Muhsin Özel

Fine structure of human immunodeficiency virus (HIV) and immunolocalization of structural proteins.

Ultrathin section and surface replica electron microscopy were applied in combination with immunoelectron microscopy to elucidate the fine structure of HIV. The shell of the tubular core shows p24 antigenicity, while p17 is located at the inner leaflet of the lipid membrane. The virus particle is studded with 70–80 protrusions. These knobs have a diameter of 15 nm, a height of 9 nm, and are probably arranged in a T = 7 I symmetry. The major envelope protein gpl20 is spontaneously shed from the viral surface. A possible role of released gpl20 in pathogenesis is discussed.

Morphogenesis and morphology of HIV structure-function relations

SummaryFine structure and antigenic make-up analysis of HIV were combined in a 2D model, from which functional aspects can be deduced. On the envelope 72 probably trimeric surface knobs (gp 120) are connected to the virion via the transmembrane protein gp41. Gp120 is shed during ageing of the virion, but host cell antigens stay firmly anchored to the envelope. Underneath the envelope, p17 forms the matrix protein layer, while the capsid of the double cone shaped core is built up of p24. The relation between biochemical findings and morphogensis and maturation of HIV as well as aspects of pathogenesis and vaccination are discussed.

Quaternary structure of the immunostimulating complex (iscom)

Proteins of either HIV-1, hepatitis B, or rabies virus were incorporated with the adjuvant substance Quil A and cholesterol into the immunostimulating complex: iscom. Formation and symmetry of this regular complex were analyzed by electron microscopy. Micellar structures with a diameter of about 12 nm, occasionally with a 7-nm stain-filled center, were formed in a 0.03% water suspension of Quil A. Cavities or holes appeared in the smooth structures of cholesterol upon the addition of Quil A, and after mixing Quil A and cholesterol 1:1 fragile and flattened structures of matrix were produced with a diameter of about 40 nm. By freeze-drying the matrix was preserved as a cage-like, isometric particle. Stable iscom particles composed of Quil A, cholesterol, and selected viral proteins had an approximate diameter of 32 nm. The particles had an uniform, cage-like structure, exhibiting icosahedral symmetry, irrespective of the viral proteins incorporated. Tilting experiments and rotational image analysis indicated that the iscoms were composed of 20 morphological subunits assembled in a pentagonal dodecahedron with a hole on each of the 12 pentagonal faces. The symmetrical shape of the iscom might explain both its remarkable stability and its capacity to efficiently present antigens to the immune system.

The organization of the envelope projections on the surface of HIV

SummaryThe organization of envelope projections (knobs) of four different isolates of the human immunodeficiency virus types 1 and 2 (HIV-1 and -2) was studied using surface replica and thin section electron microscopy (EM) in combination with rotational image enhancement. All HIV strains show an identical organization of knobs on the virus envelope. The surface of an “ideal”, well-preserved HIV particle is studded with 72 knobs arranged in a T=7 laevo symmetry. The role of the p17 protein, which is coating the inner leaflet of the viral envelope, is discussed as a matrix protein functioning as a scaffold for the envelope and its projections during morphogenesis as well as with mature virions.

Highly Infectious Purified Preparations of Disease-Specific Amyloid of Transmissible Spongiform Encephalopathies Are Not Devoid of Nucleic Acids of Viral Size

An efficient purification protocol for infectivity causing a transmissible spongiform encephalopathy (TSE) is described. From fractions purified by this protocol about 3 x 10(8) LD50 but only 3 ng of nucleic acids per gram of brain material can be isolated from all TSE-affected brains (hamster, human, sheep, cattle). By PAGE such fractions from brains of infected and control hamsters contained only one distinct nucleic acid band of 1.5 kg together with some broader smear of nucleic acid material. Although distilled water was used for such purifications, quite often a similar nucleic acid band was isolated from blanks containing no brain material. In all instances this material proved to be DNA. The result challenges the potentially important claim that purified infectious preparations of TSE-specific amyloid are free of nucleic acids of viral size. Nucleic acids isolated by other groups from diseased brain were not detected in preparations isolated by the new protocol. The application of this purification protocol in future studies will be helpful to decide whether TSEs are caused by agents containing nucleic acid or by protein only.

Fine structure of human immunodeficiency virus (HIV), immunolocalization of structural proteins and virus-cell relation

Abstract The classification of a new agent, i.e. its grouping to a defined virus family, is facilitated by electron microscopical investigations. Depending on the method used, the different distinct features of a virus can be evaluated. Combining immunological and electron microscopical techniques we received detailed information on the morphogenesis and fine structure of the human immunodeficiency virus (HIV). Two strains of HIV-1 and HIV-2 were studied. Light microscopy and SEM of virus infected cultures allowed a rapid overview on the cell cultures and gave information on cell morphology and virus production. The more laborious surface replica preparation technique permitted, in addition to an overview, the observation of structural details on the surface of the virion. Rotational image analysis of surface knobs revealed a T = 7 laevo symmetry, indicating that icosahedral principles are governing the envelope architecture. Thin section TEM of tannic acid treated HIV- producing H9 cells proved to be the most rewarding technique for the analysis of the labile HIV. No morphological differences between HIV-1 and -2 strains were detectable. The combination of structural and immuno-electron microscopical findings led to the establishment of a structural model of HIV. Whether the submembrane p17 protein serves as a morphopoetic factor and scaffolding matrix for the envelope is discussed. The viral core is built up as a double cone with a long and a short axis. The core shell containing the major viral capsid protein p24 encloses the electron dense ribonucleoprotein complex. Structural features, including the loss of the viral surface protein gp120 and the incorporation of MHC class I and II antigents into the viral envelope are discussed as factors involved in the pathogenesis of AIDS.

Negative staining in diagnostic virology

Abstract Negative staining has been quickly developed since the early 1960s to a world wide used technique for the direct visualization of viruses and macromolecules by electron microscopy (see Hayat and Miller, 1990). In particular, negative staining has become an indispensable method in the rapid diagnosis of viral diseases. In this article we describe briefly the history and development and discuss some methodological and diagnostic aspects of negative staining in virology.

Cytopathogenicity of Balamuthia mandrillaris, an Opportunistic Causative Agent of Granulomatous Amebic Encephalitis

ABSTRACT. Balamuthia mandrillaris is a free‐living ameba and an opportunistic agent of lethal granulomatous amebic encephalitis in humans and other mammals. Balamuthia mandrillaris is highly cytopathic but, in contrast to the related Acanthamoeba, does not feed on bacteria and seems to feed only on eukaryotic cells instead. Most likely, the cytopathogenicity of B. mandrillaris is inseparable from its infectivity and pathogenicity. To better understand the mechanisms of B. mandrillaris cytopathogenicity, an assay for measuring amebic cytolytic activity was adapted that is based on the release of a reporter enzyme by damaged target cells. The ameba is shown to lyse murine mastocytoma cells very efficiently in a time‐ and dose‐related manner. Furthermore, experiments involving semipermeable membranes and phagocytosis inhibitors indicate that the cytolytic activity of B. mandrillaris is essentially cell contact‐dependent. Standard and fluorescence light microscopy, as well as scanning and transmission electron microscopy support and extend these findings at the ultrastructural level.

Chronic basidiomycetous endophthalmitis after extracapsular cataract extraction and intraocular lens implantation.

Abstract• Background: Basidiomycetes are known as rare pathogens for meningitis, sinusitis, pneumonia, ulcerative lesions of the hard palate and onychomycosis. To our knowledge, no filamentous basidiomycete has been reported from a case of fungal endophthalmitis. • Patient: We report on a 67-year-old man with delayed-onset endophthalmitis caused by an opportunistic basidiomycete. Tissue obtained during vitrectomy was cultured and examined by light and transmission electron microscopy. After enucleation the eye was examined by light microscopy. • Conclusion: The patient had endophthalmitis from a sterile hyphomycete, harboured in remnants of lens capsule and a granuloma on the ciliary body. It was recognized as a Holobasidiomycete on the basis of its dolipore structure with perforated pore cap, seen with transmission electron microscopy. Species identification was not possible because fruiting bodies were absent. The patient failed to respond to intravitreal and systemic amphotericin B and systemic itraconazole. The eye was enucleated. This case demonstrates that filamentous basidiomycetes can cause endophthalmitis when inoculated during cataract extraction.

Cellular and humoral immunogenicity of hamster polyomavirus-derived virus-like particles harboring a mucin 1 cytotoxic T-cell epitope

In this study, we examined hamster polyomavirus (HaPyV) major capsid protein VP1-derived virus-like particles (VLPs) as a carrier for a human tumor-associated cytotoxic T lymphocyte (CTL) epitope. The VP1 tolerated the insertion of an HLA-*A2-restricted CTL epitope from human mucin 1 (MUC1) into two sites independently and simultaneously, without interfering with assembly of chimeric VLPs. Chimeric VLPs did not differ in the entry pathway or maturation potential of human dendritic cells (hDCs) compared to unmodified VLPs. Recently we demonstrated that immunization of BALB/c mice with chimeric VLPs harboring two MUC1 insertions resulted in the generation of MUC1-specific monoclonal antibodies. Here we demonstrate that the monoclonal antibodies generated react specifically with human tumor cells. Co-cultivation of chimeric VLP-primed hDCs with autologous peripheral blood leukocytes resulted in the activation of MUC1 epitope-specific CD8(+) T cells. This was evidenced by IFN-gamma secretion of an expanded MUC1-specific CD8(+) T-cell pool. The induction of epitope-specific T cells in a human in vitro model and of murine MUC1-reactive antibodies in vivo indicate the potential of chimeric HaPyV VP1-derived VLPs as a delivery vehicle for immunotherapeutic targets.