Microinjected interferon does not promote an antiviral response in Hela cells

Human fibroblast interferon (Hu IFN beta) was directly introduced with glass micropipets into the cytoplasm of Hela cells. Such an injection of more than 10(4) molecules per cell failed to induce any antiviral state when challenged with vesicular stomatitis virus (VSV). These findings are discussed in relation to the possible role of internalization in the mechanism of antiviral action of interferon.     

Microinjection of anticlathrin antibodies into fibroblasts does not interfere with the receptor-mediated endocytosis of alpha2-macroglobulin

Affinity-purified antibodies prepared against the major coat protein of brain coated vesicles, clathrin, were microinjected into cultured fibroblasts, and their intracellular distribution was followed by immunofluorescence microscopy and ultrastructural immunocytochemistry. Microinjected anticlathrin antibodies were concentrated on coated regions of the plasma membrane and the GERL apparatus. When an excess of anticlathrin antibodies was injected into the cytosol, coated pits on the plasma membrane were covered by anticlathrin antibody but still functioned to cluster an internalize alpha2-macroglobulin. These results are discussed in terms of the role of clathrin in the pathway of receptor-mediated endocytosis. Our data indicate that in cultured fibroblasts coated pits are stable elements permanently attached to the plasma membrane.     

Microinjection into Acanthamoeba castellanii of monoclonal antibodies to myosin-II slows but does not stop cell locomotion

To study the in vivo role of myosin-II in Acanthamoeba castellanii, motile cells were microinjected with monoclonal antibodies raised against the myosin-II heavy chain. All injected cells underwent a transient shock response. It was found that although injection of buffer alone or of an endogenous Acanthamoeba protein decreased the motility of injected cells from 7 microns/min to approximately 3 microns/min, injection of monoclonal antibodies specific for myosin-II decreased motility further to approximately 0.8 micron/min. This effect was seen whether or not the monoclonal antibody to myosin-II inhibited the actomyosin-II MgATPase activity in vitro. Levels of antibody far in excess of endogenous myosin-II concentrations could not completely block amoeboid movement. The morphology of moving antimyosin-II-injected cells was unusual, suggesting a greater defect in the ability to retract the trailing edge of the cell rather than to extend the leading edge. Endosomes frequently disappeared from injected cells, and although buffer-injected cells rapidly recovered visible endosomes (50% recovery at 5 min), endosomes were not seen in antimyosin-II-injected cells until, on the average, approximately 50 min after injection. Injection of a nonspecific antibody or of a nonspecific exogenous protein (ovalbumin) also decreased the mobility of the injected cells beyond that of buffer-injected cells (to approximately 1 micron/min). These cells tended to recover endosomes more rapidly (approximately 25 min) than cells injected with antimyosin-II monoclonal antibodies. The inability of antibodies to myosin-II to inhibit completely any of the movements studied suggests that although myosin-II probably plays a role in these motilities, the cell either routinely uses or can draw upon another cytoplasmic motor to maintain locomotion, organelle movement, contractile vacuole activity, and endocytosis.     

Microinjection of antibodies to centromere protein CENP-A arrests cells in interphase but does not prevent mitosis

Centromere protein CENP-A is a histone H3-like protein associated specifically with the centromere and represents one of the human autoantigens identified by sera taken from patients with the CREST variant of progressive systemic sclerosis. Injection of whole human autoimmune serum to the centromere into interphase cells disrupts some mitotic events. It has been assumed that this effect is due to CENP-E and CENP-C autoantigens, because of the effects of injecting monospecific sera to those proteins into culture cells. Here we have used an antibody raised against an N-terminal peptide of the human autoantigen CENP-A to determine its function in mitosis and during cell cycle progression. Affinity-purified anti-CENP-A antibodies injected into the nucleus during the early replication stages of the cell cycle caused cells to arrest in interphase before mitosis. These cells showed highly condensed small nuclei, a granular cytoplasm and loss of their division capability. On the other hand, microinjection of nocodazole-blocked HeLa cells in mitosis resulted in the typical punctate staining pattern of CENP-A for centromeres during different stages of mitosis and apparently normal cell division. This was corroborated by time-lapse imaging microscopy analysis of mid-interphase-injected cells, revealing that they undergo mitosis and divide properly. However, a significant delay throughout the progression of mitotic stages was observed. These results suggest that CENP-A is involved predominantly in an essential interphase event at the centromere before mitosis. This may include chromatin assembly at the kinetochore coordinate with late replication of satellite DNA to form an active centromere. Received: 3 August 1998 / Accepted: 18 September 1998     

Highly pathogenic avian influenza viruses do not inhibit interferon synthesis in infected chickens but can override the interferon-induced antiviral state

From infection studies with cultured chicken cells and experimental mammalian hosts, it is well known that influenza viruses use the nonstructural protein 1 (NS1) to suppress the synthesis of interferon (IFN). However, our current knowledge regarding the in vivo role of virus-encoded NS1 in chickens is much more limited. Here, we report that highly pathogenic avian influenza viruses of subtypes H5N1 and H7N7 lacking fully functional NS1 genes were attenuated in 5-week-old chickens. Surprisingly, in diseased birds infected with NS1 mutants, the IFN levels were not higher than in diseased birds infected with wild-type virus, suggesting that NS1 cannot suppress IFN gene expression in at least one cell population of infected chickens that produces large amounts of the cytokine in vivo. To address the question of why influenza viruses are highly pathogenic in chickens although they strongly activate the innate immune system, we determined whether recombinant chicken alpha interferon (IFN-α) can inhibit the growth of highly pathogenic avian influenza viruses in cultured chicken cells and whether it can ameliorate virus-induced disease in 5-week-old birds. We found that IFN treatment failed to confer substantial protection against challenge with highly pathogenic viruses, although it was effective against viruses with low pathogenic potential. Taken together, our data demonstrate that preventing the synthesis of IFN is not the primary role of the viral NS1 protein during infection of chickens. Our results further suggest that virus-induced IFN does not contribute substantially to resistance of chickens against highly pathogenic influenza viruses.     

Mouse peritoneal cells confer an antiviral state on mouse cell monolayers: role of interferon.

Vesicular stomatitis virus and encephalomyocarditis virus do not multiply in the majority of peritoneal macrophages freshly explanted from 4- to 8-week-old male or female mice. However, when peritoneal macrophages were cultivated in vitro for 3 to 5 days, these cells became permissive for both viruses. The loss of antiviral state in "aged" macrophages paralleled a significant decrease in the intracellular levels of (2'-5')oligo-adenylate synthetase activity. Although biologically active interferon was not detected in the nutrient medium of macrophage cultures, freshly harvested peritoneal cells could confer an antiviral state on monolayer cultures of mouse cells (aged macrophages, embryonic fibroblasts, and L cells) but not on heterologous chicken embryo, rabbit kidney, or human cells infected with vesicular stomatitis virus or encephalomyocarditis virus. The conferred antiviral state required at least 7 h to develop in target cells and was totally inhibited by the presence of antibody to mouse interferon alpha/beta but not to interferon gamma in the cocultures. Heterologous guinea pig and rabbit peritoneal cells could not transfer an antiviral state to target mouse cells. Donor peritoneal cells from mice preinjected with antibody to interferon alpha/beta could not transfer an antiviral state to target mouse cells. This ensemble of results indicating that freshly harvested peritoneal cells transfer interferon (which is responsible for inducing an antiviral state in susceptible mouse target cells) adds further experimental evidence that interferon is spontaneously expressed in normal mice and plays an important role in maintaining some host cells in an antiviral state.     

Deoxycytidine is salvaged not only into DNA but also into phospholipid precursors. II. Ara-C does not inhibit the later process in lymphoid cells

dCTP formed from exogenous deoxycytidine via the salvage pathways was previously shown to serve deoxyliponucleotide synthesis in lymphocytes (Spasokukotskaja et al, Biochem. Biophys. Res. Commun. (1988) 155, 923-929) and now in lymphoma cells. After treatment with 1-beta-D-arabino-furanosylcytosine (ara-C), much more araCTP as well as araCDP-choline was formed in lymphoma cells than in lymphocytes explaining the high sensitivity of lymphoma cells to this drug. Ara-C did not inhibit labeling of 5-3H-dCDP-choline from exogenous 5-3H-deoxycytidine while inhibiting DNA synthesis. Excess of exogenous ribocytidine diminished labeling of araCDP-choline, without any effect on dCDP-choline. These data suggest that araCDP-choline and dCDP-choline were synthesized from separate pools in these cells.     

Mouse interferon produced in vivo does not inhibit the development of preimplantation mouse embryos

Mouse embryos were cultured in vitro in medium with serum containing interferon which had been induced in vivo by intravenous administration of polyinosine-polycytidylic acid. Two-cell and blastocyst-stage embryos were incubated for 72 and 24 h respectively before embryo transfer, or fixation to determine cell number. Further, blastocysts were outgrown on coverslips in embryo culture medium with fetal calf serum and interferon. Expression of an intermediate filament protein (Mr 55 000) in blastocyst outgrowths was examined with a monoclonal antibody. Embryos appeared morphologically normal and after treatment the mean cell number did not differ from that of controls. Implantation was unaffected by any of the treatments, but culture before transfer in medium containing mouse serum reduced the number of normal fetuses recovered on Day 14 of gestation compared to those cultured in medium without serum. Exposure to interferon did not modify the expression of filaments in the outgrown blastocyst. It is therefore unlikely that interferon induced by viral infection during pregnancy is responsible for preimplantation embryonic loss.     

Adenovirus infection reverses the antiviral state induced by human interferon

HeLa cells treated with human lymphoblastoid interferon do not synthesize poliovirus proteins. The antiviral state against poliovirus is reversed if cells are previously infected with adenovirus type 5. A late gene product seems to be involved in this reversion, since no effect is observed at early stages of infection or in the presence of aphidicolin.     

Microinjection of villin into cultured cells induces rapid and long- lasting changes in cell morphology but does not inhibit cytokinesis, cell motility, or membrane ruffling

Villin, a Ca2(+)-regulated F-actin bundling, severing, capping, and nucleating protein, is a major component of the core of microvilli of the intestinal brush border. Its actin binding properties, tissue specificity, and expression during cell differentiation suggest that it might be involved in the organization of the microfilaments in intestinal epithelial cells to form a brush border. Recently, Friederich et al., (Friederich, E., C. Huet, M. Arpin, and D. Louvard. 1989. Cell. 59:461-475) showed that villin expression in transiently transfected fibroblasts resulted in the loss of stress fibers and the appearance of large cell surface microvilli on some cells. Here, we describe the effect of villin microinjection into cells that normally lack this protein, which has allowed us to examine the immediate and long-term effects of introducing different concentrations of villin on microfilament organization and function. Microinjected cells rapidly lost their stress fibers and the actin was reorganized into abundant villin containing cortical structures, including microspikes and, in about half the cells, large surface microvilli. This change in actin organization persisted in cells for at least 24 h, during which time they had gone through two or three cell divisions. Microinjection of villin core, that lacks the bundling activity of villin but retains all the Ca2(+)-dependent properties, disrupted the stress fiber system and had no effect on cell surface morphology. Thus, the Ca2(+)-dependent activities of villin are responsible for stress fiber disruption, and the generation of cell surface structures is a consequence of its bundling activity. Microinjection of villin led to the reorganization of myosin, tropomyosin, and alpha-actinin, proteins normally associated with stress fibers, whereas both fimbrin and ezrin, which are also components of microvillar core filaments, were readily recruited into the induced surface structures. Vinculin was also redistributed from its normal location in focal adhesions. Despite these changes in the actin cytoskeleton, cells were able to divide and undergo cytokinesis, move, spread on a substratum, and ruffle. Thus, we show that a single microfilament-associated protein can reorganize the entire microfilament structure of a cell, without interfering with general microfilament-based functions like cytokinesis, cell locomotion, and membrane ruffling.     

Dissociation of protein kinase activity and the induction of the antiviral state in a cell line responsive to the antiviral effects of interferon

Interferons or oxidized glutathione were found to induce double-stranded RNA-dependent protein kinase activity in mouse L cells that phosphorylates the α subunit of eukaryotic peptide initiation factor 2. A mixture of leukocyte/fibroblast interferons as well as immune interferon induced the protein kinase and also suppressed virus replication in the L cells. Oxidized glutathione was equally effective in inducing protein kinase activity, but it did not induce an antiviral state in these cells. The data suggest that a simple cause and effect relationship does not exist between protein kinase induction and the establishment of the antiviral state in a cell that is responsive to the antiviral effects of interferon.