Effect of ammonium salts on the interferon-induced antiviral state in mouse L cells.

The addition of ammonium salts to cells treated with interferon prevents the development of the antiviral state and destroys it when already established. This treatment does not seem to act on the binding of interferon to the cells but blocks a further step of activation on the cell membrane. The anti-interferon effect of ammonium salts is reversible with a complete recovery of the antiviral state. It is postulated that these salts may stabilize the interferon-receptor complex and thus prevent the changes in configuration necessary for the establishment and maintenance of its biological functions.     

Resistance of pokeweed mitogen-stimulated B cells to inhibition by deoxyadenosine.

Deoxyadenosine (dAdo) levels above 2 microM inhibit plasma cell (PC) differentiation by human blood lymphocytes in pokeweed mitogen (PWM) stimulated cultures containing deoxycoformycin (dCF), a potent inhibitor of adenosine deaminase (ADA). ADA inhibition by dCF alone did not suppress PC differentiation. Thymidine uptake by T cell blasts continuously cultured in conditioned medium was inhibited by dAdo and dCF; two of five EBV-infected B cell lines were also inhibited while three were resistant. Inhibition of PWM-induced PC differentiation of B cells by dCF and dAdo was reversed when conditioned medium (a source of T cell helper factors) was added to the cultures, and dAdo and dCF added to PWM-stimulated cultures 48 hr after their initiation did not inhibit PC differentiation, though thymidine uptake and the total number of cells recovered from the cultures were reduced. Removal of T cells after 48 hr of culture slightly reduced the numbers of PC in PWM-stimulated lymphocyte cultures but no further inhibition was obtained when dCF and dAdo were added to these T-depleted cultures, nor was their thymidine uptake further reduced. These results suggest that the in vitro suppression of B cell differentiation by dAdo in PWM-stimulated cultures is not due to direct toxicity of purine nucleosides to B cells but may be due to interference with T cell help. This is consistent with the view that a relative lack of helper activity by T cells contributes to the antibody deficiency of patients with ADA deficiency.     

Suppression of interferon-induced antiviral activity in cells expressing hepatitis C virus proteins.

To elucidate the mechanism of the persistent nature of hepatitis C virus (HCV) infection, we examined whether the expression of HCV proteins affect the antiviral activity of interferon (IFN). Antiviral activity of IFN in HepG2 cells expressing all HCV (type 1b) proteins was much lower than vector control (VC) HepG2 cells when encephalomyocarditis virus (EMCV) was used as a challenge virus. Lesser sensitivity to IFN was also observed in cells expressing NS3, NS4, and NS5 and in cells expressing only NS5A. In contrast, HepG2 cells expressing core, E1, E2, NS2, and NS3 proteins were equally sensitive to IFN as VC cells. We then tested the antiviral activity by IFN in two human amnion-derived FL cell lines expressing NS5A from two different clones, one with an intact sequence of IFN sensitivity-determining region (ISDR) and the other with a mutated ISDR sequence. They were almost equally insensitive to IFN treatment when EMCV was challenged. HCV thus has functional protein(s), possibly NS5A, to suppress IFN-induced antiviral activity and plays an important role in virus-cell interaction and regulation of viral replication.     

Hepatitis C virus: evasion of the interferon-induced antiviral response

Hepatitis C virus (HCV) is an emerging virus of great medical significance. A low drug-response rate and a high frequency of persistent infection have caused HCV to reach pandemic proportions. Many infected individuals go on to develop liver cirrhosis and hepatocellular carcinoma, and HCV is now the leading reason for liver transplants in the United States. Differences in genotype response to interferon therapy suggests that one or more viral genes may participate in evasion of the interferon-mediated cellular antiviral response. This review focuses on the viral genes that interact with the host cell to evade the interferon response and on the insights that these interactions may provide into HCV pathogenesis.     

Gamma interferon-induced inhibition of Toxoplasma gondii in astrocytes is mediated by IGTP

Toxoplasma gondii is an important pathogen in the central nervous system, causing a severe and often fatal encephalitis in patients with AIDS. Gamma interferon (IFN-gamma) is the main cytokine preventing reactivation of Toxoplasma encephalitis in the brain. Microglia are important IFN-gamma-activated effector cells controlling the growth of T. gondii in the brain via a nitric oxide (NO)-mediated mechanism. IFN-gamma can also activate astrocytes to inhibit the growth of T. gondii. Previous studies found that the mechanism in murine astrocytes is independent of NO and all other known anti-Toxoplasma mechanisms. In this study we investigated the role of IGTP, a recently identified IFN-gamma-regulated gene, in IFN-gamma inhibition of T. gondii in murine astrocytes. Primary astrocytes were cultivated from IGTP-deficient mice, treated with IFN-gamma, and then tested for anti-Toxoplasma activity. In wild-type astrocytes T. gondii growth was significantly inhibited by IFN-gamma, whereas in astrocytes from IGTP-deficient mice IFN-gamma did not cause a significant inhibition of growth. Immunoblot analysis confirmed that IFN-gamma induced significant levels of IGTP in wild-type murine astrocytes within 24 h. These results indicate that IGTP plays a central role in the IFN-gamma-induced inhibition of T. gondii in murine astrocytes.     

Interferon externalization by producing cell before induction of antiviral state.

The mechanism by which poly(I · C) induces the antiviral state in human fibroblast cells was studied using antisera which were selectively reactive with the inducer [poly(I · C)], the product (human fibroblast interferon), and the fibroblast cell surface. Anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Furthermore, when added to the medium of the cells in which interferon synthesis had already been induced by poly(I · C), interferon antiserum prevented the intracellular antiviral state from developing. Antibodies specific to double stranded RNA inhibited the antiviral activity of poly(I · C) whether it was in solution or bound to the cells, but the same antibodies were without effect when added after the induction of the antiviral state was initiated. Treatment with serum directed against the cell surface of human fibroblasts failed to inhibit the antiviral activity of poly (I · C) or of human interferon in these cells. These results indicate that the antiviral effect of poly(I · C) is interferon mediated, and that for the development of the antiviral state, interferon has to interact with the external part of the cellular membrane of the producing cell.     

Reversible inhibition by phosphonoacetic acid of human B lymphocyte transformation by Epstein-Barr virus.

B lymphocytes purified by immunoabsorbent chromatography from the peripheral blood lymphocytes of adults provide highly effective targets for infection and transformation by Epstein-Barr virus (EBV). Using this system, the kinetics of DNA synthesis induction due to EBV infection have been characterized. The kinetics show two phases: an early phase, lasting 3 to 4 days, the rate and absolute level of which are dependent upon multiplicity of infection; and a later phase, representing normal exponential growth, the level but not rate of which is dependent upon multiplicity of infection. The induction of DNA synthesis begins at a time (24 hr) which agrees well with the published times for the appearance of the Epstein-Barr virus nuclear antigen (EBNA). Absorption and penetration of the cells by EBV appear to require 1 to 2 hr. The induction of DNA synthesis proceeds normally during the first phase in the presence of phosphonoacetic acid (PAA; 200 μg/ml). Thereafter, DNA synthesis remains at a plateau. Blast-transformed EBNA-positive cells may be isolated from these cultures. Removal of PAA at any time up to 28 days postinfection results in resumed proliferation and outgrowth. These cells express four phenotypic properties of transformation by EBV: (1) induction of DNA synthesis, (2) EBNA expression, (3) blast transformation, and (4) immortalization (survival for at least 28 days). However, they cannot grow out. It is therefore proposed that the phenomenon of EBV infection in the presence of PAA may be termed “abortive transformation.”     

Reversible inhibition of a thyroid-specific trans-acting factor by Ras

Exposure of rat thyroid cells for 1 week to a temperature-sensitive variant of Kirsten murine sarcoma virus (KiMSV) Ras inactivated the thyroglobulin promoter (pTg). Cellular dedifferentiation was paralleled by the loss of the thyroid-specific trans-acting factor, TgTF1, which binds to pTg. When Ras was denatured by shifting cells to 39 degrees C, TgTF1 binding and pTg function recovered rapidly without the synthesis of new protein. TgTF1 could be reactivated in vitro by treating nuclear extracts with protein kinase A. After 4 weeks of exposure to the oncogene, denaturation of Ras no longer restored TgTF1 binding or reactivated pTg. Incubation of nuclear extracts with protein kinase A likewise did not reactivate TgTF1. Cells chronically exposed to Ras did, however, yield differentiated clones after treatment with 5-azacytidine. We suggest that Ras induces dedifferentiation in two sequential steps: (1) Ras reduces PKA activity; TgTF1 (or an auxiliary protein) becomes dephosphorylated, and binding to pTg is abolished. (2) The effects of Ras become imprinted by methylation, possibly of the TgTF1 gene.     

Role of vacuolar H(+)-ATPase in interferon-induced inhibition of viral glycoprotein transport.

We have shown previously that interferon-beta (IFN-beta) induces the alkalinization of trans-Golgi network (TGN) and inhibits the transport of G protein of vesicular stomatitis virus (VSV) in L(B) cells and gD protein of herpes simplex virus (HSV-1) in LMtk- cells transfected with gD cDNA. The vacuolar H(+)-ATPase (V-ATPase) is responsible for maintaining pH in TGN, and V-ATPase-mediated acidification is required for normal transport of proteins. To examine whether alkalinization caused by IFN is mediated through V-ATPase, the activity of V-ATPase was determined in IFN-treated cells by coupling ATP hydrolysis to NADH oxidation. Bafilomycin (Baf) was used as positive control, as it specifically inhibits V-ATPase. The activity of V-ATPase was reduced in IFN-treated or Baf-treated cells compared with untreated cells. Doses of IFN-beta or Baf that neither alter pHi nor inhibit the transport of viral glycoproteins concomitantly inhibited the transport of G and gD proteins in TGN, as demonstrated by indirect immunofluorescence studies, and raised the pH of TGN as demonstrated by a decrease in the uptake of DAMP. Further, the effect of Baf on IFN-induced antiviral activity against VSV was examined to correlate the biologic significance of these findings. Data showed that Baf significantly enhances (5-50-fold) the IFN-induced antiviral activity as demonstrated by viral titers from supernatants. These findings suggest that the inhibition of transport of G and gD proteins by IFN-beta, may be related to the inhibition of V-ATPase-mediated acidification of TGN.     

Abrogation of gamma interferon-induced inhibition of Ehrlichia chaffeensis infection in human monocytes with iron-transferrin.

Ehrlichia chaffeensis is an obligate intracellular bacterium which infects cells of the macrophage/monocyte lineage. To test whether gamma interferon (IFN-gamma) inhibits infection of monocytes with E. chaffeensis, human peripheral blood monocytes were incubated with recombinant human IFN-gamma for 3 h and then exposed to E. chaffeensis. With 2,000 U of IFN-gamma per ml, maximal inhibition of infection by E. chaffeensis was observed. THP-1 cells, a human monocyte cell line, pretreated with phorbol myristic acetate or not pretreated, were incubated with various concentrations of IFN-gamma. Maximum inhibition was obtained at 1,000 U of IFN-gamma per ml with phorbol myristic acetate-treated THP-1 cells. However, nontreated cells did not achieve a similar level of anti-ehrlichial activity even with 10,000 U of IFN-gamma per ml. IFN-gamma given within 6 h postinfection was effective in inhibiting E. chaffeensis. Nitric oxide production was not demonstrated in the monocyte medium incubated with IFN-gamma and E. chaffeensis. None of the reactive oxygen intermediate scavengers tested blocked the IFN-gamma-induced anti-ehrlichial activity. Deferoxamine, an intracellular iron chelator, at 15 microM completely inhibited the survival of E. chaffeensis. Iron-saturated transferrin at 1.67 mg/ml completely reversed the IFN-gamma-induced ehrlichial killing. These results indicate that (i) E. chaffeensis is sensitive to intracytoplasmic iron depletion, (ii) E. chaffeensis is sensitive to IFN-gamma-induced killing, and (iii) the anti-ehrlichial activity induced in human monocytes by IFN-gamma is mediated by limitation of available cytoplasmic iron and is not due to the generation of reactive oxygen intermediates or nitric oxide.     

Reversible inhibition of cellular aggregation by cytochalasin B and colcemid

Aggregation of normal mouse fibroblast-like cells, placed on the surface of a Millipore filter, was investigated quantitatively. The addition of 10 g/ml of cytochalasin B (a substance blocking active cell movements) or 0.1 g/ml of colcemid (a substance disturbing polarization processes in cells and, consequently, interfering with directional cell movements) completely inhibits such aggregation. The inhibition is reversible—after removal of the agents the cells again aggregate. It is concluded that aggregation of cells in this experimental system requires active cell movement and cellular polarization.Laboratory of Mechanisms of Carcinogenesis, Institute of Experimental and Clinical Oncology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 1, pp. 49–51, January, 1976.     

Reversible inhibition of poliovirus ribonucleic acid

Summary Monkey kidney tissue culture (MKTC) cell homogenate depressed the infectivity of poliovirus RNA. This depression, however, was not due to RNAase activity of the cell homogenate, since after the depressed RNA solution was diluted and fresh facilitator was added, the infectivity was restored. Yeastolate, egg white, casein hydrolysate, and commercial RNA and DNA were found to have the same inhibitory effect on infectious RNA. The inhibitors destroyed the facilitating capacity of the facilitator. They could be easily removed by adsorbing them to large quantities of facilitator. The 7 per cent phenol treatment and the 50 per cent phenol extraction of poliovirus to obtain infectious RNA were compared. If the resulting RNA solution was inoculated undiluted, the 50 per cent method was superior because this method removed the proteinaceous material and hence part of the inhibitors in the crude virus suspension.Aided by a grant from the National Foundation.     

The antiviral potential of interferon-induced cotton rat Mx proteins against orthomyxovirus (influenza), rhabdovirus, and bunyavirus.

Influenza A virus (FLUAV) is an important human pathogen able to cause devastating pandemics. Recently, cotton rats have been proposed as an animal model to study the innate immune response against FLUAV and other human pathogens. The interferon (IFN)-induced Mx GTPases are part of the cell-autonomous innate immune response against viruses. We, therefore, tested the antiviral activity of the two cotton rat Mx proteins that were recently identified. The nuclear cotton rat Mx1 protein was found to be a strong inhibitor of FLUAV, whereas the cytoplasmic cotton rat Mx2 protein was inactive. Cotton rat Mx2, but not cotton rat Mx1, was able to inhibit the rhabdovirus vesicular stomatitis virus (VSV) and the bunyavirus Rift Valley fever virus (RVFV) known to replicate in the cytoplasm of infected cells. Thus, cotton rats possess two Mx proteins that have selective antiviral activity that depends on their intracellular localization. We conclude that the Mx status of cotton rats differs from that of conventional inbred mouse strains, which are known to have defective Mx genes. Therefore, cotton rats are a suitable animal model to study experimental infections with FLUAV and other RNA viruses.     

Kinetics of antiviral state in cells activated by interferons in vitro

It was found that development of antiviral state in cells activated by IFN alpha and beta were quite rapid. After 6-7 h treatment IFN alpha and IFN beta protected cells completely and their effects lasted for another 9-12 h after removing them from the cultures. Then the effect declined, but some protective action remained after 48 h of incubation. IFN-gamma was different from IFN alpha or beta. It activated cells much more slowly, and could not protect cells completely before 24 h of treatment. When we compared three concentrations of IFN-alpha, the cells could not be protected completely by 5 IU/ml, and kinetics were similar with 250 and 25 IU/ml. When we added anti-IFN-alpha serum before IFN treatment, the development of the antiviral state was inhibited. The results suggested that one should adopt different ways when titrating and using different forms of IFN, and it might not be needed to use maximal tolerated doses and daily administrations, and to keep the high level of IFN for a long time. We think that these results might be useful for the clinicians considering the optimal schedule for IFN treatment.