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C M Preston

Publications and source records attributed to C M Preston.

At least 55 records · Page 3Linked to original sources
PubMed1993

Specific transcriptional activation in vitro by the herpes simplex virus protein VP16.

The herpes simplex virus protein VP16 interacts with cellular factors, including the protein Oct-1, to activate viral immediate early (IE) gene transcription. We have reproduced this effect by addition of purified, full-length VP16 and the DNA-binding 'POU' domain of Oct-1 (Oct-1/POU) to a HeLa cell in vitro transcription system. Stimulation of transcription was dependent on the IE-specific element, TAATGARAT. In agreement with earlier observations from electrophoretic mobility shift assays, activation was not observed when Oct-2/POU, the DNA-binding domain from the Oct-2 protein, was substituted for Oct-1/POU. Single round transcription assays revealed that, together, VP16 and Oct-1/POU facilitate the assembly of pre-initiation complexes at target gene promoters.

Base Sequence↗
PubMed1993

Neurons containing latency-associated transcripts are numerous and widespread in dorsal root ganglia following footpad inoculation of mice with herpes simplex virus type 1 mutant in1814.

The herpes simplex virus type 1 (HSV-1) mutant in1814 lacks the ability to trans-activate immediate early gene transcription and enter lytic replication but it can establish and reactivate from latency. We therefore investigated the number of neurons that expressed latency-associated transcripts (LATs) in animals latently infected with in1814, the rescued revertant (1814R), or wild-type (wt) HSV-1. The percentage of LAT+ neurons increased with increasing doses of each of the viruses. After inoculation of equal amounts of infectious virus many more LAT+ neurons were observed in animals infected with in1814 than with 1814R or wt HSV-1. Whereas the LAT+ neurons in animals infected with 1814R or wt HSV-1 were largely confined to lumbar dorsal root ganglia (DRG) L4/L5/L6 (those which innervate the lower leg), in animals infected with in1814 they were also present in DRG not directly involved with such innervation (thoracic 12 and 13, L1, L2 and L3). We concluded that the large number of LAT+ neurons observed with in1814 was related to the high particle numbers in the inoculum and that spread of virus was related to limited replication as well as to the low neurovirulence of in1814. This spread was not unique to in1814 but when it occurred with more virulent viruses such as 1814R or wt HSV-1, it resulted in the death of the host.

Animals↗
PubMed1993

Reactivation in vivo and in vitro of herpes simplex virus from mouse dorsal root ganglia which contain different levels of latency-associated transcripts.

In the dorsal root ganglia (DRG) of mice latently infected with the herpes simplex virus type 1 mutant in1814, there are more neurons that contain latency-associated transcripts (LATs) than in DRG of mice infected with a dose of equal infectivity of either a revertant or a wild-type virus. We investigated whether higher levels of LAT+ neurons resulted in more extensive reactivation either in vivo following neurectomy of the sciatic nerve or in vitro after explantation into culture. Neurectomy appeared to induce expression of immediate early 1 mRNA (IE1mRNA) in neurons of mice latently infected with each of three viruses. However IE1mRNA was detected in no more than 0.25% of the neurons of DRG from animals 2 to 4 days after neurectomy, irrespective of the percentage of LAT+ neurons present. Of the 22 neurons shown to express IE1mRNA, none expressed LATs also. However the lack of expression of viral antigen and the absence of a reduced potential for reactivation on explanation suggested that neurectomy had not induced full reactivation involving lytic replication leading to the death of the latently infected neurons. When DRG were explanted into culture, the distribution of the frequency of reactivation was similar to the distribution of DRG that contained LAT+ neurons. The presence of a high proportion of LAT+ neurons was not directly associated with earlier detection of reactivation but such experiments cannot be regarded as quantitative. We therefore concluded that neurectomy did not result in a reduced reactivation potential as described by others and that the frequency of expression of IE1mRNA following neurectomy did not correlate with the number of LAT+ neurons present.

Animals↗
PubMed1992

Herpes simplex virus immediate early gene expression in the absence of transinduction by Vmw65 varies during the cell cycle.

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.

Base Sequence↗
PubMed1992

Hexamethylene bisacetamide stimulates herpes simplex virus immediate early gene expression in the absence of trans-induction by Vmw65.

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.

Acetamides↗
PubMed1991

Identification of two protein binding sites within the varicella-zoster virus major immediate early gene promoter.

Binding sites for cellular proteins in the promoter of the varicella-zoster virus (VZV) major immediate early (IE) gene were investigated. Protein binding was detected at sequence motifs possessing homology to the CCAAT element and an ATF/AP-1-like binding site, and recognition of the ATF/AP-1 site was apparently facilitated by occupation of the CCAAT site. Gene expression directed by the VZV major IE promoter was stimulated by the adenovirus 5, 289 amino acid EIA gene product. The implications of the results for VZV gene expression and replication are discussed.

Base Sequence↗
PubMed1991

Investigation of herpes simplex virus type 1 (HSV-1) gene expression and DNA synthesis during the establishment of latent infection by an HSV-1 mutant, in1814, that does not replicate in mouse trigeminal ganglia.

In previous studies, the herpes simplex virus type 1 (HSV-1) mutant, in1814, which lacks the trans-inducing function of Vmw65, did not replicate in the trigeminal ganglia of mice following corneal inoculation but did establish a reactivatable latent infection in the ganglia 12 to 24 h after ocular infection. Since in1814 did not replicate in vivo, the molecular events during the establishment phase of latent HSV-1 infection could be characterized without the complications of concurrent productive viral infection. In comparison to parental HSV-1 strain 17+, the expression of viral immediate early (IE), early and late genes and the levels of viral DNA in the trigeminal ganglia of mice following in1814 infection were greatly reduced. However, accumulation of latency-associated transcripts, a prominent feature of latent HSV-1 infection, occurred in a wild-type fashion. Furthermore, low levels of viral gene expression and an increase in the level of viral DNA in the in1814-infected ganglia were not detected until 1 to 2 days after the establishment of HSV-1 latency. Thus, IE gene expression and replication of viral DNA in the trigeminal ganglia are not prerequisites for the establishment of HSV-1 latency. These results suggest that the pathways leading to productive and latent infections in neurons may diverge at an early stage of the host-HSV-1 interaction and that the level of viral IE gene expression has a key role in determining the outcome of infection.

Animals↗
PubMed1991

Establishment of latency in vitro by the herpes simplex virus type 1 mutant in1814.

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.

Cell Line↗
PubMed1991

Retention of nonlinear viral DNA during herpes simplex virus latency in vitro.

We investigated the state of the herpes simplex virus type 2 (HSV-2) genome during latency in vitro. Latent DNA was present in the cell nucleus in a nonlinear configuration. The joint fragment was represented at approximately double the molar concentration of fragments from unique regions, indicating that genome termini had fused. The HSV-2 genome copy number was estimated to be between 1.5 and 8, with a mean value of 4.5 per latently infected cell. Nonlinear HSV DNA can therefore exist during latency both in vivo and in vitro.

DNA, Viral↗
PubMed1990

The product of varicella-zoster virus gene 62 autoregulates its own promoter.

Varicella-zoster (VZV) gene 62 encodes a protein with a predicted Mr of 140,000 (140K) which has considerable amino acid identity with the major immediate early (IE) protein Vmw175 (ICP4) of herpes simplex virus type I (HSV-1). Vmw175 is an essential virus polypeptide with a pivotal role in the activation of early and late viral gene expression and also in the repression of IE gene expression. The VZV 140K protein has been shown to function as a strong transcriptional activator in transfection assays and largely complements for the loss of Vmw175 function in HSV-1. We report the results of cotransfection experiments which demonstrate that the 140K protein strongly represses expression from its own promoter, that of gene 62, thus establishing further functional similarity between it and Vmw175. However, whereas Vmw175 can substitute for the 140K protein in repression of the gene 62 promoter, the 140K protein does not repress the HSV-1 IE3 promoter in the reciprocal experiment. The integrity of a domain of Vmw175 (designated region 2), previously shown to be crucial for repression of the HSV-1 IE3 promoter, is also required for repression of the gene 62 promoter. Moreover, a similar requirement for the highly similar region 2 of the 140K protein for repression is demonstrated, suggesting that VZV 140K protein and HSV-1 Vmw175 autoregulate IE gene expression by a related mechanism.

Animals↗
PubMed1990

Control of expression of the varicella-zoster virus major immediate early gene.

The cis-acting DNA sequences and trans-acting proteins that control the expression of the major immediate early (IE) gene of varicella-zoster virus (VZV) were investigated. The location of the IE mRNA 5' terminus was determined by primer extension and S1 nuclease analyses and the functional activities of DNA sequences upstream of this site were analysed by a transfection assay. The VZV IE promoter exhibited low activity in BHK and HeLa cells, but was transactivated by the herpes simplex virus type 1 (HSV-1) virion protein Vmw65. DNA sequences between positions -131 and +57 were responsible for promoter activity, whereas sequences between -410 and -131 mediated the response to Vmw65. Two short elements in the -410 to -131 region formed protein-DNA complexes with HeLa cell nuclear proteins and formed a ternary complex when Vmw65 was added. One of the elements, ATGTAAATGAAAT, possessed a strong similarity to the HSV-1 TAATGARAT. The VZV homologue of Vmw65, encoded by open reading frame (ORF) 10, failed to trans-activate expression from HSV-1 or VZV IE promoters and did not form a ternary complex with functional TAATGARAT elements and HeLa cell proteins. Therefore, stimulation of VZV IE transcription by Vmw65 can occur by a mechanism similar to that employed by HSV-1, but VZV ORF 10 does not function as a trans-activator of IE gene expression.

Animals↗
PubMed1990

A herpes simplex virus type 1 mutant containing a nontransinducing Vmw65 protein establishes latent infection in vivo in the absence of viral replication and reactivates efficiently from explanted trigeminal ganglia.

Vmw65, a herpes simplex virus type 1 (HSV-1) tegument protein, in association with cellular proteins, transactivates viral immediate early genes. In order to examine the role of Vmw65 during acute and latent infection in vivo, a mutant virus (in1814), containing a 12-base-pair insertion in the Vmw65 gene, which lacks the transactivating function of Vmw65 (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Cameron, and C. M. Preston, J. Virol. 63:2260-2269, 1989) was examined in mice. Following corneal inoculation, the parental virus (17+) and the revertant (1814R) replicated effectively in eyes and trigeminal ganglia with 30 to 60% mortality. At either equal PFU or equal particle numbers, in1814 did not replicate in trigeminal ganglia and none of the infected mice died. Although in1814 did not replicate following corneal inoculation, it established latent infection in trigeminal ganglia. HSV-1 in1814 reactivated at explant as efficiently and rapidly as did 17+ and 1814R. Even low amounts of inoculated in1814 (10(2) PFU) were sufficient to establish latent infection in some animals. Since infectious in1814 was not detected at any time in mouse trigeminal ganglia, in1814 provided a unique opportunity to determine how soon after primary infection latency begins. Latent in1814 infection was detected shortly after virus reached the sensory ganglia, between 24 to 48 h postinfection. Thus, though Vmw65 may be required for lytic infection in vivo, it is dispensable for the establishment of and reactivation from latent infection. These data support the hypotheses that the latent and lytic pathways of HSV-1 are distinct and that latency is established soon after infection without a requirement for viral replication. However, the levels of Vmw65 reaching neuronal nuclei may be a critical determinant of whether HSV-1 forms a lytic or latent infection.

Animals↗
PubMed1989

Construction and characterization of a herpes simplex virus type 1 mutant unable to transinduce immediate-early gene expression.

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.

Animals↗
PubMed1989

Herpes simplex virus type 1 immediate-early protein Vmw110 reactivates latent herpes simplex virus type 2 in an in vitro latency system.

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.

Adenoviridae↗
PubMed1988

A complex formed between cell components and an HSV structural polypeptide binds to a viral immediate early gene regulatory DNA sequence.

Transcription of HSV immediate early genes is stimulated by a virus structural protein, Vmw65, in a process that requires specific recognition of the sequence TAATGARAT (R = purine). Upon incubation of nuclear extracts of HSV-infected cells with a short DNA fragment containing TAATGARAT, a novel virus-induced protein-DNA complex (named IEC) was detected. Addition of virion extract, containing Vmw65, to nuclear extract from uninfected cells also resulted in the formation of IEC. Vmw65 is a component of IEC, which contains proteins bound specifically to TAATGARAT. Thus, Vmw65 and cellular factors combine to form a sequence-specific DNA-binding complex. This system provides a model for studies of the regulation of inducible cellular genes.

DNA, Viral↗
PubMed1988

Mutational analysis of the herpes simplex virus type 1 trans-inducing factor Vmw65.

The herpes simplex virus type 1 (HSV-1) polypeptide Vmw65 is a structural component of the virus particle and is also responsible for trans-induction of immediate early (IE) transcription. Functional domains of this polypeptide were investigated by constructing a series of 10 plasmids each with a 12 bp insertion in the gene encoding Vmw65. Plasmids were analysed for their ability to stimulate IE transcription in short term transfection assays, and the altered Vmw65 polypeptides were assayed for the ability to form an IE-specific protein-DNA complex (IEC) in vitro. A direct correlation was observed between stimulation of transcription and formation of IEC, strongly suggesting that IEC is an important intermediate in transcription activation. Plasmids were also tested for their ability to rescue the temperature-sensitive mutation in the HSV-2 assembly mutant ts2203, since marker rescue analysis indicated that this mutation maps within the gene encoding Vmw65. Five plasmids failed to rescue ts2203, thereby defining regions of Vmw65 required for virus assembly. The results show that distinct domains exist in Vmw65 for activation of transcription and assembly of virus.

Animals↗