive temperature. Therefore, while the synthesis of new transcripts continued, the accumulation of late mRNAs and their translation into protein was reduced when ICP27 was defective, whereas, the converse was found for immediate early products. That is, the synthesis of new transcripts was reduced yet mRNA and protein accumulated to high levels. These results suggest that ICP27 acts at least in part post-transcriptionally to regulate the expression of immediate early and late gene products.

Animals↗

PubMed1992

The herpes simplex virus immediate early protein ICP27 encodes a potential metal binding domain and binds zinc in vitro.

The herpes simplex virus type 1 (HSV-1) immediate-early regulatory proteins ICP27 and ICP0 each encode putative zinc-finger metal-binding domains. We utilized the technique of metal chelate affinity chromatography to demonstrate that ICP27 and ICP0 were able to bind to zinc in vitro. This property was further exploited to purify ICP27 from extracts of HSV-1-infected cells. The purification procedure also revealed that ICP27 possessed single-stranded DNA-binding activity. Analysis of ICP27 truncated peptides produced by in vitro translation verified that the zinc-binding region of ICP27 resides in the carboxy terminal 105 amino acids spanning the putative metal binding motif. However, a specific configuration of cysteine and histidine residues in this region was not required for binding to occur as demonstrated by the ability of a frame-shift mutation to bind with an efficiency similar to wild type. The mutated peptide retained four histidine and cysteine residues but in a configuration different from the consensus proposed for zinc-finger motifs. Therefore, while the region spanning the metal binding domain of ICP27 is essential for both the activator and repressor functions, and ICP27 binds zinc in vitro, it is not clear whether zinc binding in vivo is necessary for function.

Amino Acid Sequence↗

PubMed1991

Mutations in the activation region of herpes simplex virus regulatory protein ICP27 can be trans dominant.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an essential regulatory protein which is required for virus replication. Transfection experiments have demonstrated that ICP27 along with the HSV-1 transactivators ICP4 and ICP0 can positively regulate the expression of some late HSV-1 target plasmids and can negatively regulate the expression of some immediate-early and early target plasmids. We previously showed that mutants defective in the activation of a late target plasmid mapped to the carboxy-terminal half of the protein, whereas mutants defective in the repression of an early target plasmid mapped within the C-terminal 78 amino acids of ICP27 (M. A. Hardwicke, P. J. Vaughan, R. E. Sekulovich, R. O'Conner, and R. M. Sandri-Goldin, J. Virol. 63:4590-4602, 1989). In this study, we cotransfected ICP27 activator and repressor mutants along with wild-type ICP27 plasmid to determine whether these mutants could interfere with the wild-type activities. Mutants which were defective only in the activation function were dominant to the wild-type protein and inhibited the activation of the late target plasmid pVP5-CAT, whereas mutants defective in the repressor function did not inhibit either the activation of pVP5-CAT or the repression of the early target plasmid pTK-CAT. Furthermore, cell lines which stably carried three different activator mutants were impaired in their ability to support the growth of wild-type HSV-1 strain KOS, resulting in virus yields 5- to 40-fold lower than in control cells. The defect in virus replication appeared to stem from a decrease in the expression of HSV-1 late gene products during infection as measured by steady-state mRNA levels and by immunoprecipitation analysis of specific polypeptides. These results indicate that ICP27 activator mutations specifically interfere with the activation function of the protein both in transfection and during infection. Moreover, these results suggest that the repressor region may be important for binding of the polypeptide, since mutations in this region did not interfere with the activities of wild-type ICP27 and therefore presumably could not compete for binding.

Amino Acid Sequence↗

PubMed1989

The regions important for the activator and repressor functions of herpes simplex virus type 1 alpha protein ICP27 map to the C-terminal half of the molecule.

The herpes simplex virus type 1 (HSV-1) alpha or immediate-early proteins ICP4 (IE175), ICP0 (IE110), and ICP27 (IE63) are trans-acting proteins which affect HSV-1 gene expression. We previously showed that ICP27 in combination with ICP4 and ICP0 could act as a repressor or an activator in transfection assays, depending on the target gene (R. E. Sekulovich, K. Leary, and R. M. Sandri-Goldin, J. Virol. 62:4510-4522, 1988). To investigate the regions of the ICP27 protein which specify these functions, we constructed a series of in-frame insertion and deletion mutants in the ICP27 gene. These mutants were analyzed in transient expression assays for the ability to repress or to activate two different target genes. The target plasmids used consisted of the promoter regions from the HSV-1 beta or early gene which encodes thymidine kinase and from the beta-gamma or leaky late gene. VP5, which encodes the major capsid protein, each fused to the chloramphenicol acetyltransferase gene. Our previous studies showed that induction of pTK-CAT expression by ICP4 and ICP0 was repressed by ICP27, whereas the stimulation of pVP5-CAT expression seen with ICP4 and ICP0 was significantly increased when ICP27 was also added. In this study, a series of transfection assays was performed with each of the ICP27 mutant plasmids in combination with plasmids containing the ICP4 and ICP0 genes with each target. The results of these experiments showed that mutants containing insertions or deletions in the region from amino acids 262 to 406 in the carboxy-terminal half of the protein were unable to stimulate expression of pVP5-CAT but were able to repress induction of pTK-CAT activity by ICP4 and ICP0. Mutants in the carboxy-terminal 78 amino acids lost both activities; that is, these mutants did not show repression of pTK-CAT activity or stimulation of pVP5-CAT activity, whereas mutants in the hydrophilic amino-terminal half of ICP27 were able to perform both functions. These results show that the carboxy-terminal half of ICP27 is important for the activation and repression functions. Furthermore, the carboxy-terminal 62 amino acids are required for the repressor activity, because mutants with this region intact were able to repress. Analysis of the DNA sequence showed that there are a number of cysteine and histidine residues encoded by this region which have some similarity to zinc finger metal-binding regions found in other eucaryotic regulatory proteins. These results suggest that the structural integrity of this region is important for the function of ICP27.

Amino Acid Sequence↗