Induction of interferon and erythropoietic differentiation in cells transformed by Friend virus.

Two lines of Friend virus (FV)-transformed mouse spleen cells have been analyzed in respect to their interferon production capacity: neither F4 cells, which liberate infectious FV when kept under tissue culture conditions, nor the thymidine kinase-deficient B8 cells, which do not produce significant amounts of FV, release detectable amounts of autogenous interferon into cell supernatants. However, interferon is produced in these cells in amounts comparable to that in L-929 cells when interferon induction is initiated with UV-inactivated Newcastle disease virus. Conversely poly(I)-poly(C), a potent interferon inducer in L-929 cells, proved ineffective in this capacity in F4 or B8 cells. When erythropoietic differentiation is induced in these cells by dimethyl sulfoxide, no autogenous interferon production occurs, but with NDV-induction a four- to fivefode increase of interferon production is observed. A similar elevation of interferon production is achieved during 5-bromodeoxyuridine stimulation of differentiation in the thymidine kinase-deficient B8 cells. The refractiveness against poly(I)-poly(C) displayed in unstimulated cells is not overcome at any stage of differentiation, indicating major differences of Newcastle disease virus and poly(I)-poly(C) induction mechanisms.     

Induction of Interferon and Erythropoietic Differentiation in Cells Transformed by Friend Virus

Two lines of Friend virus (FV)-transformed mouse spleen cells have been analyzed in respect to their interferon production capacity: neither F4 cells, which liberate infectious FV when kept under tissue culture conditions, nor the thymidine kinase-deficient B8 cells, which do not produce significant amounts of FV, release detectable amounts of autogenous interferon into cell supernatants. However, interferon is produced in these cells in amounts comparable to that in L-929 cells when interferon induction is initiated with UV-inactivated Newcastle disease virus. Conversely poly(I) · poly(C), a potent interferon inducer in L-929 cells, proved ineffective in this capacity in F4 or B8 cells. When erythropoietic differentiation is induced in these cells by dimethyl sulfoxide, no autogenous interferon production occurs, but with NDV-induction a four- to fivefold increase of interferon production is observed. A similar elevation of interferon production is achieved during 5-bromodeoxyuridine stimulation of differentiation in the thymidine kinase-deficient B8 cells. The refractiveness against poly(I) · poly(C) displayed in unstimulated cells is not overcome at any stage of differentiation, indicating major differences of Newcastle disease virus and poly(I) · poly(C) induction mechanisms.     

Effects of interferon on hemoglobin synthesis and leukemia virus production in Friend cells

Establishment of the antiviral state by interferon does not impair differentiation of Friend cells. Interferon actually produces an increase in dimethylsulfoxide-induced hemoglobin synthesis. However, both the constitutive production and the induction of leukemia virus in these cells are inhibited by interferon.     

Differential response of type C and intracisternal type A particle markers in cells treated with iododeoxyuridine and dexamethasone.

Mouse neuroblastoma cells containing intracisternal type A particles were treated with iododeoxyuridine and dexamethasone to induce the release of type C oncornavirus particles. For 5 days after treatment, antigenic markers and DNA polymerase activities specific to particles of each of the two types were assayed in the cells and in pellets obtained by high-speed centrifugation of the culture fluid. There was a marked release of C-particle antigen (p30) and DNA polymerase activity in extracellular particulate form, reaching a maximum on day 3 after treatment and falling thereafter. In contrast, no extracellular A-particle antigen was detected, and A-particle-specific DNA polymerase activity in the medium pellets did not increase from the original very low level. Electron microscopy confirmed the presence of free type C virus particles, but not intracisternal type A particles, in the culture fluid. Although intracellular levels of C-particle antigen rose 20- to 30-fold per milligram of cell protein, intracellular A-particle antigen and DNA polymerase activity did not vary more than two-fold. The relative rate of A-particle synthesis in the treated cells, as judged by incorporation of radioactive amino acids into the major structural protein (P73), was also unchanged over the period of observation. Thus, the induction of type C virus particle formation in cultured neuroblastoma cells had no detectable effect on the quantity, synthesis rate, or location of intracisternal type A particles.     

Effects of monensin on morphogenesis and infectivity of Friend murine leukemia virus.

The transport of the gp70 glycoprotein to the cell surface and concomitant release of infectious virus was inhibited by treatment of Friend murine leukemia virus-infected Eveline cells with the sodium ionophore monensin. Virus yields were reduced more than 50-fold by 10(-5) M monensin, whereas particle production was reduced by 50% in monensin-treated cells. The resulting particles failed to incorporate newly synthesized gp70 and p15(E), whereas the other structural proteins, p30, p15, p12, and p10, were incorporated into virions. However, monensin did not inhibit the incorporation into virions of preformed gp70. A reduction in the efficiency of cleavage of the PrENV glycoprotein precursor and a defect in the processing of simple endo-H-sensitive to complex endo-H-resistant oligosaccharides suggest that intracellular transport of gp70 may be blocked before its entry into the Golgi apparatus. Fewer particles were found to bud from the cell surface, but intracellular vacuoles with budding virions were detected. Ferritin labeling and pulse-chase studies suggested a cell surface origin for these vacuoles. These experiments indicate that monensin inhibits the transport of Friend murine leukemia virus glycoproteins at an early stage, with a resultant block in the assembly and release of infectious virus.     

Characterization of DNA polymerase and RNA associated with A-type particles from murine myeloma cells.

The RNA-dependent DNA polymerase present in intracisternal A-type particles from mouse myeloma tumor cells has been studied. This polymerase can use either endogenous A particle RNA or an exogenous synthetic polynucleotide [poly (rA)] as a template. The DNA reaction product is small (4S-10S) and over 90% of it hybridizes to A particle RNA, whereas up to 50% of it hybridizes to murine sarcoma-leukemia virus RNAs. The RNA isolated from purified A particles is generally of low molecular weight (5S-15S) but contains small amount of 70S and 35S components. These results suggest that A-type particles may be related to C-type oncornaviruses.     

Inhibition of friend murine leukemia virus production by low-ionic-strength medium.

The effect of medium of low ionic strength on the release of virus from Friend leukemia cells has been studied. The release of infectious Friend leukemia virus is almost completely inhibited in medium of low ionic strength, as measured by a focus-forming assay (XC assay), by endogenous RNA-dependent DNA polymerase activity of released virus particles, and by electron microscope studies of the production of C-type particles. Friend leukemia virus-transformed proerythroblasts undergo extensive morphological changes in low-ionic-strength medium. The cells are viable in this medium, but they can no longer be stimulated with dimethyl sulfoxide to produce hemoglobin and increase virus production. Infectious virus is released between 30 and 120 min of resuspension of inhibited cells in normal medium. The rate of virus release after reversal of the inhibition is much greater than the rate of virus release during normal cell growth. The morphological changes occurring after dimethyl sulfoxide stimulation of Friend leukemia cells are compared with those resulting from resuspension in normal medium of cells inhibited by low ionic strength.     

Expression of a transformation-related protein (p53) in the malignant stage of Friend virus-induced diseases.

Stage 1 (pre-malignant) and stage 2 (malignant) cells derived from mice infected with Friend murine leukemia virus or polycythemia-inducing Friend virus complex were examined and compared for the expression of a transformation-related cellular protein, p53. Stage 2 cells were found to express high levels of p53, whereas stage 1 cells did not express detectable levels of this protein. These results indicate that p53 may be a marker for transformed cells present in the second stage of diseases induced by Friend murine leukemia virus or polycythemia-inducing Friend virus complex.     

Viral glycoproteins synthesis in Friend cell lines producing polycythemic (FV-P) and anemic (FV-A) Friend viruses

Two tumor Friend cell lines producing anemia-inducing virus (TF-A line) or polycythemia-inducing virus (TF-P line) were compared for their viral-encoded glycoproteins. The envelope glycoproteins of the two viral populations differ by their electrophoretic mobilities. The gpr^env precursors also differ by their relative mobilities. The TF-P cells contain the typical gp50–52 molecular species, which is coded for by Spleen Focus Forming sequences (SFFV) present in the genome of the polycythemia-inducing virus. The TF-A cells do not contain the gp50–52, but express in small amounts a species with a higher apparent molecular weight. This species which has been named FV-A gp55 could be equivalent to the gp50–52 coded for by the SFFV sequences. Very similar results were obtained with leukemic cells prepared from enlarged spleens of mice infected with the anemic or polycythemic Friend viruses.     

AtT20 pituitary tumour cells contain mouse mammary tumour virus and intracisternal A-type particles in addition to murine leukemia virus

By electron microscopy and immunocytochemistry we have examined the retroviruses endogenous to AtT20 D16V cells, a cloned line of murine pituitary tumour cells. In addition to the C-type retrovirus particles related to Rauscher murine leukemia virus (MuLV) previously reported to bud from these cells we observed cytoplasmic A-type particles and intracisternal A-type particles (IAP). In the cytoplasm the A-type particles occur in large clusters often associated with sheets of material with a fine structure resembling the shells of the particles. At the plasma membrane individual A-type particles bud to give rise to extracellular virions. The IAP are restricted to the rough endoplasmic reticulum (RER) into which they bud: they are not transported out of the RER to the Golgi apparatus and beyond. We describe a new monoclonal antibody (designated 83E7) which is specific for an epitope of the major core protein (MTVp27) of mouse mammary tumour virus (MMTV). Using immunogold labelling procedures we have specifically labelled both the A-type particles and the associated sheets of material with this antibody. We conclude that the A-type particles and the virions they give rise to are MMTV. The sheets of material must also at least in part be made up of the major core protein of MMTV or its precursor polypeptide. AtT20 cells, therefore, contain endogenous MuLV and MMTV as well as IAP.     

Effect of interferon on growth and division cycle of Friend erythroleukemic murine cells in vitro

The administration of appropriate doses of interferon to cultures of Friend leukemia cells causes a pronounced inhibition of cell growth. Several lines of evidence indicate that this effect is due to interferon itself, rather than to unknown contaminants of interferon preparations. Autoradiograph analysis of growth parameters of Friend leukemia cells during treatment with interferon demonstrates that the rate of entry into the S phase, the percent decline of unlabeled mitoses, and the mitotic indexes are significantly lower in interferon- treated cell cultures than in control untreated cultures when tritiated thymidine was added 12 h after the administration of interferon. These data indicate that fractions of interferon-treated cell population are delayed in both G1 and in G2 phases of the cell cycle. This was confirmed by exact measurements of the length of the various phases of the cycle. The interferon-induced inhibition of growth of Friend leukemia cells is reversible after removal of the compound. Autoradiograph data obtained from control cultures and from cultures previously treated with interferon that had been washed free of interferon and reseeded in interferon-free medium, demonstrate that during the first 12 h after removal of interferon, a large majority of the cells previously treated with interferon had a deranged flow into the S phase, a high number of unlabeled mitoses, and a low mitotic index. These data provide further evidence for the above-mentioned prolongations of G1 and G2 phases of the cell cycle. All growth parameters tested reverted to normal values within 12 h after washing out interferon.     

Broad-Spectrum Inhibition of Retroviral and Filoviral Particle Release by Tetherin

The expression of many putative antiviral genes is upregulated when cells encounter type I interferon (IFN), but the actual mechanisms by which many IFN-induced gene products inhibit virus replication are poorly understood. A recently identified IFN-induced antiretroviral protein, termed tetherin (previously known as BST-2 or CD317), blocks the release of nascent human immunodeficiency virus type 1 (HIV-1) particles from infected cells, and an HIV-1 accessory protein, Vpu, acts as a viral antagonist of tetherin. Here, we show that tetherin is capable of blocking not only the release of HIV-1 particles but also the release of particles assembled using the major structural proteins of a variety of prototype retroviruses, including members of the alpharetrovirus, betaretrovirus, deltaretrovirus, lentivirus, and spumaretrovirus families. Moreover, we show that the release of particles assembled using filovirus matrix proteins from Marburg virus and Ebola virus is also sensitive to inhibition by tetherin. These findings indicate that tetherin is a broadly specific inhibitor of enveloped particle release, and therefore, inhibition is unlikely to require specific interactions with viral proteins. Nonetheless, tetherin colocalized with nascent virus-like particles generated by several retroviral and filoviral structural proteins, indicating that it is present at, or recruited to, sites of particle assembly. Overall, tetherin is potentially active against many enveloped viruses and likely to be an important component of the antiviral innate immune defense.     

Impaired macrophage function in Friend virus leukemia: restoration by statolon.

Phagocytic and migratory functions of peritoneal macrophages from Friend virus (FV) leukemic mice are significantly depressed as compared with normal controls. Leukemic macrophages exposed in vivo and in vitro to statolon, an extract of the mold Penicillium stoloniferum, shown previously to suppress FV erythroleukemia, regain normal function and release reduced amounts of FV. Statolon's in vivo restoration of leukemic macrophage function is paralleled by restoration of humoral immune competence. Statolon induces interferon in vitro but its effects on leukemic macrophages are probably direct, since restoration of macrophage function occurs at dosage levels far below those that induce interferon. These studies suggest that macrophages play an integral role in both the pathogenesis and the statolon-induced suppression of FV disease.     

Intracellular production of virus particles and viral components in NIH/3T3 cells chronically infected with Moloney murine leukemia virus: effect of interferon.

The effect of interferon on the biochemical properties and the maturation process of intracellular viral particles isolated from the cytoplasmic fraction of NIH/3T3 cells chronically infected with Moloney murine leukemia virus was investigated. By labeling these virions with either [35S]methionine or [3H]glucosamine, we demonstrated that they contain the same viral proteins and glycoproteins found in extracellular virions. Interferon treatment was found to reduce the rate of intracellular virus assembly. This effect was not a consequence of an interferon inhibition of viral RNA synthesis or its translation or a consequence of an interference with the posttranslational cleavage processing of viral precursor proteins, since all of these steps were not affected by interferon. However, the reduced rate of virus assembly could be attributed to the inhibition of viral protein glycosylation observed in interferon-treated cells. Nevertheless, despite this reduced rate, virus particles accumulated in interferon-treated cells. This accumulation was probably due to the strong inhibition of their final release from such cells.     

T-cells inhibit Friend murine leukemia virus infection of B-cells in vitro

The ability of splenic T-cells to regulate Friend murine leukemia virus replication in lipopolysaccharide-activated target B-cells infected in vitro was investigated. Removal of the T-cell fraction from spleen cells resulted in an 8- to 10-fold enhancement in the number of productively infected cells in the remaining B-cell-enriched fraction, as compared with unseparated spleen cells, and the addition of increasing numbers of purified T-cells to isolated B-cells prior to infection resulted in a directly proportional reduction in the number of B-cells releasing infectious progeny virus. Separation of splenic T-cells into Lyt 2- and Lyt 2+ T-cells before addition to infected B-cell cultures resulted in inhibition of infection only with the Lyt 2- T-cells; Lyt 2+ T-cells did not inhibit infection, even at high 1:1 ratios. Similarly, separation of splenic T-cells into L3T4+ and L3T4- T-cells before addition resulted in inhibition by L3T4+ but not L3T4- T-cells. Also, cytotoxic treatment of splenic T-cells with monoclonal anti-L3T4 antibody and complement before addition to B-cell cultures destroyed the regulatory effects. Finally, depletion of macrophages from both T-cells and B-cells before infection and coculture had no effect on the ability of T-cells to regulate B-cell infection. Collectively these results demonstrate that L3T4+ T-cells can inhibit Friend murine leukemia virus replication in target B-cells. Culture of isolated splenic T-cells with Friend murine leukemia virus in vitro resulted in the induction of alpha/beta but not interferon-gamma synthesis and in some experiments interferon-containing supernatants from T-cell-virus cultures were able to mediate suppression of B-cell infection with Friend helper virus; the addition of antibody specific for interferon-alpha/beta to cultures inhibited the ability of T-cells to regulate B-cell infection.