Wendell A. Daniel

Replication of polyoma virus in mouse embryo cells: Electron microscopic observations

Abstract Single step replication of polyoma virus in mouse embryo (ME) cells has been examined by electron microscopy. Cells phagocytose individual virus particles or membrane bound aggregates into the cytoplasm. Individual particles appear in the cytoplasm as 50–60 mμ membrane-bounded viruses. Either 38 mμ or 50–60 mμ particles may be found in the larger phagocytic inclusions. As early as the eighth hour of infection, virus particles have been occasionally observed between the two nuclear membranes; however, at later periods they are frequently observed in this location. By the twentieth hour of infection, small, densely staining, bundles of filaments are observed in multiple loci in nuclei. These bundles increase in number with time and are often surrounded by typical 38 mμ “nuclear” virus particles which may form intranuclear crystals as early as 24 hours after infection. Virus in cells whose nuclei are degenerating exhibit a remarkable affinity for nuclear, cytoplasmic and cell surface membranes. These observations support the suggestion of Bernard et al. (1959) that the nuclear filament is a precursor of polyoma virus.

Replication of poliovirus in HeLa cells: Electron microscopic observations

Abstract Poliovirus-infected HeLa cells were examined by electron microscopy. Both nuclear and cytoplasmic changes were observed during the first 4 hours of infection. Nuclei of cells infected from 2 to 6 hours developed progressively more numerous extrusions which contained ribosome-like particles. These extrusions appeared to eventually become cytoplasmic inclusions. Changes in the cytoplasm were observed at about the fourth hour of infection, at which time clusters of small cytoplasmic vesicles appeared; occasionally they contained viruslike particles. By the sixth hour arrays of virus particles were found within cytoplasmic vesicles. From the sixth to the eighth hour these vesicles appeared to coalesce into large crystalline aggregates of virus. Eventually the large crystals of virus appeared to “melt.” The possible significance of these observations is discussed in relation to numerous other approaches to understanding the nature of replication of small RNA virus.

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.

A comparison of Knobby (K+) and Knobless (K−) parasites from two strains of Plasmodium falciparum

Erythrocytes infected with Plasmodium falciparum develop knob-like protrusions on their membranes. Knobby (K+) parasites of the FCR-3 (Gambian) strain have been shown to possess a histidine-labelled protein of apparent molecular weight 80 000 which is absent from knobless (K-) variants of the same strain. Here we report similar findings with K+ and K- parasites of another strain, the Malayan Camp strain, and also with cloned K+ and K- parasites of the FCR-3 strain. A histidine-labelled protein unique to the two K+ parasites was identified as a broad band with an apparent molecular weight of 89 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of this protein in both K+ Malayan Camp parasites and K+ FCR-3 (Gambian) parasites and its absence from K- parasites of both strains is consistent with this protein being a major component of knobs.

Radioiodination of new protein antigens on the surface of Plasmodium knowlesi schizont-infected erythrocytes

Abstract Schizont-infected red blood cells (SI-RBCs) from Plasmodium knowlesi -infected rhesus monkeys ( Macaca mulatta ) have been radioiodinated and the 125 I-proteins and 125 I-antigens compared with those of uninfected RBCs. New 125 I-proteins of M r 230 000, 180 000, 165 000, 155 000, 135 000, 107 000, 72 000 and 65 000 were shown to be parasite-dependent components of SI-RBCs, the M r 230 000 and 72 000 bands being quantitatively the major new components. New 125 I-antigens of SI-RBCs that were absent from uninfected cells and were only immunoprecipitated by sera from infected monkeys had M r 230 000, 200 000, 180 000, 165 000, 155 000, 135 000, 130 000, 107 000, 72 000, 65 000 and 47 000. The following approaches were used to test which new radioiodinated components are on the SI-RBC outer membrane: analysis of radioactivity in haemoglobin; electron microscopic analysis of the integrity and purity of the SI-RBCs; treatment of intact 125 I-SI-RBCs with trypsin to ascertain the sensitivity of new proteins to exogenous protease; immunoprecipitation of antigen-antibody complexes after addition of antibody to intact 125 I-SI-RBCs. Although each test has inherent disadvantages, the accumulated evidence suggests that many, if not all of the new antigens identified for the first time in this report are on the SI-RBC outer membrane. The SICA variant antigen on P. knowlesi SI-RBCs was not identified by this approach.

Export of PlasmodiumFalciparum Proteins to the Host Erythrocyte Membrane: Special Problems of Protein Trafficking and Topogenesis

Asexual blood-stage malaria parasites induce several morphological, antigenic and functional changes of the infected erythrocytes membrane. In the human malaria Plasmodium falciparum these changes include the following: 1. expression of knob-like protrusions of the host cell membrane and underlying electron dense material (EDM), together called a ‘knob’. 2. expression of the capacity of infected erythrocytes to cytoadhere specifically to capillary endothelial cells. Knobs bear the ligand(s) responsible for cytoadherence. 3. expression on the cell surface of a very large (Mr~300,000) malarial protein called the cytoadherence protein, or, PfEMP1 (P. falciparum erythrocyte membrane protein 1). A body of indirect evidence, summarized below, suggests that PfEMP1 either bears the ligand(s) responsible for recognition of endothelial cells, or, is located in close physical proximity to the cytoadherence ligand(s). 4. insertion of another very large malarial protein (Mr~300,000) under the surface membrane at the EDM of knobs. This protein can be distinguished from PfEMP1 not only by its submembrane location, but by specific reaction with monoclonal antibodies. We designate this molecule PfEMP2. 5. insertion of a histidine-rich protein, called the knob-protein or PfHRP1 (P. falciparum histidine-rich-protein 1), into the EDM at knobs.

Morphologic changes in the rabbit SIRC cell line induced by simian sarcoma-associated virus

Abstract Rabbit cornea (SIRC) cells were altered morphologically upon simian sarcoma-associated virus infection. These focal alterations could be used for virus assay. Unusual small cytoplasmic particles of uncertain origin were observed by electron microscopy in addition to typical C-type particles.