Ecological dynamics of the bivalve-killing dinoflagellate Heterocapsa circularisquama and its infectious viruses in different locations of western Japan

We studied the ecological relationships between the bloom-forming dinoflagellate Heterocapsa circularisquama and its infectious viruses in field surveys conducted in western Japan. The occurrence of H. circularisquama blooms in Imari Bay during 2002 and in Ago Bay during 2002 and 2004 was accompanied by specific increase in abundance of viruses lytic to H. circularisquama. Using northern dot-blot analysis, approximately 96% of the clonal virus isolates collected in the field surveys positively reacted with a molecular probe specific for HcRNAV (H. circularisquama RNA virus); hence, viral impacts on H. circularisquama population observed in these field surveys are considered largely due to HcRNAV and/or its closely related viruses. The dynamics of type UA viruses and type CY viruses having complementary host ranges to H. circularisquama clones were different in each survey and considered to reflect fluctuations in abundance of their suitable host cells in situ. The dynamics of H. circularisquama and its viruses in Ago Bay from 2002 to 2004 suggests the concentration of HcRNAV in the sediment prior to the host's blooming season is a significant factor in determining the size and length of the H. circularisquama blooms. These results support the hypothesis that HcRNAV infection is one of the significant factors affecting the population dynamics of H. circularisquama in both quantity (biomass) and quality (clonal composition).     

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.     

Interferon inhibits the in vitro accumulation of virus specific RNA in nuclei isolated from SV40 infected cells.

Nuclei prepared from Vero cells infected with SV40 in the presence of cytosine arabinoside synthesize [³H]RNA in vitro, a small proportion of which is virus specific. This synthesis is sensitive to low doses of α-amanitin. The accumulation of viral RNA is blocked in nuclei prepared from cells pretreated with interferon prior to infection and incubated in a reaction mixture containing 100 mM KCl. This resembles the situation found in intact cells. However, when the reaction mixture contains 300 mM KCl, the interferon-induced block in viral RNA accumulation is reversed.     

Diverse responses of the bivalve-killing dinoflagellate Heterocapsa circularisquama to infection by a single-stranded RNA virus

Viruses are believed to be significant pathogens for phytoplankton. Usually, they infect a single algal species, and often their infection is highly strain specific. However, the detailed molecular background of the strain specificity and its ecological significance have not been sufficiently understood. Here, we investigated the temporal changes in viral RNA accumulation and virus-induced cell lysis using a bloom-forming dinoflagellate Heterocapsa circularisquama and its single-stranded RNA virus, HcRNAV. We observed at least three host response patterns to virus inoculation: sensitive, resistant, and delayed lysis. In the sensitive response, the host cell culture was permissive for viral RNA replication and apparent cell lysis was observed; in contrast, resistant cell culture was nonpermissive for viral RNA replication and not lysed. In the delayed-lysis response, although viral RNA replication occurred, virus-induced cell lysis was faint and remarkably delayed. In addition, the number of infectious virus particles released to the culture supernatant at 12 days postinoculation was comparable to that of the sensitive strain. By further analysis, a few strains were characterized as variants of the delayed-lysis strain. These observations indicate that the response of H. circularisquama to HcRNAV infection is highly diverse.     

Analysis of Nuclear Accumulation of Influenza NP Antigen in von Magnus Virus-Infected Cells

When 1–5C-4 cells were infected with von Magnus virus derived from influenza A/RI/5⁺ virus by four successive undiluted passages in chick embryos, virus-specific proteins were synthesized but production of infectious virus was inhibited. In these cells the synthesis of viral RNA was suppressed and the nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to standard virus-infected cells in which the antigen was distributed throughout the whole cell. The intracellular location and migration of NP were determined by isotope labeling and sucrose gradient centrifugation of subcellular fractions. In standard virus-infected cells NP polypeptide was present predominantly in the cytoplasm in the form of viral ribonucleoprotein (RNP) and intranuclear RNP was detected in reduced amounts. In contrast, in von Magnus virus-infected cells NP polypeptide was present predominantly in the nucleus in a nonassembled, soluble form and the amount of cytoplasmic RNP was considerably reduced. After short-pulse labeling NP was detected exclusively in the cytoplasm in a soluble form and after a chase a large proportion of such soluble NP was seen in the nucleus. It is suggested that a large proportion of the NP synthesized in von Magnus virus-infected cells is not assembled into cytoplasmic RNP because of the lack of available RNA and the NP migrated into the nucleus and remained there.     

Dinoflagellates,diatoms, and their viruses

Since the first discovery of the very high virus abundance in marine environments, a number of researchers were fascinated with the world of "marine viruses", which had previously been mostly overlooked in studies on marine ecosystems. In the present paper, the possible role of viruses infecting marine eukaryotic microalgae is enlightened, especially summarizing the most up-to-the-minute information of marine viruses infecting bloom-forming dinoflagellates and diatoms. To author's knowledge, approximately 40 viruses infecting marine eukaryotic algae have been isolated and characterized to different extents. Among them, a double-stranded DNA (dsDNA) virus "HcV" and a single-stranded RNA (ssRNA) virus "HcRNAV" are the only dinoflagellate-infecting (lytic) viruses that were made into culture; their hosts are a bivalve-killing dinoflagellate Heterocapsa circularisquama. In this article, ecological relationship between H. circularisquama and its viruses is focused. On the other hand, several diatom-infecting viruses were recently isolated and partially characterized; among them, one is infectious to a pen-shaped bloom-forming diatom species Rhizosolenia setigera; some viruses are infectious to genus Chaetoceros which is one of the most abundant and diverse diatom group. Although the ecological relationships between diatoms and their viruses have not been sufficiently elucidated, viral infection is considered to be one of the significant factors affecting dynamics of diatoms in nature. Besides, both the dinoflagellate-infecting viruses and diatom-infecting viruses are so unique from the viewpoint of virus taxonomy; they are remarkably different from any other viruses ever reported. Studies on these viruses lead to an idea that ocean may be a treasury of novel viruses equipped with fascinating functions and ecological roles.     

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.     

Viral genome integration with nuclear DNA in chronic viral infection]

A possibility of the integration of tick-borne encephalitis virus RNA in the host cell genome is studied under the condition of long-term chronic infection of HEp-2 cells. Molecular hybridization of viral RNA, labelled with 3H-uridine, and of DNA from intact and chronically infected cells was performed in 50% formamide with 0,4 M NaCl with subsequent investigation of the hybridization product by means of equilibrium centrifugation in CsSO4. 0,3 and 0,5 mg of DNA were used in hybridization experiments. It is shown that RNA obviously forms hybrids with DNA from chronically infected cells, whose density varied from 1,55 to 1,46 g/ml, depending on the proportion of RNA and DNA in the hybrids. When the amount of DNA increased, the number of DNA-RNA hybrids increased as well. It is concluded that in nuclear DNA of cells chronically infected with tick-borne encephalitis virus, there are DNA sites, homologous to viral RNA, which are absent in intact cells.     

The use of immunosorption for the analysis of influenza virus RNA in intracellular viral ribonucleoproteins

Protein A-containing formaldehyde-fixed S. aureus (strain Cowan) was incubated with an antiviral serum or with a monospecific serum against NP protein, washed, and used as immunosorbent in order to isolate viral ribonucleoproteins (nucleocapsids) containing intact viral RNA from the extracts of influenza virus infected [3H]-uridine-labelled cells.     

Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line.

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.     

Membrane-Associated Replication Complex in Arbovirus Infection

Cytoplasmic extracts of chicken embryo fibroblast cells infected with Semliki Forest virus were subjected to isopycnic centrifugation in discontinuous sucrose gradients. Seven distinct bands were usually formed. The four upper bands contained predominantly smooth membranes and the lowest band was enriched in rough endoplasmic reticulum. One fraction (fraction 5), banding at a density of 1.16 g/cm(3), was found to be heavily enriched in pulse-labeled ribonucleic acid (RNA), viral RNA polymerase, and viral RNA forms associated with RNA replication. Thus, fraction 5 evidently contained a membrane-associated viral replication complex of a type previously defined in picornavirus infections. Fraction 5 was also consistently enriched with unique membranous structures previously observed in intact cells as type 1 cytopathic vacuoles (CPV-1). When the CPV-1 in fraction 5 were isolated from cells briefly incubated with (3)H-uridine and (3)H-adenosine prior to cell disruption, a large proportion was found to be labeled by high-resolution autoradiography. Thus, ultrastructural, biochemical, and biological evidence were all consistent with the interpretation that the CPV-1 membranes represent a significant element of the viral replication complex.     

Imaging and characterizing influenza A virus mRNA transport in living cells

The mechanisms of influenza A virus mRNA intracellular transport are still not clearly understood. Here, we visualized the distribution and transport of influenza A virus mRNA in living cells using molecular beacon (MB) technology. Confocal-FRAP measurements determined that the transport of influenza A virus intronless mRNA, in both nucleus and cytoplasm, was energy dependent, being similar to that of Poly(A)⁺ RNA. Drug inhibition studies in living cells revealed that the export of influenza A virus mRNA is independent of the CRM1 pathway, while the function of RNA polymerase II (RNAP-II) may be needed. In addition, viral NS1 protein and cellular TAP protein were found associated with influenza A virus mRNA in the cell nucleus. These findings characterize influenza A virus mRNA transport in living cells and suggest that influenza A virus mRNA may be exported from the nucleus by the cellular TAP/p15 pathway with NS1 protein and RNAP-II participation.     

Nuclear localization of flavivirus RNA synthesis in infected cells

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.     

Maturation Defects in Temperature-sensitive Mutants of Sindbis Virus

Temperature-sensitive mutants of Sindbis virus, which synthesize viral ribonucleic acid (RNA) but not mature virus at the nonpermissible temperature, were selected for the study of viral maturation. Of these, three mutants which complement each other genetically were used. Two major proteins, the nucleocapsid and membrane proteins, located, respectively, in the viral nucleoid and membrane, were found in intact virions. In cells infected with wild-type Sindbis virus, four distinct types of viral RNA with sedimentation coefficients of 40S, 26S, 20S, and 15S were detected in constant distribution. The 20S RNA was ribonuclease-resistant, whereas the other types were ribonuclease-sensitive. The 40S RNA, identical to that obtained from the virion, was found associated with nucleocapsid protein as a subviral particle, which was assumed to be the nucleoid. Viral materials from cells infected with the mutants under nonpermissive conditions were compared with those from cells infected with wild-type virus, in terms of (i) the distribution of the different types of RNA, (ii) the association of infectious viral RNA into subviral particles, and (iii) the ability of infected cells to hemadsorb goose erythrocytes. According to these criteria, each of the three mutants demonstrated different maturation defects. Defective nucleocapsid proteins and membrane proteins may each account for one of the above mutants. The thrid mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus.     

Development of antigens in human cells infected with Simian virus 40

Bissett, Marjorie L. (University of Michigan, Ann Arbor), and Francis E. Payne. Development of antigens in human cells infected with simian virus 40. J. Bacteriol. 91:743-749. 1966.-An explanation for the apparent infrequency with which human cells transform in response to exposure to simian virus 40 (SV40) was sought by following the development of virus-induced antigens in human euploid cells, strain CR. For about 8 weeks after exposure to a high multiplicity of SV40, only a small proportion of the cells produced tumor (T) or viral (V) antigen detected by immunofluorescence. Double-tracer staining techniques revealed that the development of T and V antigen in about 1% of the CR cells resembled that in green monkey kidney cells, strain BS-C-1, in which SV40 replicates and destroys all the cells. T antigen was detected before V antigen; both antigens were detected in the nucleus, but only V antigen appeared later in the cytoplasm. All intact cells that contained V antigen also contained T antigen. Infected CR cell cultures, before and after transformation or when in "crisis," contained only 0.1 to 1.0% of cells with both V and T antigen. Some CR cells contained only T antigen, and by 8 days after exposure to virus these cells were present as loose foci associated with an occasional cell containing V antigen. The proportion of CR cells with only T antigen increased from about 1% during the first 4 weeks to 8% at 7 weeks, and to nearly 100% at 11 weeks, when essentially all of the cells were epithelioid. Foci of epithelioid cells were first recognized in the 9th week. It was concluded that those CR cells that contained T antigen at any given time represented (i) a few cells that subsequently produced V antigen and lysed, and (ii) a progressively increasing population that produced only T antigen. If the latter population, in whole or in part, gave rise to the epithelioid transformed cells, then its initial size could account, at least in part, for the apparent infrequency with which human cells transform in response to SV40.