Variants of Defective Simian Papovavirus 40 (PARA) Characterized by Cytoplasmic Localization of Simian Papovavirus 40 Tumor Antigen

Three isolates of PARA (particle aiding replication of adenovirus)-adenovirus 7 out of a total of 112 clonal progeny derived by two successive plaque purifications in green monkey kidney cells (GMK) were found to induce the synthesis of simian papovavirus40 (SV 40) tumor (T) antigen in the cytoplasm of infected cells. The variant viruses induced plaque formation in human embryonic kidney cells which followed one-hit kinetics. In GMK cells, plaque formation followed two-hit kinetics which converted to first-order kinetics in the presence of additional helper adenovirus type 7. Analysis of plaque progeny from human cells showed that the progeny could replicate only in human cells, whereas progeny from monkey cells could multiply in both human and monkey cells. Heterologous human adenoviruses were able to enhance plaque formation by the variant viruses in monkey kidney cells. Neutralization tests indicated that both components of the populations had a type 7 adenovirus capsid. All three viruses were capable of inducing SV40 transplantation immunity in weanling hamsters. These results indicate the three variants are PARA-adenovirus 7 populations. Response of the induction of the synthesis of the cytoplasmic antigen to metabolic inhibitors was the same as for the synthesis of the nuclear SV40 T antigen. Different pools of sera which reacted with the intranuclear SV40 T antigen also detected the cytoplasmic antigen induced by the variant viruses. An adsorption experiment with cells containing either nuclear or cytoplasmic T antigen to remove tumor antibody from hamster sera also indicated that it is probably SV40 T antigen which is responsible for the cytoplasmic reaction. The species of the host cell-human, simian, or rabbit-appeared to play no role in the altered localization of this antigen. It is postulated that these PARA variants are further defective in some virus-mediated transport mechanism which shifts the T antigen from the cytoplasm to the nucleus.     

Modulation of the Cellular Distribution of Human Cytomegalovirus Helicase by Cellular Factor Snapin

Controlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV UL105 is believed to encode the helicase of the DNA replication machinery that needs to localize in the nuclei, the site of viral DNA synthesis. No host factors that interact with UL105 have been identified. In this study, we show that UL105 specifically interacts with Snapin, a human protein that is predominantly localized in the cytoplasm and associated with cellular vesicles. UL105 was found to interact with Snapin in both the yeast two-hybrid screen and coimmunoprecipitation experiments in HCMV-infected cells. The nuclear and cytoplasmic levels of UL105 were decreased and increased in cells overexpressing Snapin, respectively, while the levels of UL105 in the nuclei and cytoplasm were increased and decreased in cells in which the expression of Snapin was downregulated with anti-Snapin small interfering RNA (siRNA) molecules, respectively. Furthermore, viral DNA synthesis and progeny production were decreased in cells overexpressing Snapin and increased in the anti-Snapin siRNA-treated cells, respectively. Our results provide the first direct evidence to suggest that Snapin interacts with UL105 and alters its cellular distribution, leading to modulation of viral DNA synthesis and progeny production. Our study further suggests that modulation of the cellular distribution of viral helicase by Snapin may represent a possible mechanism for regulating HCMV genomic DNA synthesis, a key step during herpesvirus lytic and persistent infections.     

Electron microscope studies on the intracellular growth of PL-1 phage of Lactobacillus casei

Ultrathin sections of the cells of Lactobacillus casei infected with or without PL-1 phages were observed by the rapid-freezing and substitution-fixation method. Phage-head-like particles were first observed in the nuclear region. The region was seen more widely dispersed in the cytoplasm than that observed by the conventional chemical fixation method. The features of cells just broken open by the infected phages were observed by the sedimentation method devised by us. The bursting occurred in more than one place in the cells with liberation of progeny phages.     

Paternal inheritance of mitochondria in rapeseed (Brassica napus)

Summary Transfer of a mitochondrially associated plasmid following sexual crosses in Brassica napus rapeseed suggested that paternal mitochondria were being transferred to the cytoplasm of the egg. To examine this possibility further, plants carrying the chloroplast (cp) marker of triazine resistance, but which had lost the plasmid associated with the mitochondria of this cytoplasm, were crossed as females to males carrying the polima cytoplasm. The males carried a nuclear fertility restorer gene on an extra chromosome to overcome the male sterility marker conferred by the mitochondria of this cytoplasm. Approximately 10% of the F₁ progeny displayed the male sterility and flower morphology of the male parent. Mitochondrial (mt) DNA from the progeny showed the combined restriction patterns of both parents, but this rut heterogeneity did not continue into subsequent generations. All progeny retained the cp DNA restriction patterns of the maternal plant as well as resistance to the herbicide atrazine. To date, sexually mediated cybrid plants have shown no morphological abnormalities and have maintained their unique combination of cp and mt traits through several sexual generations.     

Variation in Properties of Plaque Progeny of PARA (Defective Simian Papovavirus 40)-Adenovirus 7

One hundred and twelve progeny from double plaque-purified clones were derived from the original PARA (defective simian papovavirus 40)-adenovirus 7 population. These progeny were found to differ in their oncogenic potential in newborn hamsters with progeny from 20 clones not inducing any tumors during 1 year of observation. The varying tumorigenicity of the individual clonal progeny was not related to the titer of PARA (particle aiding replication of adenovirus) in the inoculum. There was a perfect correlation between the tumor antigen content of the tumor cells and the antibody response of the tumor-bearing host. The tumors containing both adenovirus and simian papovavirus 40 (SV40) tumor antigens appeared earlier than those carrying only SV40 tumor antigen. Progeny from clones which induced mixed tumors also produced tumors which contained only SV40 tumor antigen. Three variants of PARA were isolated which induced the synthesis of SV40 tumor antigen in the cytoplasm of infected simian cells; all other clones yielded progeny which induced synthesis of SV40 tumor antigen in the nucleus.     

Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.     

The expression of hex A and hex B isozymes of hexosaminidase in parental and experimental human fibroblast cells and their components

The expression of the two major isozyme forms of hexosaminidase (EC 3.2.1.30), hesoxaminidase A and hexosaminidase B, has been examined. The parental cells and/or cellular components of parental cells are individually fused using inactivated Sendai virus with the aid of a micromanipulator. The progeny cells produced from such hybrids are subjected to a microenzymatic assay which allows measurements at the single cell level. The lysosomal-deficient cells used in this study are Tay-Sachs and Sandhoff fibroblasts, and the normal cells used are WI-38 (fetal lung fibroblasts), amniotic fluid cells (GM 473), and JASD₃ (normal human foreskin). The results show that the ratio of cell components which are fused to form the experimental cell affects the percentage of hexosaminidase A expressed in the progeny cells. Furthermore, our results imply the presence of a “factor” in the Sandhoff cell's cytoplasm which, together with the Tay-Sachs nucleus, is necessary for hexosaminidase A expression in the experimental cell's progeny.     

Intrachromosomal changes and genomic instability in site-specific microbeam-irradiated and bystander human-hamster hybrid cells

Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (A(L)) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.     

草鱼呼肠孤病毒在CIK细胞中复制及形态发生的研究

以草鱼呼肠孤病毒（ＧＣＲＶ）感染的草鱼肾细胞系（ＣＩＫ）为模型,进行了草鱼呼肠孤病毒在细胞内的形态发生的研究。当病毒以感染复数为５￣１０ＰＦＵ／ＣＥＬＬ感染ＣＩＫ细胞时,在病毒感染细胞４ｈ以内的切片中,可观察到脱去部分外层衣壳的不完整病毒颗粒。感染细胞８ｈ,可观察到浆胞内病毒发生基质,其内含有大量的直径约５０ｎｍ的亚病毒颗粒,无外层蛋白结构。感染１２￣１６ｈ后,这些亚病毒颗粒装配上外层蛋白结构,形     

Assembly of Rickettsia tsutsugamushi progeny in irradiated L cells.

In the assembly of Rickettsia tsutsugamushi progeny in irradiated L cells, nascent forms first appear as undemarcated foci in the host cell granular cytoplasm, in which electron-lucent filamentous (f) and electron-dense granular (g) areas differentiate. Morphological observations indicated that the assembly involves formation of a filamentous network in the f area, manufacture of rickettsial ribosomes in the g area, and formation of mildly electron-dense fuzzy zones, along which a double membrane assembles.     

Genome packaging of reovirus is mediated by the scaffolding property of the microtubule network

Reovirus replication occurs in the cytoplasm of the host cell, in virally induced mini‐organelles called virus factories. On the basis of the serotype of the virus, the virus factories can manifest as filamentous (type 1 Lang strain) or globular structures (type 3 Dearing strain). The filamentous factories morphology is dependent on the microtubule cytoskeleton; however, the exact function of the microtubule network in virus replication remains unknown. Using a combination of fluorescent microscopy, electron microscopy, and tomography of high‐pressure frozen and freeze‐substituted cells, we determined the ultrastructural organisation of reovirus factories. Cells infected with the reovirus microtubule‐dependent strain display paracrystalline arrays of progeny virions resulting from their tiered organisation around microtubule filaments. On the contrary, in cells infected with the microtubule‐independent strain, progeny virions lacked organisation. Conversely to the microtubule‐dependent strain, around half of the viral particles present in these viral factories did not contain genomes (genome‐less particles). Complementarily, interference with the microtubule filaments in cells infected with the microtubule‐dependent strain resulted in a significant increase of genome‐less particle number. This decrease of genome packaging efficiency could be rescued by rerouting viral factories on the actin cytoskeleton. These findings demonstrate that the scaffolding properties of the microtubule, and not biochemical nature of tubulin, are critical determinants for reovirus efficient genome packaging. This work establishes, for the first time, a functional correlation between ultrastructural organisation of reovirus factories with genome packaging efficiency and provides novel information on how viruses coordinate assembly of progeny particles.     

Modulation of Nuclear Localization of the Influenza Virus Nucleoprotein through Interaction with Actin Filaments

The influenza virus genome is transcribed in the nuclei of infected cells but assembled into progeny virions in the cytoplasm. This is reflected in the cellular distribution of the virus nucleoprotein (NP), a protein which encapsidates genomic RNA to form ribonucleoprotein structures. At early times postinfection NP is found in the nucleus, but at later times it is found predominantly in the cytoplasm. NP contains several sequences proposed to act as nuclear localization signals (NLSs), and it is not clear how these are overridden to allow cytoplasmic accumulation of the protein. We find that NP binds tightly to filamentous actin in vitro and have identified a cluster of residues in NP essential for the interaction. Complexes containing RNA, NP, and actin could be formed, suggesting that viral ribonucleoproteins also bind actin. In cells, exogenously expressed NP when expressed at a high level partitioned to the cytoplasm, where it associated with F-actin stress fibers. In contrast, mutants unable to bind F-actin efficiently were imported into the nucleus even under conditions of high-level expression. Similarly, nuclear import of NLS-deficient NP molecules was restored by concomitant disruption of F-actin binding. We propose that the interaction of NP with F-actin causes the cytoplasmic retention of influenza virus ribonucleoproteins.     

Temporal events in the invasion of the codling moth,Cydia pomonella,by a granulosis virus: An electron microscope study

The replication cycle of the granulosis virus of Cydia pomonella, the codling moth, was studied at the cellular and tissue level. Membranelike complexes were observed forming within the remnants of the nucleolus in the cytoplasm of infected cells. Differences in cell polarity relative to the sites of virus entry assembly and budding as well as differences in the temporal aspects of replication were observed between midgut, fat body, and epidermal cells. The progressive spread of virus throughout larval tissues was studied at 24, 32, 48, 56, and 72 hr postinfection. The basal lamina seemed to be an effective barrier for the release of budded progeny virus into the hemocoel and large numbers of budded virus were produced.     

Morphogenesis of the Nucleoprotein of Vesicular Stomatitis Virus

Accumulation of the nucleoprotein of vesicular stomatitis virus (VSV) in the cytoplasm of BHK-21 cells and in two of four human cell lines was demonstrated. Appearance and progression of the nucleoprotein inclusions paralleled development of virus-specific immunofluorescence and production of virus progeny. The inclusions appeared early as discrete foci of filamentous material which eventually increased in size to form large masses which replaced normal cytoplasmic constituents. The filamentous strands were found in close proximity to budding virions. The inclusion material was extracted from infected cells and purified in cesium chloride gradients. The isolated filaments resembled the ribonucleoprotein isolated from purified virions. They incorporated (3)H-uridine, exhibited virus-specific complement-fixing activity, had a buoyant density of 1.32 g/cm(3), and appeared as single wavy strands the width of which varied from 2.5 to 8.5 nm, depending on the angle of viewing.     

In vitro exposure of mammalian cells to radon: dosimetric considerations

We have developed a model to calculate the dose to the cell nucleus in cells exposed in suspension to radon and/or radon progeny. The model addresses the influence of (1) different radiation qualities and energies in the irradiation milieu; (2) the contribution to dose from radioactivity in the medium surrounding the cell after exposure to the radon gas as well as that from excess radon progeny associated with the cell; (3) the geometry of the cell and of the radiosensitive target, the cell nucleus; (4) the intracellular localization of the radionuclides; (5) attenuation of the alpha particles by the cytoplasm; (6) the radionuclide concentrations in the medium; and (7) the length of exposure. Investigation of the influence of these various parameters was made using an irradiation system in which cells were exposed to 212Bi, which decays to stability with the emission of an alpha particle (either 6.05 or 8.78 MeV). The information from these studies was then used to develop the system further for more complex systems in which 222Rn and its progeny are present. The model takes into account the contribution of dose from different radiation sources using scintillation counts of the medium and the cells, and it is useful for calculations of dose in situations where cells are exposed in suspension culture.     

Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.     

The human immunodeficiency virus type 1-specific protein vpu is required for efficient virus maturation and release.

A deletion mutation affecting vpu was introduced into an infectious molecular clone of human immunodeficiency virus type 1, and the resultant phenotype was examined after infection of human T lymphocytes. The absence of vpu resulted in an accumulation of cell-associated viral proteins and impaired the release of progeny virions. Both electron microscopic and biochemical analyses indicated that a large proportion of the mutant particles was attached to the surface of infected cells. Significant variation in the size and shape of these progeny virions was observed. In addition, intracytoplasmic particles, some of which formed aberrant budding structures, were visualized in T cells infected with the vpu mutant. Indirect immunofluorescence analyses of cultures inoculated with wild-type virus with use of a vpu-specific antiserum demonstrated that vpu is mainly localized to a perinuclear region in the cytoplasm of virus-producing cells.     

MITOSIS AND CYTOKINESIS IN THE CHLOROMONADOPHYCEAN ALGA GONYOSTOMUM SEMEN1

Mitosis and cytokinesis in Gonyostomum semen (Ehrenberg) Diesing have been investigated with the light microscope. During prophase nucleoli disappear and the chromatid structure of the chromosomes becomes apparent. Separation of chromatids at anaphase is accompanied by progressive fusion of the progeny chromosomes. This process continues into telophase by which stage the progeny nuclei consist of dense masses of chromatin with occasional chromosomes extending from their equatorial surfaces. By the end of telophase, nucleoli are reforming and the interphase nuclear morphology is reestablished. Mitosis is followed by cytokinesis, which is a relatively lengthy phase. In early cytokinesis the 2 interphase nuclei are present, and there is no indication of the forthcoming division of the cytoplasm. Later in cytokinesis a membrane is formed between the 2 nuclei. Final separation of the progeny individuals is accomplished by vigorous movements of swimming cells or, in the case of palmelloid cells, by the deposition of a mucilaginous layer.     

Electron microscopic study of the division processes in cysts of the sarcosporidian, Sarcocystis, ovifelis

The metrocyte stage in the cysts of S. ovifelis has been found to divide not by endodyogeny, but by a different mode, not yet completely understood. The nucleus of the dividing metrocyte displays no nuclear envelope, the cytoplasm is full of large vacuoles. During the metrocyte division, some kind of nucleoplasm spreading occurs over the cytoplasm and between the large vacuoles, thus making any morphological limits between the cytoplasm and the nucleus invisible. The cytokinesis is accomplished due to the deep invaginations of the metrocyte pellicle. The metrocyte division first gives rise to a large multicellular (4--6) body lying in the peripheral zone of the cyst. The very impossibility of endodyogenetic division in the metrocyte stage may be due to its vaery peculiar morphofunctional organization, much different from what is characteristic of any asexual penetrative stage (= zoite) of Apicomplexa. The progeny of a metrocyte are interstitial cells which eventually, through the number of generations, evolve towards the metrozoite stage. Step by step, the interstitial cells aquire polarity due to the establishment of a conoid, rhoptries and micronnemes on the anterior pole, the pellicle becoming more rigid thus making, together with subpellicular microtubules, the cytoskeleton of the parasite. The study performed enables us to conclude that it is interstitial cells of advanced generations that divide by endodogeny. Merozoites being homologous to gamonts of other coccidia undergo no asexual division at all.     

Brefeldin A arrests the maturation and egress of herpes simplex virus particles during infection.

Herpes simplex virus (HSV) requires the host cell secretory apparatus for transport and processing of membrane glycoproteins during the course of virus assembly. Brefeldin A (BFA) has been reported to induce retrograde movement of molecules from the Golgi to the endoplasmic reticulum and to cause disassembly of the Golgi complex. We examined the effects of BFA on propagation of HSV type 1. Release of virions into the extracellular medium was blocked by as little as 0.3 microgram of BFA per ml when present from 2 h postinfection. Characterization of infected cells revealed that BFA inhibited infectious viral particle formation without affecting nucleocapsid formation. Electron microscopic analyses of BFA-treated and untreated cells (as in control cells) demonstrated that viral particles were enveloped at the inner nuclear membrane in BFA-treated cells and accumulated aberrantly in this region. Most of the progeny virus particles observed in the cytoplasm of control cells, but not that of BFA-treated cells, were enveloped and contained within membrane vesicles, whereas many unenveloped nucleocapsids were detected in the cytoplasm of BFA-treated cells. This suggests that BFA prevents the transport of enveloped particles from the perinuclear space to the cytoplasmic vesicles. These findings indicate that BFA-induced retrograde movement of molecules from the Golgi complex to the endoplasmic reticulum early in infection arrests the ability of host cells to support maturation and egress of enveloped viral particles. Furthermore, we demonstrate that the effects of BFA on HSV propagation are not fully reversible, indicating that maturation and egress of HSV type 1 particles relies on a series of events which cannot be easily reconstituted after the block to secretion is relieved.