A Study of the In Vitro Replication of Adenovirus Type 4 DNA

The work described in this thesis concerns the development and characterization of an in vitro system for the initiation of adenovirus type 4 DNA replication and the first steps towards the purification and characterization of the individual components of the system. The study was suggested by the differences observed in the minimal DNA sequence requirements for adenovirus type 2 (Ad.2) and adenovirus type 4 (Ad.4) DNA replication in vivo (Hay,1985a,b). The minimal Ad.2 origin of replication consisted of the terminal 45b.p. of the genome containing two important sequence elements: The 10b.p. AT-rich sequence conserved in all the human adenoviruses (Stillman et al, 1982) between nucleotides 9-18 and the consensus binding site for the cellular protein nuclear factor I (NFI) between nucleotides 19-45 (Nagata et al,1983). In contrast the Ad.4 origin of replication comprised only the terminal 18b.p. of the genome, which were identical to the terminal 18b.p. of Ad.2 (Hay,1985b). An in vitro study was seen as the best way to further define the mechanistic differences between Ad.2 and Ad.4 DNA replication. Initially full-length Ad.4 DNA was isolated from virions by a number of treatments leaving it associated with either the terminal protein and core polypeptides (Ad.4 cores), the terminal protein alone (Ad.4 DNA-prot.), or completely protein-free (Ad.4 DNA) and it was shown that these templates could support initiation of DNA replication in vitro. Initiation was defined as the formation of a covalent complex between 5'-dCMP and the 80,000-dalton precursor terminal protein (pTP-dCMP complex). In Ad.2 the formation of this complex is catalysed by the viral DNA polymerase which is tightly associated with the pTP. Use of (a32P)dCTP enabled the detection of this product by SDS-polyacrylamide gel electrophoresis and autoradiography. Subsequent experiments utilized a plasmid containing the terminal 140b.p. of Ad.4 as a template for reasons of stability and reproducibility of results. Formation of a pTP-dCMP complex was shown to be catalysed by either nuclear or cytoplasmic extract of Ad.4 infected cells alone. An inhibitory activity in nuclear extracts meant that cytoplasmic extracts were used exclusively to characterize the reaction. Various parameters of the reaction were studied including the formation of pTP-dCMP complex at different protein concentrations, the time course of pTP-dCMP complex formation and the nucleotide specificity of the reaction. The activity of cytoplasmic extract in forming pTP-dCMP complex was studied at varying concentrations of ATP and divalent cations and in varying conditions of ionic strength. By including in the reaction dATP, dTTP and ddGTP it was shown that the pTP-dCMP complex could be elongated, presumably by the Ad.4 specific DNA polymerase, to the first dG residue in the sequence - the 30th nucleotide. This resulted in the formation of an additional product with an apparent molecular weight of 90,000-daltons. The template requirements for the formation of a pTP-dCMP complex were studied: Both the DNA sequence and template conformation requirements were investigated using both single- and double-stranded DNA. It was shown that the double-stranded DNA sequence requirements were the same as in vivo. Finally the Ad.4 specific DNA polymerase was partially purified from Ad.4 infected HeLa cells and the response of this DNA polymerase to various inhibitors studied. A factor from uninfected HeLa cell nuclei that stimulated pTP-dCMP complex formation by cytoplasmic extract of Ad.4 infected cells was partially purified. This factor was shown to stimulate pTP-dCMP complex formation on a template containing only 18b.p. from the Ad.4 terminus, indicating that it might specifically recognize sequences within that region

Synergy between HIV-1 Tat and adenovirus E1A is principally due to stabilization of transcriptional elongation

We studied the combined effects of Tat and general trans-activators, such as E1A and phorbol esters, on human immunodeficiency virus-1 (HIV-1) gene expression. Interaction between these two types of trans-activators may be involved in the transition from transcriptional quiesence during viral latency to active gene expression during productive infection. E1A cooperated with Tat to produce a fourfold greater increase in accumulation of full-length, cytoplasmic HIV-1-directed RNA than is expected if they were acting additively to increase RNA accumulation. Similarly, phorbol 12-myristate 13-acetate (PMA) also cooperated with Tat to elevate HIV RNA levels synergistically. Analysis of transcription rates across the HIV-1-directed transcription unit indicated, unexpectedly, that synergy between Tat and E1A could not be accounted for by increased promoter proximal transcription rates that were merely additive. However, Tat and E1A produced a greater than additive increase in transcription rates in the 3' end of the gene. These findings imply that synergy between Tat and E1A (or other general transcriptional activators) is due principally to stabilization of transcriptional elongation. Furthermore, the observation that Tat elicits only a small increase in promoter proximal transcription in the presence of E1A suggests that the magnitude of the effect of Tat on initiation is decreased when the basal level of transcription is increased. These findings underscore the importance of the ability of Tat to stabilize elongation, as well as to stimulate initiation, in an HIV-1-directed transcription unit

YB-1 dependent oncolytic adenovirus efficiently inhibits tumor growth of glioma cancer stem like cells

Background: The brain cancer stem cell (CSC) model describes a small subset of glioma cells as being responsible for tumor initiation, conferring therapy resistance and tumor recurrence. In brain CSC, the PI3-K/AKT and the RAS/mitogen activated protein kinase (MAPK) pathways are found to be activated. In consequence, the human transcription factor YB-1, knowing to be responsible for the emergence of drug resistance and driving adenoviral replication, is phosphorylated and activated. With this knowledge, YB-1 was established in the past as a biomarker for disease progression and prognosis. This study determines the expression of YB-1 in glioblastoma (GBM) specimen in vivo and in brain CSC lines. In addition, the capacity of Ad-Delo3-RGD, an YB-1 dependent oncolytic adenovirus, to eradicate CSC was evaluated both in vitro and in vivo. Methods: YB-1 expression was investigated by immunoblot and immuno-histochemistry. In vitro, viral replication as well as the capacity of Ad-Delo3-RGD to replicate in and, in consequence, to kill CSC was determined by real-time PCR and clonogenic dilution assays. In vivo, Ad-Delo3-RGD-mediated tumor growth inhibition was evaluated in an orthotopic mouse GBM model. Safety and specificity of Ad-Delo3-RGD were investigated in immortalized human astrocytes and by siRNA-mediated downregulation of YB-1. Results: YB-1 is highly expressed in brain CSC lines and in GBM specimen. Efficient viral replication in and virus-mediated lysis of CSC was observed in vitro. Experiments addressing safety aspects of Ad-Delo3-RGD showed that (i) virus production in human astrocytes was significantly reduced compared to wild type adenovirus (Ad-WT) and (ii) knockdown of YB-1 significantly reduced virus replication. Mice harboring othotopic GBM developed from a temozolomide (TMZ)-resistant GBM derived CSC line which was intratumorally injected with Ad-Delo3-RGD survived significantly longer than mice receiving PBS-injections or TMZ treatment. Conclusion: The results of this study supported YB-1 based virotherapy as an attractive therapeutic strategy for GBM treatment which will be exploited further in multimodal treatment concepts

Adenovirus serotype 5 L4-22K and L4-33K proteins have distinct functions in regulating late gene expression

Adenoviruses express up to 20 distinct mRNAs from five major late transcription unit (MLTU) regions, L1 to L5, by differential splicing and polyadenylation of the primary transcript. MLTU expression is regulated at transcriptional and posttranscriptional levels. The L4-33K protein acts as a splicing factor to upregulate several MLTU splice acceptor sites as the late phase progresses. The L4 region also expresses a 22K protein whose sequence is related to the sequence of L4-33K. L4-22K is shown here also to have an important role in regulating the pattern of MLTU gene expression. An adenovirus genome containing a stop codon in the L4-22K open reading frame expressed low levels of both structural and nonstructural late proteins compared to the wild-type (wt) adenovirus genome; a decrease in intermediate proteins, IVa2 and IX, was also observed. However, early protein synthesis and replication were unaffected by the absence of L4-22K. Intermediate and late protein expression was restored to wt levels by L4-22K expressed in trans but not by L4-33K. Increased MLTU promoter activity, resulting from stabilization of the transcriptional activator IVa2 by L4-22K, made a small contribution to this restoration of late gene expression. However, the principal effect of L4-22K was on the processing of MLTU RNA into specific cytoplasmic mRNA. L4-22K selectively increased expression of penton mRNA and protein, whereas splicing to create penton mRNA is known not to be increased by L4-33K. These results indicate that L4-22K plays a key role in the early-late switch in MLTU expression, additional to and distinct from the role of L4-33K

The Control of Herpes Simplex Virus Late Gene Transcription

The coordinate temporal control of the HSV-1 transcriptional programme involves three main phases of gene expression; immediate-early (IE), early (E), and late (L). The features which distinguish IE genes are well defined. IE genes are transcribed in the absence of de novo protein synthesis. Their transcription is stimulated by a component of the virus particle, Vmw65, and this requires the presence of a "far-upstream region" which includes one or more copies of the consensus sequence, 5'-TAATGARATTC-3'. IE gene products are required for the activation of the later classes of viral genes, whose expression in the absence of DNA replication is either maximal (E genes), sub-maximal (EL genes) or very poor (L genes). The organization of the promoter sequences of IE and E genes resembles that of many other eukaryotic (non-"housekeeping") genes, characterized by (i) a proximal element bearing a TATA box homology, and (ii) distal elements which are recognized by cellular transcription factors such as Spl and CTF. E genes do not possess far-upstream regulatory elements. Until recently, very little was known about late gene expression: What distinguishes a late promoter from an early promoter; why is efficient late gene expression dependent on DNA replication? The work in this thesis has been directed at the control of expression of an HSV-1 late gene, US11. It was shown that the US11 gene was regulated in the virus with "true-late" kinetics, in terms of the time-course of appearance of US11 RNA and the sensitivity of US11 gene expression to inhibition of DNA replication. Under conditions of DNA replication inhibition, the accumulation of US11 RNA was reduced by 50 to 100-fold. In contrast, the accumulation of RNA from an EL gene, glycoprotein D (gD), was reduced by only 5 to 10-fold. Thus there is a clear difference between the regulation of transcription of gD, an EL gene, and US11, a "true-late" gene. A plasmid system was developed in order to study the regulation of the US11 promoter. Plasmid DNA was introduced into tissue culture cells by a short-term transfection procedure, followed by infection with virus to provide the necessary factors for activation of US11. The US11 promoter was therefore linked to the coding portion of the rabbit beta-globin gene, in order to distinguish between transcripts derived from the plasmid and those from the virus. The activity of the plasmid-borne US11 promoter in constructs containing or lacking a functional HSV-1 origin of DNA replication (ORIs) was analysed by quantitative SI mapping of correctly initiated hybrid transcripts. Following HSV-1 infection of transfected HeLa cells, the US11 promoter in ORI+ plasmids was expressed with similar kinetics to its viral counterpart. US11 promoter activity was first detected at the same time as the onset of DNA template replication. Expression of US11 RNA was detectable from non-replicating ORI- plasmids, although transcript accumulation was reduced by greater than 90%. Sequences containing the IE gene 5 promoter (a 3' co-terminal gene whose transcription starts 5' of US11) also played a positive role in achieving normal US11 gene expression, which suggested that both replication and through-transcription may act by inducing structural changes to the late promoter DNA. The DNA sequence requirements for US11 gene expression were investigated using plasmids which contain ORIs. The sequences necessary for fully efficient regulated expression of US11 lie within the region -31 to +39 relative to the RNA start sites. It appeared that a late promoter might consist only of a proximal TATA box-cap site region. This hypothesis was tested by removing the distal upstream region of the gD promoter (which is required for its normal regulation as an EL promoter) and linking this truncated promoter to ORIs. The result was the conversion of gD promoter regulation to late gene kinetics during virus super infection. It therefore seems unlikely that late promoters are distinguished by late promoter-specific sequences. Co-transfection of the US11 promoter with plasmids bearing HSV-1 IE genes showed that US11 could be activated by Vmw175 and Vmw110 and most efficiently, by the two in combination. This suggests that the target sequence for trans-activation by HSV 1E gene products may be in the TATA box region. These results are discussed in terms of the possible mechanisms of regulation of late genes, and of transactivation of transcription in general, and also in comparison with late gene regulation in other viral systems

Effects of DNA tumour virus infection on host nuclear proteins

Immunofluorescence techniques have been used to investigate the distributions of a number of host cell proteins upon infection with three different DNA tumour viruses: herpes simplex virus type 1 (HSV-1), adenovirus (types 2, 4 and 5) and simian virus 40 (SV40). A number of host DNA replication proteins were shown to specifically redistribute to viral replication "compartments" labelled by antibody to the HSV-1 major DNA-binding protein (ICP8) during productive HSV-1 infection. These host proteins also colocalised to varying extents with ICP8 in the presence of a specific inhibitor of viral DNA replication which caused a marked alteration in the location of intranuclear ICPB. Two anti- oncogenic proteins, retinoblastoma and p53 were shown to relocate in the same manner as the cellular DNA replication proteins during both productive and abortive HSV-1 infections, raising the possibility that these proteins may be associated with DNA replication complexes in uninfected cells. Transfection experiments showed that the seven HSV-1 proteins shown to be essential for DNA replication can induce replication compartment formation in uninfected cells. Host DNA replication proteins were also redistributed during productive (but not abortive) infection with adenovirus, although, of those tested, only proliferating cell nuclear antigen (PCNA) was detected in viral replication compartments. A specific population of PCNA was also detected at novel discrete nuclear foci. SV40 infection resulted in the redistribution of singlestranded DNA-binding protein (SSB) to foci which enlarged as infection progressed and also contained SV40 large T antigen. SV40 infection of adenovirus-transformed 293 cells caused p53 to move from the cytoplasm to the nucleus. SV40 co-infections with adenovirus and HSV-1 were also studied. The production and characterisation of monoclonal antibodies to the HSV-1 major DNA binding protein (ICP8) and polymerase accessory factor (UL42) are also described

HIV induces expression of complement component C3 in astrocytes by NF-κB-dependent activation of interleukin-6 synthesis

Background Abnormal activation of the complement system contributes to some central nervous system diseases but the role of complement in HIV-associated neurocognitive disorder (HAND) is unclear. Methods We used real-time PCR and immunohistochemistry to detect complement expression in postmortem brain tissue from HAND patients and controls. To further investigate the basis for viral induction of gene expression in the brain, we studied the effect of HIV on C3 expression by astrocytes, innate immune effector cells, and targets of HIV. Human fetal astrocytes (HFA) were infected with HIV in culture and cellular pathways and factors involved in signaling to C3 expression were elucidated using pharmacological pathway inhibitors, antisense RNA, promoter mutational analysis, and fluorescence microscopy. Results We found significantly increased expression of complement components including C3 in brain tissues from patients with HAND and C3 was identified by immunocytochemistry in astrocytes and neurons. Exposure of HFA to HIV in culture-induced C3 promoter activity, mRNA expression, and protein production. Use of pharmacological inhibitors indicated that induction of C3 expression by HIV requires NF-κB and protein kinase signaling. The relevance of NF-κB regulation to C3 induction was confirmed through detection of NF-κB translocation into nuclei and inhibition through overexpression of the physiological NF-κB inhibitor, I-κBα. C3 promoter mutation analysis revealed that the NF-κB and SP binding sites are dispensable for the induction by HIV, while the proximal IL-1β/IL-6 responsive element is essential. HIV-treated HFA secreted IL-6, exogenous IL-6 activated the C3 promoter, and anti-IL-6 antibodies blocked HIV activation of the C3 promoter. The activation of IL-6 transcription by HIV was dependent upon an NF-κB element within the IL-6 promoter. Conclusions These results suggest that HIV activates C3 expression in primary astrocytes indirectly, through NF-κB-dependent induction of IL-6, which in turn activates the C3 promoter. HIV induction of C3 and IL-6 in astrocytes may contribute to HIV-mediated inflammation in the brain and cognitive dysfunction

Late phase inhibition of murine cytomegalovirus replication by synergistic action of interferon-gamma and tumour necrosis factor

We have shown previously that the antiviral function of CD4+ T lymphocytes against murine cytomegalovirus (MCMV) is associated with the release of interferon- (IFN-). We now demonstrate that IFN- and tumour necrosis factor alpha (TNF-) display synergism in their antiviral activity. As little as 2 ng/ml of IFN- and TNF- reduced the virus yield by about three orders of magnitude. There was no effect on immediate early (IE) and early (E) gene expression as far as the candidate genes IE1, E1 and those encoding the major DNA-binding protein and the DNA polymerase were concerned. Late gene transcription, assayed by the candidate genes encoding glycoprotein B and the MCMV homologue of ICP 18.5, was blocked and MCMV DNA replication was found to be reduced but not halted. The most prominent finding of the cytokine effect, seen by electron microscopy, was an alteration of nucleocapsid formation. Altogether, the synergism is multifaceted and acts at more than one stage during viral morphogenesis. Because the cytokines clearly do not act at an early stage of infection we conclude that the mode of cytokine activity differs between alpha- and betaherpesviruses