Biomedical subjects
Publications and source records attributed to A J Bellett.
Mycoplasma contamination of two alphavirus isolates and adenovirus 5 was completely removed using a very simple, rapid protocol based on solvent extraction. Treated virus preparations remained free of mycoplasma after 6 months in culture.
The mechanism for down-regulation of E1a expression by products encoded in the E3 transcription unit of human adenovirus types 2 and 5, that occurs in infected L929 cells, has been investigated further. We show that the phenomenon occurs in different mouse cells and also in some human cells suggesting that the observations have relevance to natural human infections. We also provide evidence that probably all viral proteins are down-regulated by E3 products, although to different extents, but that host proteins are unaffected. Whereas E1a protein levels and synthesis are reduced in the presence of E3 products, E1a protein half-life and polysomal E1a RNA levels and size distribution are not. These data suggest that E3 products down-regulate E1a protein levels by interfering with the translation of E1a-specific mRNA. Studies were additionally carried out with mutant adenoviruses containing different defects in the E3 transcription unit. Based on these studies it seems likely that the E3 14.5K and 10.4K proteins are crucially involved in E1a down-regulation. Our data are discussed in terms of strategies for immune evasion by group C human adenoviruses.
Biomedical research was once an unquestioned good, and generous funding for a short time allowed researchers to work on whatever interested them. Two contradictory pressures have changed this. As costs have risen and economic rationalism has become politically dominant, governments, private corporations and granting agencies have increasingly demanded compliance with their own priorities, instrumental values and performance criteria. On the other hand, social and ethical critics have characterized biomedical research as being out of touch with real health needs and community values and as being an agent of social control that entrenches the power of a technocratic hegemony. The profession has largely acquiesced in bureaucratic and corporate intervention in exchange for continued funding, and assumed that social concerns could be allayed by 'top down' paternalistic education of the public. However, this response tends to add weight to the criticism that biomedicine is an agent of social control. What is needed is a spirited defence of the value of independent scholarship and research that is not limited to science but includes the humanities. Equally important is a process of community education in which scientists not only transmit their knowledge and enthusiasm to the public, but themselves become open to the social and ethical concerns of the community.
Effects of mutations in the adenovirus 5 (Ad 5) E3 transcription unit on the immune Tc cell response to Ad 5 were investigated. We observed enhanced lysis of L929 target cells infected with the E3 defective mutant viruses dl 327 and dl 355 compared to wild-type (wt) Ad 5 by Ad 5 immune Tc cells. This enhanced lysability was not due to E3 effects on the cell surface expression of class I MHC H-2Kk molecules as determined by monoclonal antibody binding or alloreactive Tc cell recognition. Furthermore MHC class I molecules were able to efficiently present vaccinia virus antigens in the presence of the Ad 5 E3 genes, excluding functional modification of class I MHC antigens by E3 gene products. When levels of the Ad 5 immunodominant antigen E1a were compared between wt and E3 mutant viruses, we observed an 8- to 10-fold increase in E1a levels in E3 mutant-infected cells over wt Ad 5-infected cells. No differences were observed between these viruses at the mRNA level. We conclude that E3 products interfere with Ad 5 immune Tc cell responses by some post-transcriptional mechanism which reduces expression of the E1a immunodominant antigen.
Products encoded in the E1a oncogene of adenoviruses are required to activate transcription of all viral early genes and some cellular genes. A current interpretation of experimental data supports the hypothesis that this "trans-activation" is mediated solely by a block of amino acids known as conserved domain 3, which is unique to the largest E1a protein, while the remaining E1a protein sequences contain discrete domains required for functions other than trans-activation. However, there is also considerable evidence inconsistent with this simple model of E1a structure and function. Both of the major E1a proteins appear to participate in trans-activation by three different types of interaction with cellular transcription factors and other regulatory proteins. In this review we attempt to rationalize the experimental data and provide a more integrated view of E1a structure and function.
We show using mild extraction procedures that the p53 proto-oncogene forms a complex with adenovirus 5 E1b-58 kD during infection. These complexes are detected as coimmunoprecipitates from radiolabeled extracts of adenovirus infected cells on SDS-PAGE. Furthermore, adenovirus mutants with defects in E1b-58 kD fail to form complexes, whereas mutants in other early region genes still show evidence of complex. Using a panel of monoclonal antibodies to mouse p53, we show that antibodies reacting with N-terminal epitopes on p53, displace E1b-58 kD. This result suggests that E1b-58 kD binds to an N-terminal region of mouse p53. In addition, in a transient transfection assay in monkey COS cells, we show that an N-terminal deletion mutant of mouse p53 does not bind to E1b-58 kD but wild-type mouse p53 does bind. This result again suggests that E1b-58 kD binds an N-terminal determinant on p53.
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Some undifferentiated F9 embryonal carcinoma cells allow adenovirus genes to be expressed independently of the E1a oncogene normally required for their activation; this has been attributed to a cellular equivalent of E1a in F9 cells. However, transcription of all early genes was low in undifferentiated OTF963 embryonic carcinoma cells during the first 48 hr after infection with adenovirus type 5 (Ad5). Transcription then increased to about the level seen 16 hr after infection of cells induced to differentiate by retinoic acid (RA) (referred to as RA-dF9 cells), but this increase did not occur in cells infected by the E1a deletion mutant dl312. Addition of E1a in trans, or of RA, had no immediate effect on viral transcription in OTF963 cells, but viral transcription increased about 48 hr after these additions. Ad5 induced transcription of several differentiation-specific genes in OTF963 cells with about the same kinetics as their induction by RA. These genes were superinduced in RA-dF9 cells by cAMP or infection by adenovirus. We suggest the small amount of E1a produced early in infection of OTF963 cells activates cellular genes, some of which are differentiation specific and required for efficient transcription of viral genes, so that E1a both induces and is induced by differentiation. The simple hypothesis of a cellular equivalent to E1a does not adequately explain the complex interactions between viral and cellular genes in OTF963 embryonic carcinoma cells.
Adenovirus infection was compared in F9 (OTF963) cells and cells induced to differentiate with retinoic acid, in order to study expression of early genes under the control of the reported "E1a-like factor" in F9 cells. However, not only was transcription of the viral E1a gene defective in undifferentiated cells but expression of all the other early genes was found to be reduced in OTF963 cells in comparison to differentiated cells. The defect in early gene expression was detected at the level of transcriptional initiation during the first 48 h of infection and resulted in similarly low levels of viral cytoplasmic mRNA and viral protein synthesis. Viral DNA replication was delayed and reduced. After 48 h of infection, the defect in transcription in OTF963 cells of E1a and other early genes was relieved, so that by 72 h postinfection the level of transcription was similar to that 16 h after infection of differentiated cells. At no time did adenovirus early gene expression occur independently of viral E1a. These results suggest limits to the generality and explanatory power of the hypothesis that F9 embryonal carcinoma cells contain an E1a-like factor.
We have characterized the cellular immune response to human adenovirus 5 (Ad-5) in mice, as a basis for future study of responses to foreign antigens in recombinant adenoviruses. Primary in vivo lytic effector cells contained both virus immune cytolytic T (Tc) cells and natural killer cells. The Tc effectors could be boosted in vitro to give a secondary Tc cell response. The Tc cell response to Ad-5 was major histocompatibility complex (MHC) restricted, and in CBA/H (H-2k) mice mapped to the K end of MHC. Kinetic experiments suggested the Tc cell response was directed against early rather than late viral proteins. Experiments with viral mutants showed that the main responses were to the E1A and E1B proteins, with some involvement of E2. Expression of E3 and E4 in infected targets was not required, in fact lysis of target cells infected by viruses with a deletion in E3 was augmented.
Rat embryo cells were infected with adenovirus type 5 mutants that code for only one of the two early E1A proteins, mutants with defects in one of the two conserved regions common to the two proteins, or mutants with defects in the 46-amino-acid region unique to the 289-amino-acid E1A protein. Cells were scored for altered cell cycle progression, disruption of actin stress fibers, and activation of E2A expression. Mutants lacking either E1A protein were able to cause all of these effects; but mutants lacking a 243-amino-acid protein had less effect, and mutants lacking a 289-amino-acid protein much less effect, than wild-type virus. A mutation in any of the three conserved regions caused a defect in each E1A effect. To investigate the reported function of conserved domain 2 in mitosis, we monitored by fluorescence-activated cell sorter the reduction in Hoechst 33342 fluorescence that occurs when cells divide after undergoing a round of DNA replication in 5-bromodeoxyuridine. A smaller percentage of adenovirus-infected cells than mock-infected cells divided within a given period after completing a round of DNA replication. Viruses with mutations in conserved domain 2 were defective for initiation of cellular DNA replication, as were all other E1A mutants we have examined, but had no specific defect in cell division compared with wild-type virus. Thus, although there may be some specialization of function between the two E1A proteins and between their conserved domains, it was not apparent in the aspects of E1A function and the mutants that we examined.
Flow cytometry and staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin were used to investigate organization of the actin cytoskeleton in rat embryo cells at different stages of normal and adenovirus E1A-induced cell cycles. In uninfected cells in G0-G1 and S phases, actin was predominantly in the form of stress fibers. In G2, this organization changed to peripheral rings of thin filaments, while during mitosis, actin had a diffuse distribution. Infection of quiescent rat cells by adenovirus caused them to enter the cell cycle and replicate DNA and also caused disruption of stress fibers. Rapid disappearance of stress fibers and the appearance of peripheral rings of actin filaments began from 13 h after infection and closely followed synthesis of the E1A proteins. Infected cells began S phase at about 24 h after infection, and cells in G2 and mitosis were seen from 30 to 50 h. Thus, disruption of the actin cytoskeleton is an early effect of E1A and not an indirect consequence of the entry of infected cells into the cell cycle.
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The actin microfilament organization in rat embryo cells was examined by fluorescence microscopy with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and by electron microscopy, after mock infection or infection with adenovirus type 5 (Ad5). Infected cells showed severely reduced numbers of actin microfilaments and stress fibers, detectable early after infection. Mutants defective in Ad5 early genes were used to show that reduced microfilament organization was a function of the Ad5 transformation early gene 1a (E1a) and did not require expression of any other viral gene. The product of the E1a 13s mRNA was essential for the effect, although the 12s mRNA product appeared to contribute. Ad5 infection of the cells had no observable effect on total cell actin levels or on the ratio of monomeric to polymeric actin. E1a, therefore, affected only the higher-order organization of actin.
Altered control of the rat cell cycle induced by adenovirus requires expression of transformation region E1A, but not of E1B, E2A, E2B, or late genes. We show here that neither E3 nor E4 is required, so the effect results directly from an E1A product. Mutants with defects in the 289-amino-acid (aa) E1A product had little or no effect on the rat cell cycle even at 1,000 IU per cell. A mutant (pm975) lacking the 243-aa E1A product altered cell cycle progression, but less efficiently than did wild-type virus. The 289-aa E1A protein is therefore essential for cell cycle effects; the 243-aa protein is also necessary for the full effect but cannot act alone. Mutants with altered 289-aa E1A proteins showed different extents of leak expression of viral early region E2A as the multiplicity was increased; each leaked more in human than in rat cells. dl312, with no E1A products, failed to produce E2A mRNA or protein at 1,000 IU per cell in rat cells but did so in some experiments in human cells. There appears to be a very strict dependence of viral early gene expression on E1A in rat cells, whereas dependence on E1A is more relaxed in HeLa cells, perhaps due to a cellular E1A-like function. Altered cell cycle control is more dependent on E1A function than is early viral gene expression.
Chick embryo lethal orphan (CELO) virus (fowl adenovirus type 1) contains at least 14 structural proteins with polypeptide molecular weights ranging from 100K to about 6K. A nomenclature of the CELO virion polypeptides is presented and the molar proportion of each polypeptide has been estimated. The CELO virus pentons were specifically released from the virion by dialysis against borate-based calcium-magnesium saline. The penton base (polypeptide III, mol. wt. 92K) and two fibres were separated, characterized and their polypeptides were correlated with their morphological positions in the virion. Peptide mapping suggested that the long fibre (polypeptide IV, mol. wt. 65K), and the short fibre (polypeptide VII, mol. wt. 44.5K) were not related in their primary sequences and are therefore probably encoded by separate genes. The time course of synthesis of the CELO virion polypeptides indicated that, like their mammalian adenovirus counterparts, they are synthesized late (after viral DNA replication).