Electron spectroscopic imaging of encapsidated DNA in vaccinia virus

Harauz, George; Evans, David H.; Beniac, Daniel R.; Arsenault, A. Larry; Rutherford, Brenda; Ottensmeyer, F. P.

doi:10.1139/m95-122

Cited by 4 publications

(5 citation statements)

References 34 publications

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“…First, our ultrastructural localization of the F18 protein within mature virions places it in a ring that surrounds the central, lucent core; in fact, the pattern of F18 localization within mature virions is quite similar to that seen for the A14 protein. Second, electron spectroscopic imaging of encapsidated DNA has also suggested that the genome may be localized in a ring that surrounds the central, lucent core of the virion (21). Taken together, these data suggest that the nucleoprotein complex may associate with proteins located on the inner face the viral membrane.…”

Section: Discussionmentioning

confidence: 91%

Elucidating the Essential Role of the A14 Phosphoprotein in Vaccinia Virus Morphogenesis: Construction and Characterization of a Tetracycline-Inducible Recombinant

Traktman

Liu

DeMasi

et al. 2000

J Virol

140

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We have previously reported the construction and characterization of vindH1, an inducible recombinant in which expression of the vaccinia virus H1 phosphatase is regulated experimentally by IPTG (isopropyl-␤-Dthiogalactopyranoside) (35). In the absence of H1 expression, the transcriptional competence and infectivity of nascent virions are severely compromised. We have sought to identify H1 substrates by characterizing proteins that are hyperphosphorylated in H1-deficient virions. Here, we demonstrate that the A14 protein, a component of the virion membrane, is indeed an H1 phosphatase substrate in vivo and in vitro. A14 is hyperphosphorylated on serine residues in the absence of H1 expression. To enable a genetic analysis of A14's function during the viral life cycle, we have adopted the regulatory components of the tetracycline (TET) operon and created new reagents for the construction of TET-inducible vaccinia virus recombinants. In the context of a virus expressing the TET repressor (tetR), insertion of the TET operator between the transcriptional and translational start sites of a late viral gene enables its expression to be tightly regulated by TET. We constructed a TET-inducible recombinant for the A14 gene, vindA14. In the absence of TET, vindA14 fails to form plaques and the 24-h yield of infectious progeny is reduced by 3 orders of magnitude. The infection arrests early during viral morphogenesis, with the accumulation of large numbers of vesicles and the appearance of "empty" crescents that appear to adhere only loosely to virosomes. This phenotype corresponds closely to that observed for an IPTG-inducible A14 recombinant whose construction and characterization were reported while our work was ongoing (47). The consistency in the phenotypes seen for the IPTG-and TET-inducible recombinants confirms the efficacy of the TET-inducible system and reinforces the value of having a second, independent system available for generating inducible recombinants.Vaccinia virus is a complex DNA virus characterized by a high degree of genetic and physical autonomy from the host cell. Among the functions of the 200 gene products encoded by the 192-kb genome are those required for the expression of three temporal classes of genes, the replication of viral DNA, and the morphogenesis of nascent virions (27). The last is a complex process which remains poorly understood but is the subject of intense study. Electron microscopy, in conjunction with pharmacological intervention and the study of conditionally lethal mutants, has been useful in generating models of virion assembly (7,9,12,20,24,25,28,31,41,44,47,49,53,55,58,59,62,63). Within the cytoplasm, morphogenesis is concentrated around areas of increased electron density which have been shown to contain viral DNA and the proteins to be encapsidated. Membrane crescents, which appear to elongate and curve until they enclose the electron-dense material within oval immature virions (IV), are the first hallmark of morphogenesis. The origin of these membranes is still a matter ...

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Section: Discussionmentioning

confidence: 91%

Elucidating the Essential Role of the A14 Phosphoprotein in Vaccinia Virus Morphogenesis: Construction and Characterization of a Tetracycline-Inducible Recombinant

Traktman

Liu

DeMasi

et al. 2000

J Virol

140

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“…Energy-filtered images were recorded and elemental maps constructed, either by the three energy window method [17,22] (with two images recorded below and one image recorded above the ionization edge energy), or with the jump ratio method [19,23] (just using one image recorded at energies below, and one image recorded above the edge energy of interest).…”

Section: Energy-filtered Transmission Electron Microscopymentioning

confidence: 99%

“…For EFTEM, it is thus most important to make the samples as thin as possible and omit additions of any heavy elements during the preparation. EFTEM has previously been used to study viral morphogenesis in infected cells [13,[17][18][19] but has to our knowledge never been applied in the study of viral structures without extensive preparations for imaging including embedding, staining, and sectioning.…”

Section: Introductionmentioning

confidence: 99%

Chemical mapping of DNA and counter-ion content inside phage by energy-filtered TEM

2011

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Double-stranded DNA in many bacterial viruses (phage) is strongly confined, which results in internal genome pressures of tens of atmospheres. This pressure is strongly dependent on local ion concentration and distribution within the viral capsid. Here, we have used electron energy loss spectroscopy (EELS), energy-filtered TEM (EFTEM) and X-ray energy dispersive spectroscopy to provide such chemical information from the capsid and the phage tail through which DNA is injected into the cell. To achieve this, we have developed a method to prepare thin monolayers of self-supporting virus/buffer films, suitable for EELS and EFTEM analysis. The method is based on entrapment of virus particles at air-liquid interfaces; thus, the commonly used method of staining by heavy metal salts can be avoided, eliminating the risk for chemical artifacts. We found that Mg(2 + ) concentration was approximately 2-4 times higher in the DNA-filled capsid than in the surrounding TM buffer (containing 10 mM Mg(2 + )). Furthermore, we also analyzed the DNA content inside the phage tail by mapping phosphorus and magnesium.

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“…The localization of chemically defined components in ultrathin sections of biological material has been a central subject of interest in structural biology for many years. In particular, the role of phosphorus as a position marker of nucleic acids is a topic that has deserved considerable effort (Ottensmeyer and Andrew, 1980;Bazett-Jones and Ottensmeyer, 1981;Adamson-Sharpe and Ottensmeyer, 1981;Korn et al, 1983;Ottensmeyer, 1984: Ottensmeyer et al, 1988;Rattner and Bazett-Jones, 1989;Heng et al, 1990;Ozel et al, 1990;Bazett-Jones, 1993: Harauz et al, 1995Abolhassani-Dadras et al, 1994, 1996Olins et al, 1996;Vazquez-Nin et al, 1996;Beniac et al, 1997a, b).…”

Section: Introductionmentioning

confidence: 99%

“…Electron spectroscopic imaging of sectioned viruses has provided high-contrast imaging and an improvement of the resolution of the viral particles, also allowing a better detectability of the immunolabeling markers (Ozel et al, 1990). Also, P-maps were obtained using a two-window method to study the organization of encapsidated DNA in vaccinia virus (Harauz et al, 1995). Interpretation problems arose in this case due to the fact that the samples were conventionally fixed and embedded.…”

Section: Introductionmentioning

confidence: 99%

Optimization of phosphorus localization by EFTEM of nucleic acid containing structures

Quintana

Marco

Bonnet

et al. 1998

Micron

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Energy Filtered Transmission Electron Microscopy (EFTEM) has been used to study nucleic acids localization in unstained thin sections of virus-infected cells. For this purpose, phosphorus maps (P-maps) have been obtained by applying the N-windows Egerton model for background subtraction from data acquired by a non-dedicated TEM Jeol 1200EXII equipped with a post-column PEELS Gatan 666-9000 and a Gatan Image Filter (GIF-100). To prevent possible errors in the evaluation of elemental maps and thus incorrect nucleic acid localization, we have studied different regions of swine testis (ST) cells with similar local density containing either high concentration of nucleic acids (condensed chromatin and ribosomes) or a very low concentration (mitochondria). Special care was taken to optimize the sample preparation conditions to avoid as much as possible the traditional artifacts derived from this source. Selection of the best set of pre-edge images for background fitting was also considered in order to produce "true P-maps". A new software for interactive processing of images series has been applied to estimate this set. Multivariate Statistical Analysis was used as a filtering tool to separate the "useful information" present in the inelastic image series (characteristic signal) from the "non-useful information" (noise and acquisition artifacts). The reconstitution of the original image series preserving mainly the useful information allowed the computation of P-maps with improved signal-to-noise ratio (SNR). This methodology has been applied to study the RNA content of maturation intermediate coronavirus particles found inside infected cells.

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Electron spectroscopic imaging of encapsidated DNA in vaccinia virus

Cited by 4 publications

References 34 publications

Elucidating the Essential Role of the A14 Phosphoprotein in Vaccinia Virus Morphogenesis: Construction and Characterization of a Tetracycline-Inducible Recombinant

Elucidating the Essential Role of the A14 Phosphoprotein in Vaccinia Virus Morphogenesis: Construction and Characterization of a Tetracycline-Inducible Recombinant

Chemical mapping of DNA and counter-ion content inside phage by energy-filtered TEM

Optimization of phosphorus localization by EFTEM of nucleic acid containing structures

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