Laser cytophotometric detection of variations in spatial distribution of concanavalin-A cell surface receptors following viral infection.

Human cells in culture (H.Ep. 2) were infected with herpes simplex virus type 2 at a multiplicity of infection (m.o.i.) of 3 for 3, 18 and 36 hr. Spatial redistribution of concanavalin (Con-A) cell surface receptor sites was detected by an indirect double-antibody (peroxidase-conjugated goat anti-rabbit to rabbit anti-Con-A) immunoenzymatic staining reaction which rendered the Con-A sites visible to light microscopy and to flow laser cytophotometry. Significant spatial redistribution of cell surface Con-A receptor sites occurred within the first 3 hr after infection with herpes simplex virus type 2. The infected cells must be in the presence of Con-A for the redistribution to occur, and significant redistribution occurs only at 37 degrees C. Fixation of infected cells prior to application of Con-A prevented this spatial redistribution of Con-A receptor sites.     

Ferrocyanide enhancement of concanavalin A-ferritin and cationized ferritin staining blood cell surface glycoconjugates

Synopsis Ferrocyanide was used to enhance cationized ferritin and concanavalin A-ferritin (Con A-ferritin) staining of surface glycoconjugates of peripheral blood and bone marrow cells from rabbits and humans. The glutaraldehyde-fixed cells were stained with Con A-ferritin or cationized ferritin and then exposed to a ferrocyanide solution. The resulting cuboidal and irregular stain deposits averaged 50 nm in diameter when viewed with the transmission (TEM) and scanning electron microscope (SEM). Rabbit blood cells demonstrated more Con A binding sites than human blood cells and the decrease in binding sites observed with maturation of human granulocytic and erythrocytic cells was not evident in rabbit cells. Differences in binding of cationized ferritin to rabbit and human cell surfaces were less prominent than that observed for Con A. These results extend previous studies of blood cell surface glycoconjugates and demonstrate that ferrocyanide enhancement significantly facilitates SEM evaluation of Con A-ferritin and cationized ferritin bound to cell surfaces.     

Human immunodeficiency virus type 1 and influenza virus exit via different membrane microdomains

Directed release of human immunodeficiency virus type 1 (HIV-1) into the cleft of the virological synapse that can form between infected and uninfected T cells, for example, in lymph nodes, is thought to contribute to the systemic spread of this virus. In contrast, influenza virus, which causes local infections, is shed into the airways of the respiratory tract from free surfaces of epithelial cells. We now demonstrate that such differential release of HIV-1 and influenza virus is paralleled, at the subcellular level, by viral assembly at different microsegments of the plasma membrane of HeLa cells. HIV-1, but not influenza virus, buds through microdomains containing the tetraspanins CD9 and CD63. Consequently, the anti-CD9 antibody K41, which redistributes its antigen and also other tetraspanins to cell-cell adhesion sites, interferes with HIV-1 but not with influenza virus release. Altogether, these data strongly suggest that the bimodal egress of these two pathogenic viruses, like their entry into target cells, is guided by specific sets of host cell proteins.     

Characterization of a porcine kidney cell line resistant to influenza virus infection.

A mutant cell line of porcine kidney cells that resists the cytopathic effect of influenza virus has been obtained and characterized. These cells, designated ESK-R, were originally obtained by prolonged cultivation of cells surviving influenza B/Kanagawa/73 virus infection. No infectious virus was recovered from ESK-R cells, and no evidence for the presence of virus antigens in the cells was demonstrated by immunofluorescent staining. ESK-R cells also showed a distinct resistance to various other strains of both types A and B influenza viruses. The growth of mumps, Sendai, or Newcastle disease virus was considerably restricted, but the cell line normally supported the replication of vesicular stomatitis virus. ESK-R cells were found to lack specific receptors for influenza virus as determined by fluorescence-activated cell sorter analyses. The membrane barrier of ESK-R cells was successfully overcome by nonspecific endocytosis of calcium-coprecipitated virus particles followed by production of an appreciable amount of progeny virus.     

Comparative ultrastructural study of four reticuloendothelias viruses.

The morphology and development of four members of the reticuloendotheliosis virus group were studied by transmission electron microscopy. Virions of duck spleen necrosis virus, duck infectious anemia virus, chicken syncytial virus, and reticuloendotheliosis virus strain T are sperical with a diameter of approximately 110 nm. They are covered with surface projections about 6 nm long and 10 nm in diameter. The center-to-center distance of surface projections is about 14 nm. The budding virions contain crescent-shaped electron-dense cores 73 nm in diameter with electron-lucent centers. After release of the virions the cores apparently become condensed to 67 nm in diameter. Virions were found budding at the plasma membrane and into smooth-walled, intracytoplasmic vesicles of productively infected cells. The distribution of budding reticuloendotheliosis viruses on cells appeared random over the cell surface, and occasionally aberrant multiple forms of budding virions were observed. The virions appear to resemble mammalian leukemia and sarcoma viruses more closely than avian leukosis-sarcoma viruses.     

Mathematical model of influenza A virus production in large-scale microcarrier culture

A mathematical model that describes the replication of influenza A virus in animal cells in large-scale microcarrier culture is presented. The virus is produced in a two-step process, which begins with the growth of adherent Madin-Darby canine kidney (MDCK) cells. After several washing steps serum-free virus maintenance medium is added, and the cells are infected with equine influenza virus (A/Equi 2 (H3N8), Newmarket 1/93). A time-delayed model is considered that has three state variables: the number of uninfected cells, infected cells, and free virus particles. It is assumed that uninfected cells adsorb the virus added at the time of infection. The infection rate is proportional to the number of uninfected cells and free virions. Depending on multiplicity of infection (MOI), not necessarily all cells are infected by this first step leading to the production of free virions. Newly produced viruses can infect the remaining uninfected cells in a chain reaction. To follow the time course of virus replication, infected cells were stained with fluorescent antibodies. Quantitation of influenza viruses by a hemagglutination assay (HA) enabled the estimation of the total number of new virions produced, which is relevant for the production of inactivated influenza vaccines. It takes about 4-6 h before visibly infected cells can be identified on the microcarriers followed by a strong increase in HA titers after 15-16 h in the medium. Maximum virus yield Vmax was about 1x10(10) virions/mL (2.4 log HA units/100 microL), which corresponds to a burst size ratio of about 18,755 virus particles produced per cell. The model tracks the time course of uninfected and infected cells as well as virus production. It suggests that small variations (<10%) in initial values and specific rates do not have a significant influence on Vmax. The main parameters relevant for the optimization of virus antigen yields are specific virus replication rate and specific cell death rate due to infection. Simulation studies indicate that a mathematical model that neglects the delay between virus infection and the release of new virions gives similar results with respect to overall virus dynamics compared with a time delayed model.     

Dual Function of CD81 in Influenza Virus Uncoating and Budding

As an obligatory pathogen, influenza virus co-opts host cell machinery to harbor infection and to produce progeny viruses. In order to characterize the virus-host cell interactions, several genome-wide siRNA screens and proteomic analyses have been performed recently to identify host factors involved in influenza virus infection. CD81 has emerged as one of the top candidates in two siRNA screens and one proteomic study. The exact role played by CD81 in influenza infection, however, has not been elucidated thus far. In this work, we examined the effect of CD81 depletion on the major steps of the influenza infection. We found that CD81 primarily affected virus infection at two stages: viral uncoating during entry and virus budding. CD81 marked a specific endosomal population and about half of the fused influenza virus particles underwent fusion within the CD81-positive endosomes. Depletion of CD81 resulted in a substantial defect in viral fusion and infection. During virus assembly, CD81 was recruited to virus budding site on the plasma membrane, and in particular, to specific sub-viral locations. For spherical and slightly elongated influenza virus, CD81 was localized at both the growing tip and the budding neck of the progeny viruses. CD81 knockdown led to a budding defect and resulted in elongated budding virions with a higher propensity to remain attached to the plasma membrane. Progeny virus production was markedly reduced in CD81-knockdown cells even when the uncoating defect was compensated. In filamentous virus, CD81 was distributed at multiple sites along the viral filament. Taken together, these results demonstrate important roles of CD81 in both entry and budding stages of the influenza infection cycle.     

Tamiflu-resistant but HA-mediated cell-to-cell transmission through apical membranes of cell-associated influenza viruses

The infection of viruses to a neighboring cell is considered to be beneficial in terms of evasion from host anti-virus defense systems. There are two pathways for viral infection to "right next door": one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission. Influenza viruses are believed to be transmitted as cell-free virus from infected cells to uninfected cells. Here, we demonstrated that influenza virus can utilize cell-to-cell transmission pathway through apical membranes, by handover of virions on the surface of an infected cell to adjacent host cells. Live cell imaging techniques showed that a recombinant influenza virus, in which the neuraminidase gene was replaced with the green fluorescence protein gene, spreads from an infected cell to adjacent cells forming infected cell clusters. This type of virus spreading requires HA activation by protease treatment. The cell-to-cell transmission was also blocked by amantadine, which inhibits the acidification of endosomes required for uncoating of influenza virus particles in endosomes, indicating that functional hemagglutinin and endosome acidification by M2 ion channel were essential for the cell-to-cell influenza virus transmission. Furthermore, in the cell-to-cell transmission of influenza virus, progeny virions could remain associated with the surface of infected cell even after budding, for the progeny virions to be passed on to adjacent uninfected cells. The evidence that cell-to-cell transmission occurs in influenza virus lead to the caution that local infection proceeds even when treated with neuraminidase inhibitors.     

Initial attachment of baby hamster kidney cells to an epoxy substratum. Ultrastructural analysis

In the presence of serum-containing medium, BHK cells attached and spread during a 1-h period onto a 3-5 nm thick serum layer absorbed on the substratum surface. The closest approach of the plasma membrane to the serum layer was observed to be about 9nm, which was determined by tilting the sectioned cells in a goniometer holder. Bundles of microfilaments or other cytoplasmic specializations were not observed in association with the regions of close contact. However, in the space between the plasma membrane and the adsorbed serum layer, a diffusely stained material could be visualized after fixation/staining by the tannic acid-glutaraldehyde technique. This technique also permitted increased clarity of visualization of trilaminar appearance of the plasma membrane. The distribution and mobility of anionic sites on the surfaces of attached and spreading cells was determined by labeling with polycationic ferritin. We observed movement of polycationic ferritin into large clusters on the cell surface, collapse of cell surface microextensions, and endocytosis, all of which were similar to our previous findings utilizing cells in suspension. However, the absolute amount of ferritin bound to the upper cell surface was less than that previously observed when suspended cells were put under similar labeling conditions. Also, polycationic ferritin did not appear to penetrate between the lower cell surface and the substratum.     

Site-specific maturation of enveloped viruses in L cells treated with cytochalasin B

Treatment of infected L cells with 10 micrograms/ml cytochalasin B (CB) was found to promote a rapid relocalization of viral glycoproteins on the cell surface. Whereas the vesicular stomatitis virus G protein and the influenza virus hemagglutinin were uniformly distributed on the surface of untreated cells, in CB-treated cells, they were strikingly concentrated at cell extremities in the regions of clustered blebs. Glycoprotein concentration at cell extremities was accompanied by preferential maturation of virus particles from the same sites; both vesicular stomatitis and influenza viruses budded predominantly from the vicinity of clustered blebs. This effect of CB was completely reversible. Removal of CB from the cell growth medium resulted in a return of viral glycoproteins to the uniform distribution characteristic of untreated cells and to uniform virus budding. The results of this study are interpreted in terms of a model that suggests that preferential budding of viruses from the regions of bleb clusters is due to the concentration of viral glycoproteins at these sites.     

An Anti-Influenza Virus Antibody Inhibits Viral Infection by Reducing Nucleus Entry of Influenza Nucleoprotein

To date, four main mechanisms mediating inhibition of influenza infection by anti-hemagglutinin antibodies have been reported. Anti-globular-head-domain antibodies block either influenza virus receptor binding to the host cell or progeny virion release from the host cell. Anti-stem region antibodies hinder the membrane fusion process or induce antibody-dependent cytotoxicity to infected cells. In this study we identified a human monoclonal IgG₁ antibody (CT302), which does not inhibit both the receptor binding and the membrane fusion process but efficiently reduced the nucleus entry of viral nucleoprotein suggesting a novel inhibition mechanism of viral infection by antibody. This antibody binds to the subtype-H3 hemagglutinin globular head domain of group-2 influenza viruses circulating throughout the population between 1997 and 2007.     

Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA

Primary cell cultures of African Green monkey kidney (AGMK) contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions. Influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded. When polarized primary AGMK cells were infected with recombinant SV40 viruses containing DNA coding for either an influenza virus H1 or H2 subtype hemagglutinin (HA), the HA proteins were preferentially expressed at the apical surface in a manner identical to that observed in influenza virus-infected cells. Thus, cellular mechanisms for sorting membrane glycoproteins recognize some structural feature of the HA glycoprotein itself, and other viral proteins are not necessary for this process.     

流感病毒感染诱导MDCK细胞凋亡的研究

用荧光染色、DNA凝胶电泳等方法检测了A型流感病毒株A1／京防86－1和B型流感病毒株B/沪防93－1诱导狗肾体代细胞(MDCKcells)的凋亡情况,并采用MTT法和流式细胞仪比较了这2株病毒对MMCK细胞的毒力和凋亡诱导能力水平。结果显示：病毒感染6h后,细胞DNA发生断裂,病毒感染12h后,可见明显的染色质凝聚;在一定范围内,细胞凋亡强度表现出明显的时间和剂量依赖关系;并且,A型流感病毒株的毒力和调亡诱导能力均强于B型流感病毒株。实验结果表明：流感病毒感染引起的细胞死亡主要是通过调亡实现的,毒力不同的流感病毒株诱导细胞调亡的能力不同。     

Entry of Vesicular Stomatitis Virus into L Cells

Early stages of the entry of vesicular stomatitis (VS) virus into L cells were followed by electron microscopy with the aid of ferritin antibody labeling. Cells which were infected at 0 C and incubated for 10 min at 37 C were reacted first with antiviral-antiferritin hybrid antibody and then with ferritin or fluorescein-labeled apoferritin. Extensive ferritin labeling of the cell surface was detected by both electron and fluorescence microscopy. The labeled regions of the cell surface were continuous with and indistinguishable from the rest of the host cell membrane, suggesting incorporation of viral antigens into the cell surface during viral penetration. Fusion of parental viral membrane with host cell membrane was further demonstrated by examining the localization of (3)H-labeled viral structural proteins in cells infected at 0 C and incubated for short periods at 37 C. Viral nucleoprotein was found in a soluble fraction of the cells which was derived primarily from the cytoplasm, whereas a particulate fraction from the cells was enriched in viral envelope proteins. Cytoplasmic membrane was isolated from these cells, and this membrane contained viral envelope proteins. These results suggest that penetration by VS virus occurs by fusion of the viral and cellular membranes followed by release of nucleo-protein into the cytoplasm.     

Delayed appearance of pseudotypes between vesicular stomatitis virus influenza virus during mixed infection of MDCK cells.

In intact Madin-Darby canine kidney (MDCK) cell monolayers, vesicular stomatitis virus (VSV) matures only at basolateral membranes beneath tight junctions, whereas influenza virus buds from apical cell surfaces. Early in the growth cycle, the viral glycoproteins are restricted to the membrane domain from which each virus buds. We report here that phenotypic mixing and formation of VSV pseudotypes occurred when influenza virus-infected MDCK cells were superinfected with VSV. Up to 75% of the infectious VSV particles from such experiments were neutralized by antiserum specific for influenza virus, and a smaller proportion (up to 3%) were resistant to neutralization with antiserum specific for VSV. The latter particles, which were neutralized by antiserum to influenza A/WSN virus, are designated as VSV(WSN) pseudotypes. During mixed infections, both wild-type viruses were detected 1 to 2 h before either phenotypically mixed VSV or VSV(WSN) pseudotypes. Coincident with the appearance of cytopathic effects in the monolayer, the yield of pseudotypes rose dramatically. In contrast, in doubly infected BHK-21 cells, which do not show polarity in virus maturation sites and are not connected by tight junctions, VSV(WSN) pseudotypes were detected as soon as VSV titers rose to the minimum levels which allowed detection of pseudotypes, and the proportion observed remained relatively constant at later times. Examination of thin sections of doubly infected MDCK monolayers revealed that polarity in maturation sites was preserved for both viruses until approximately 12 h after inoculation with influenza virus, when disruption of junctional complexes was evident. Even at later periods, the majority of each virus type was associated with its normal membrane domain, suggesting that the sorting mechanisms responsible for directing the glycoproteins of VSV and influenza virus to separate surface domains continue to operate in doubly infected MDCK cells. The time course of VSV(WSN) pseudotype formation and changes in virus maturation sites are compatible with progressive mixing of viral glycoproteins at either intracellular or plasma membranes of doubly infected cells.     

Resolution of diaminobenzidine for the detection of horseradish peroxidase on surfaces of cultured cells

The resolution of indirect immunoperoxidase methods for localizing antigens on the surface of plasma membranes of cultured cells was tested using dissociated monolayer cultures of ciliary ganglion neurons prelabeled with cationic ferritin. Clusters of ferritin were produced on the cell surface by warming the cells to 37 degrees C after the ferritin, rabbit anti-ferritin, and goat anti-rabbit immunoglobulin coupled to horseradish peroxidase had all been applied. Intense 3,3'-diaminobenzidine tetrahydrochloride (DAB) staining was limited to the regions immediately surrounding the ferritin clusters. The lateral spread of the DAB reaction product beyond the outer ferritin particles in each cluster averaged 54-81 nm in four experiments. A second type of increased density, coinciding with the thickness of the plasma membrane, was also seen. These stained plasma membranes extended 161-339 nm from the ferritin clusters.     

Pseudorabies virus envelope glycoproteins gp50 and gII are essential for virus penetration, but only gII is involved in membrane fusion.

To investigate the function of the envelope glycoproteins gp50 and gII of pseudorabies virus in the entry of the virus into cells, we used linker insertion mutagenesis to construct mutant viruses that are unable to express these proteins. In contrast to gD mutants of herpes simplex virus, gp50 mutants, isolated from complementing cells, were able to form plaques on noncomplementing cells. However, progeny virus released from these cells was noninfectious, although the virus was able to adsorb to cells. Thus, the virus requires gp50 to penetrate cells but does not require it in order to spread by cell fusion. This finding indicates that fusion of the virus envelope with the cell membrane is not identical to fusion of the cell membranes of infected and uninfected cells. In contrast to the gp50 mutants, the gII mutant was unable to produce plaques on noncomplementing cells. Examination by electron microscopy of cells infected by the gII mutant revealed that enveloped virus particles accumulated between the inner and outer nuclear membranes. Few noninfectious virus particles were released from the cell, and infected cells did not fuse with uninfected cells. These observations indicate that gII is involved in several membrane fusion events, such as (i) fusion of the viral envelope with the cell membrane during penetration, (ii) fusion of enveloped virus particles with the outer nuclear membrane during the release of nucleocapsids into the cytoplasm, and (iii) fusion of the cell membranes of infected and uninfected cells.     

Human ferritin: effects of antigen source and fixation on leucocyte staining by immunoperoxidase technique

The localization of ferritin was studied in peripheral blood cells and variously fixed tissues with the antibodies against ferritins isolated from human heart and spleen. The unlabelled antibody enzyme method (PAP) was used to detect the binding sites of antibodies. In peripheral blood cell smears both antisera gave rise to strong staining of polymorphonuclear (PMN) cell cytoplasm, whereas the monocytes stained relatively weakly. There were no staining differences between the two antisera. In human spleen sections the spleen ferritin antiserum stained the PMN cells and sinusoidal lining cells, whereas the heart ferritin antiserum stained only PMN cells. Neither of the two antisera stained monocytes in the spleen sections. This finding was observed in specimens fixed in Bouin's fixative, Baker's fixative and neutral formalin. However, the immunoreactivity of ferritin was totally destroyed by some other fixatives (Carnoy's fixative, formol sucrose and glutaraldehyde). These results suggest that ferritin is more readily released from monocytes than from PMN cells, and that mature spleen macrophages contain antigenic determinants of ferritin that are recognized only by anti-spleen ferritin antiserum.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae

Summary The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone wich was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.