Association of Paramecium bursaria Chlorella viruses with Paramecium bursaria cells: ultrastructural studies

Paramecium bursaria Chlorella viruses were observed by applying transmission electron microscopy in the native symbiotic system Paramecium bursaria (Ciliophora, Oligohymenophorea) and the green algae Chlorella (Chlorellaceae, Trebouxiophyceae). Virus particles were abundant and localized in the ciliary pits of the cortex and in the buccal cavity of P. bursaria. This was shown for two types of the symbiotic systems associated with two types of Chlorella viruses - Pbi or NC64A. A novel quantitative stereological approach was applied to test whether virus particles were distributed randomly on the Paramecium surface or preferentially occupied certain zones. The ability of the virus to form an association with the ciliate was investigated experimentally; virus particles were mixed with P. bursaria or with symbiont-free species P. caudatum. Our results confirmed that in the freshwater ecosystems two types of P. bursaria -Chlorella symbiotic systems exist, those without Chlorella viruses and those associated with a large amount of the viruses. The fate of Chlorella virus particles at the Paramecium surface was determined based on obtained statistical data and taking into account ciliate feeding currents and cortical reorganization during cell division. A life cycle of the viruses in the complete symbiotic system is proposed.     

Decrease in the transforming action of Rous sarcoma virus produced by avian cells grown in the presence of 5'-deoxy-5'-S-isobutyladenosine (SIBA)

Treatment of Rous Sarcoma virus transformed chick embryo fibroblasts with 1 mM 5'-deoxy-5'-S-isobutyladenosine for 24 hrs. leads to the inhibition of transforming virus production. A kinetic analysis of the inhibition of active virion production revealed that the effect of the drug was time and concentration dependent. After 24 hrs. with 1 mM SIBA, the production of transforming virus was inhibited 165 fold. However, under these conditions there was only a 2 fold inhibition in viral particle production. Thus, these viral particles were either non infective (non adsorbed on cell membrane) or non transforming. The majority of viral particles produced by cells cultured with the drug have a decreased density. Analysis of these virions showed a decrease of protein P19 and an accumulation of proteins with high molecular weight.     

Growth characteristics and intraspecies host specificity of a large virus infecting the dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30 degrees C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20 degrees C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

The effect of host Chlorella NC64A carbon : phosphorus ratio on the production of Paramecium bursaria Chlorella Virus-1

1. We used the freshwater alga Chlorella NC64A (Division Chlorophyta) and its virus Paramecium bursaria Chlorella virus‐1 (PBCV‐1) as a model system to test for potential stoichiometric constraints on a virus–host interaction. 2. Media phosphorus concentrations were manipulated to create Chlorella NC64A host cells with low (91 ± 23) or high (453 ± 246) C : P ratio. In contrast, the C : P ratio of PBCV‐1, calculated from its biochemical composition, was 17 : 1. 3. Stoichiometric theory predicts that infection success and postinfection viral production should be depressed in high C : P cultures due to insufficient intracellular P for production of P‐rich viral particles. 4. Consistent with this hypothesis, viral production was strongly affected by host C : P ratio. While host C : P ratio did not affect viral attachment or the percentage of new viral particles that were infectious, in the low C : P Chlorella NC64A treatment, nine times more viruses were produced per infected cell than in the high C : P treatment (158 ± 138 versus 18 ± 18), indicating that the low C : P cells were higher quality for PBCV‐1 proliferation. 5. This result implies that the stoichiometric quality of algal cells can have a major effect on host–virus population dynamics.     

Photosynthetic Shutdown in Chlorella NC64A Associated with the Infection Cycle of Paramecium bursaria Chlorella Virus-1

The effects of the algal virus Paramecium bursaria Chlorella virus-1 on the photosynthetic physiology of its host, Chlorella NC64A, was studied by observing changes in Chl fluorescence quenching and O2 exchange. Metabolic changes were calibrated against electron microscopic analysis of the morphological changes that occur during the infection cycle. It takes approximately 10 h from attachment of the virus to final lysis of the host cell, so a complete infection cycle can be observed continuously in one experiment. During the early stages of the infection cycle many rapid changes occurred in the host cell's metabolism and these were reflected in changes of photosynthetic and respiratory rates. The dramatic inhibition of photosynthesis in Chlorella NC64A cells by P. bursaria Chlorella virus-1 has facilitated the use of fluorescence quenching as an accurate measure of the first phase of viral infection (attachment and penetration of the host cell) and the extent to which a population of host cells is infected. Effects of temperature and cation requirement of the infection cycle are described. The relevance of our observations to the events observed during viral infection of higher plants is discussed.     

The Association of Hepatitis C Virus Glycoproteins with Apolipoproteins E and B Early in Assembly Is Conserved in Lipoviral Particles

In patients chronically infected with hepatitis C virus and in the HCV cell culture system (HCVcc), it is known that highly infectious virus particles have low to very low buoyant densities. These low densities have been attributed to the association of HCV with lipoprotein components, which occur during the viral morphogenesis. The resulting hybrid particles are known as lipoviral particles (LVP); however, very little is known about how these particles are created. In our study, we used Huh7.5 cells to investigate the intracellular association between envelope proteins and apolipoproteins B and E (ApoB and ApoE, respectively). In particular, we were interested in the role of this association in initiating LVP morphogenesis. Co-immunoprecipitation assays revealed that ApoB, ApoE, and HCV glycoproteins formed a protein complex early in the HCV lifecycle. Confocal analyses of naïve, E1E2-transduced and HCVcc-infected cells showed that HCV glycoproteins, ApoB and ApoE were found strongly colocalized only in the endoplasmic reticulum. We also found that HCV glycoproteins, ApoB and ApoE were already associated with intracellular infectious viral particles and, furthermore, that the protein complex was conserved in the infectious viral particles present in the supernatant of infected Huh7.5 cells. The association of HCV glycoproteins with ApoE was also evidenced in the HCVpp system, using the non-hepatic HEK293T cell line. We suggest that the complex formed by HCV E1E2, ApoB, and ApoE may initiate lipoviral particle morphogenesis.     

Intraspecies host specificity of a single-stranded RNA virus infecting a marine photosynthetic protist is determined at the early steps of infection

Viruses are extremely abundant in seawater and are believed to be significant pathogens to photosynthetic protists (microalgae). Recently, several novel RNA viruses were found to infect marine photosynthetic protists; one of them is HcRNAV, which infects Heterocapsa circularisquama (Dinophyceae). There are two distinct ecotypes of HcRNAV with complementary intraspecies host ranges. Nucleotide sequence comparison between them revealed remarkable differences in the coat protein coding gene resulting in a high frequency of amino acid substitutions. However, the detailed mechanism supporting this intraspecies host specificity is still unknown. In this study, virus inoculation experiments were conducted with compatible and incompatible host-virus combinations to investigate the mechanism determining intraspecies host specificity. Cells were infected by adding a virus suspension directly to a host culture or by transfecting viral RNA into host cells by particle bombardment. Virus propagation was monitored by Northern blot analysis with a negative-strand-specific RNA probe, transmission electron microscopy, and a cell lysis assay. With compatible host-virus combinations, propagation of infectious progeny occurred regardless of the inoculation method used. When incompatible combinations were used, direct addition of a virus suspension did not even result in viral RNA replication, while in host cells transfected with viral RNA, infective progeny virus particles with a host range encoded by the imported viral RNA were propagated. This indicates that the intraspecies host specificity of HcRNAV is determined by the upstream events of virus infection. This is the first report describing the reproductive steps of an RNA virus infecting a photosynthetic protist at the molecular level.     

Correlation of Sugar Release and Concanavalin A Agglutinability with Infectivity of Symbiotic Algae from Paramecium bursaria for Aposymbiotic P. bursaria

Synopsis.
Eighteen strains of algae, including 17 formerly symbiotic with Paramecium bursaria , were tested for capacity to release sugar. Detectable amounts of sugar were found in the supernatant fluids from 10 strains, including 6 strains infective for aposymbiotic P. bursaria syngen 2. The other 4 sugar-releasing strains were noninfective and released ˜26–46 g sugar/mg dry cell weight compared to ˜90–175 g sugar/mg dry cell weight for infective strains. This relationship of infectivity with capacity to release sugar supplements data that indicate a relationship of infectivity with resistance to Con A agglutination. The correlation is completed if we assume that resistance to Con A agglutination and capacity for sugar release must both be present in an algal strain for infectivity. The data thus strongly suggest that these 2 characteristics must be present for infectivity by any algal strains for aposymbiotic P. bursaria syngen 2.     

Assay of Variola Virus by the Fluorescent Cell-Counting Technique

A quantitative assay for infective variola virus particles was developed which is based on the enumeration of cells containing fluorescent viral antigen after infection of McCoy cell monolayers. The direct fluorescent-antibody technique was employed to stain cells. The efficiency of virus adsorption was markedly enhanced by centrifugation of virus inoculum onto McCoy cell monolayers at 500 x g for 15 min. By this procedure, a proportionality was obtained between the number of fluorescent cells and volume of inoculum. Observations on the sequential development of viral antigen within cells and counts of fluorescent cells showed that the optimal time for enumerating fluorescent cells was after an incubation period of 16 to 20 hr. A linear function existed between virus concentration and cell-infecting units. Fluorescent cells were distributed randomly in infected cover slip cell monolayers. The assay was demonstrated to be highly sensitive, precise, and reproducible.     

The cell surface receptor is a major determinant restricting the host range of the B-lymphotropic papovavirus.

The B-lymphotropic papovavirus (LPV) productively infects only a subset of human B-lymphoma-derived cell lines while transfection of the viral genome yields infectious viral particles in a much wider variety of human hematopoietic cell lines. We have analyzed the contribution of a putative LPV receptor on the cell surface of B-cell lines in restricting the virus host range. In order to establish a quantitative virus binding assay for LPV, infectious virus particles were highly purified by metrizamide equilibrium density centrifugation and used as immunogens to raise seven mouse monoclonal antibodies specific for LPV VP1. Virus particle binding was quantitated in an indirect, nonradioactive assay with an LPV VP1-specific enzyme-linked immunosorbent assay. Binding of LPV particles to permissive human B-lymphoma cell line BJA-B occurred within minutes. Kinetics and capacity of binding were similar at 4 and 37 degrees C. A BJA-B cell was estimated to bind approximately 600 virus particles at conditions under which 50% of the administered virus was bound. The sialidase and trypsin sensitivities of the cellular virus binding moiety show that sialylated and proteinaceous components are necessary components of the LPV receptor on BJA-B cells. Despite a high binding capacity of BJA-B cells for simian virus 40, LPV binding was not significantly affected by a 20-fold excess of simian virus 40 particles, indicating that these related polyomaviruses do not bind to the same receptor on BJA-B cells. Reduction of LPV binding to sialidase-pretreated BJA-B cells was accompanied by a similar reduction of infection, indicating that virus binding may be a limiting factor in the LPV replicative cycle. The two highly LPV-permissive human B-lymphoma cell lines BJA-B and Namalwa displayed high virus binding whereas low and nonpermissive hematopoietic cell lines showed reduced or undetectable virus binding. We conclude that the inability of LPV particles to productively infect the nonpermissive human hematopoietic cell lines analyzed is probably due to the absence or insufficient expression of a functional cell surface receptor.     

Polyamine depletion of cells reduces the infectivity of herpes simplex virus but not the infectivity of Sindbis virus

The effect of polyamines on the viral growth was examined using cell strains that could be effectively depleted of polyamines. In order to avoid the polyamines present in serum we used a polyamine auxotrophic Chinese hamster ovary cell line P22 growing in serum-free medium and Vero cells growing in low serum medium. The final yield of an enveloped RNA virus, Sindbis, in P22 cells was not decreased by depletion of cellular polyamines although the onset of the viral replication was delayed. In contrast the final yield of an enveloped DNA virus, Herpes simplex virus (HSV), was considerably reduced in Vero cells, depleted of polyamines by alpha-difluoromethylornithine, an inhibitor of polyamine synthesis. However, the number of HSV particles detected by electronmicroscopy was not decreased. Southern blot analysis of HSV-DNA from the polyamine depleted and the control cells showed changes in the relative abundance of the DNA fragments suggesting that impairment in DNA synthesis may have caused the decreased infectivity of HSV.     

Genome assembly and particle maturation of the birnavirus infectious pancreatic necrosis virus

In this study, we have analyzed the morphogenesis of the birnavirus infectious pancreatic necrosis virus throughout the infective cycle in CHSE-214 cells by using a native agarose electrophoresis system. Two types of viral particles (designated A and B) were identified, isolated, and characterized both molecularly and biologically. Together, our results are consistent with a model of morphogenesis in which the genomic double-stranded RNA is immediately assembled, after synthesis, into a large (66-nm diameter) and uninfectious particle A, where the capsid is composed of both mature and immature viral polypeptides. Upon maturation, particles A yield particles B through the proteolytic cleavage of most of the remaining viral precursors within the capsid, the compaction of the particle (60-nm diameter), and the acquisition of infectivity. These studies will provide the foundation for further analyses of birnavirus particle assembly and RNA replication.     

Virus propagation in a swine monocyte cell line

The propagation of seventeen virus strains, classified into five virus families, in a swine monocyte cell line (SW/K99) was studied in the point of infective progeny production. The viruses examined were adapted to grow in a swine epithelial cell line (KSEK6) and were proved to show clear CPE in advance. These viruses were successively passaged in the monocyte cultures regardless of CPE occurrence. The cultures at 3rd passage level were titrated for their infectivity using KSEK6 cells. Out of the viruses examined, only Aujeszky's disease virus (ADV) was able to propagate in the monocytes. Four ADV strains, two virulent and two attenuated strains, were compared for their growth in two cell lines. The final amount of infective progeny measured at 72 hours incubation at 36.5 degrees C was almost in a similar order between 2 cell lines. However, the amount of infective progeny produced at 24 hours incubation was higher in SW/K99 cells than that in KSEK6 cells. The occurrence of CPE was also more evident in SW/K99 cells in an early incubation. The data indicate that viral susceptibility of the monocytes to ADV is higher than that of epithelium. Other viruses were abortive in the monocytes.     

Analysis of extracellular West Nile virus particles produced by cell cultures from genetically resistant and susceptible mice indicates enhanced amplification of defective interfering particles by resistant cultures

[3H]uridine-labeled extracellular West Nile virus (WNV) particles produced by cell cultures obtained from genetically resistant C3H/RV and congenic susceptible C3H/HE mice were compared by sucrose density gradient centrifugation as well as by analysis of the particle RNA. Defective interfering (DI) WNV particles were observed among progeny produced during acute infections in both C3H/RV and C3H/HE cells. Although only a partial separation of standard and DI particles was achieved, the DI particles were found to be more dense than the standard virions. Particles containing several species of small RNAs consistently constituted a major proportion of the total population of virus progeny produced by C3H/RV cells, but a minor proportion of the population produced by C3H/HE cells. Decreasing the multiplicity of infection or extensive plaque purification of the WNV inoculum decreased the proportion of small RNAs found in the progeny virus. The ratio of DI particles to standard virus observed in progeny virus was determined by the cell type used to grow the virus. The ratio could be shifted by passaging virus from one cell type to the other. Homologous interference could be demonstrated with WNV produced by C3H/RV cells but not with virus produced by C3H/HE cells. Continued passage of WNV in C3H/HE cells resulted in a cycling of infectivity. However, passage in C3H/RV cells resulted in the complete loss of infectious virus. Four size classes of small viral RNA, with sedimentation coefficients of about 8, 15, 26, and 34S, were observed in the extracellular particles. A preliminary analysis of these RNAs by oligonucleotide fingerprinting indicated that the smaller RNAs were less complex than the 40S RNA and differed from each other. The data are consistent with the conclusion that WNV DI particles interfere more effectively with standard virus replication and are amplified more efficiently in C3H/RV cells than in congenic C3H/HE cells. The relevance of these findings to the further understanding of genetically controlled resistance to flaviviruses is discussed.     

Replication of Epstein-Barr virus: ultrastructural and immunofluorescent studies of P3HR1-superinfected Raji cells.

We have studied by means of electron microscopy and immunofluorescence the different steps of the replication of the P3HR1 strain of Epstein-Barr virus in Raji cells. The virus entered the cell by fusion of the viral envelope with the plasma membrane, followed by the disintegration of the capsid. In some cases, the migration of nucleocapsids toward the nuclear membrane was observed. The synthesis of new virions began as early as 7 h after infection (in the case of a high multiplicity of infection [MOI]-800 particles per cell) and took place in low-electron-density areas of the nucleus. A viral envelope was acquired by budding either through the nuclear membrane or more often through membranes of the Golgi apparatus or cytoplasmic vacuoles. Comparing immunofluorescence and electron microscopic data a good correlation was found between the presence of early antigen and ultrastructurally altered cells, as well as between the presence of viral capsid antigen and virus-producing cells. With different MOIs, different types of viral cycles were observed: at a low MOI (less than or equal to 50 particles per cell), a nonproducer cycle was induced, with early antigen synthesis only; at a higher MOI (100 particles per cell), a transient production of a small amount of virions was observed, and at a high MOI (greater than or equal to 300 particles per cell), a productive cycle was the rule.     

Quaternary assembly and crystal structure of GDP-D-mannose 4,6 dehydratase from Paramecium bursaria Chlorella virus

GDP-D-mannose 4,6 dehydratase is the first enzyme in the de novo biosynthetic pathway of GDP-L-fucose, the activated form of L-fucose, a monosaccharide found in organisms ranging from bacteria to mammals. We determined the three-dimensional structure of GDP-D-mannose 4,6 dehydratase from the Paramecium bursaria Chlorella virus at 3.8A resolution. Unlike other viruses that use the host protein machinery to glycosylate their proteins, P. bursaria Chlorella virus modifies its structural proteins using many glycosyltransferases, being the first virus known to encode enzymes involved in sugar metabolism. P. bursaria Chlorella virus GDP-D-mannose 4,6 dehydratase belongs to the short-chain dehydrogenase/reductase protein superfamily. Accordingly, the family fold and the specific Thr, Tyr, and Lys catalytic triad are well conserved in the viral enzyme.     

AcMNPVP35抑制HaSNPV诱导的Tn-Hi5细胞凋亡

苜蓿银纹夜蛾核多角体病毒(Autographa californica multicapsid nuclear polyhedrosis virus,AcMNPV)能够抑制棉铃虫核多角体病毒(Helicoverpa armigera Nucleopoly hedrovirus,HaSNPV)诱导的Tn Hi5 细胞凋亡,并能辅助HaSNPV在Tn Hi5细胞中复制,产生具有感染能力的子代病毒。瞬时表达实验证明,在Tn Hi5细胞中,p35具有明显抑制凋亡的能力,但是不能辅助HaSNPV在Tn Hi5细胞中的复制;进一步构建超表达p35 的重组病毒:vHap35,发现vHap35能够抑制Tn Hi5细胞凋亡,但是不能产生具有感染力的病毒粒子。电镜观察发现感染重组病毒的部分细胞中存在单粒包埋的病毒粒子(ODV)。