Low infectivity of vesicular stomatitis virus (VSV) particles released from interferon-treated cells is related to glycoprotein deficiency

We have investigated the mechanism for the low infectivity of vesicular stomatitis virus (VSV) released from interferon (IFN) -treated cells. With 10-30 units/ml of IFN there was an approximately 5-30 fold reduction in the production of virus particles, as measured by VSV proteins; however, the infectivity of the VSV released from IFN-treated mouse LB, JLS-V9R, or human GM2504 was drastically reduced (2 to 4 logs). The low infectivity of VSV was directly related to a deficiency in virion glycoprotein (G). IFN treatment did not change the specific infectivity of the VSV particles released by HeLa cells; their G protein was also not reduced. A further effect of IFN to reduce the amount of virion M protein appeared to be secondary and was probably not related to the reduced infectivity of VSV.     

Detection of functional interferon alpha receptors in human neuroendocrine tumor cell lines using a new monoclonal antibody.

While first described as antiviral agents, interferons (IFNs) exhibit significant antiproliferative and antitumor effects as well. IFN alpha has been successfully used in clinical trials to treat several malignancies, including leukemias and certain solid tumors. While many cell types have been studied for IFN alpha receptor expression, very little is known about receptor expression on human neuroendocrine cells. Using a novel anti-IFN alpha receptor monoclonal antibody, we examined IFN alpha receptor expression in 10 human cell lines derived from tumors of neuroendocrine origin, including neuroblastoma, neuroepithelioma and small cell lung carcinoma. All cell lines studied displayed a similar pattern of IFN alpha receptor expression and 5 of 8 cell lines demonstrated reduced thymidine incorporation following IFN alpha treatment. Addition of exogenous IFN alpha caused a decrease in IFN alpha receptor expression, while differentiating agents, such as phorbol esters and retinoic acid, induced an increase in receptor number without altering receptor affinity.     

Expression of hepatitis C virus proteins interferes with the antiviral action of interferon independently of PKR-mediated control of protein synthesis

Hepatitis C virus (HCV) of genotype 1 is the most resistant to interferon (IFN) therapy. Here, we have analyzed the response to IFN of the human cell line UHCV-11 engineered to inducibly express the entire HCV genotype 1a polyprotein. IFN-treated, induced UHCV cells were found to better support the growth of encephalomyocarditis virus (EMCV) than IFN-treated, uninduced cells. This showed that expression of the HCV proteins allowed the development of a partial resistance to the antiviral action of IFN. The nonstructural 5A (NS5A) protein of HCV has been reported to inhibit PKR, an IFN-induced kinase involved in the antiviral action of IFN, at the level of control of protein synthesis through the phosphorylation of the initiation factor eIF2alpha (M. Gale, Jr., C. M. Blakely, B. Kwieciszewski, S. L. Tan, M. Dossett, N. M. Tang, M. J. Korth, S. J. Polyak, D. R. Gretch, and M. G. Katze, Mol. Cell. Biol. 18:5208-5218, 1998). Accordingly, cell lines inducibly expressing NS5A were found to rescue EMCV growth (S. J. Polyak, D. M. Paschal, S. McArdle, M. J. Gale, Jr., D. Moradpour, and D. R. Gretch, Hepatology 29:1262-1271, 1999). In the present study we analyzed whether the resistance of UHCV-11 cells to IFN could also be attributed to inhibition of PKR. Confocal laser scanning microscopy showed no colocalization of PKR, which is diffuse throughout the cytoplasm, and the induced HCV proteins, which localize around the nucleus within the endoplasmic reticulum. The effect of expression of HCV proteins on PKR activity was assayed in a reporter assay and by direct analysis of the in vivo phosphorylation of eIF2alpha after treatment of cells with poly(I)-poly(C). We found that neither PKR activity nor eIF2alpha phosphorylation was affected by coexpression of the HCV proteins. In conclusion, expression of HCV proteins in their biological context interferes with the development of the antiviral action of IFN. Although the possibility that some inhibition of PKR (by either NS5A or another viral protein) occurs at a very localized level cannot be excluded, the resistance to IFN, resulting from the expression of the HCV proteins, cannot be explained solely by inhibition of the negative control of translation by PKR.     

Interferon acts directly on human B lymphocytes to modulate immunoglobulin synthesis

At different times of exposure, interferon (IFN) enhanced and suppressed pokeweed mitogen- (PWM) induced IgG synthesis by human peripheral blood lymphocytes (PBL). Pretreatment of PBL and IFN frequently increased antibody production by more than 100% when compared with that by untreated PBL. Results of experiments in which PBL were separated into T and B subpopulations indicated that IFN preparations acted directly on B cells. Thus, mixtures of IFN-treated B cells and untreated T cells from 5 of 7 persons tested produced 81% to 500% more IgG than untreated, matched control cells. However, IFN-treated monocytes mixed with untreated B and T cells or IFN-treated T cells mixed with untreated B cells failed to enhance IgG production significantly in similar assays. In contrast to the pretreatment protocol, when IFN was present in the incubation mixture throughout the PWM assay, IgG production decreased. Sephadex chromatography of the IFN and tests of the resulting fractions indicated that the IgG production-enhancing activity was located in the fraction carrying the antiviral activity.     

Inhibition of viral mRNA translation in interferon-treated L cells infected with reovirus.

Murine L cells were treated with interferon (IFN) concentrations which reduced by 75 to 80% the synthesis of viral mRNA after infection with reovirus. Protein synthesis was not inhibited in these cells up to 6 h after infection, but a large fraction of the viral mRNA was not associated with polyribosomes and sedimented at about 50S. In contrast, most of the reovirus mRNA was associated with polyribosomes in control infected cells. This mRNA was of similar size to non-polyribosomal mRNA from IFN-treated cells when analyzed by Northern blot hybridization with a cloned cDNA for the s2 reovirus mRNA, indicating that the non-polyribosomal mRNA was not appreciably degraded. Viral mRNA was labeled with [3H]uridine and the non-polyribosomal mRNA was isolated from IFN-treated cells. This mRNA could quantitatively bind to 80S initiation complexes when incubated in a rabbit reticulocyte cell-free system. These findings indicated that the non-polyribosomal RNA was translatable, but that its binding to functional initiation complexes was inhibited in IFN-treated cells by a discriminatory mechanism, which did not affect translation of cellular mRNA. Previous experiments showed that mRNA is blocked in 48S complexes when the alpha subunit of initiation factor eIF-2 is phosphorylated by the double-stranded RNA-dependent protein kinase induced by IFN. A localized activation of this kinase could explain the block of viral mRNA in 48S complexes. By labeling the phosphoproteins of IFN-treated cells with 32P, eIF-2 (alpha P) was shown to cosediment with non-polyribosomal mRNA, presumably in 48S complexes.     

Mechanism of interferon action. Kinetics of decay of the antiviral state and protein phosphorylation in mouse fibroblasts treated with natural and cloned interferons

The kinetics of decay of the antiviral state and protein phosphorylation induced with natural mouse interferon (IFN) and with cloned human IFN were examined in monolayer cultures of mouse Ll929 fibroblast cells. The antiviral state measured by single cycle virus yield reduction with either vesicular stomatitis virus or reovirus decayed significantly within 2 to 3 days following removal of IFN and by 5 to 8 days virus yields had returned to the level of untreated control cells. Trypsinization of IFN-treated cells did not detectably alter the rate of decay of the antiviral state; however, the decay occurred slightly more rapidly in actively growing as compared to stationary cell cultures. The decay of the IFN-induced protein kinase which catalyzes the phosphorylation of endogenous protein P1 and purified initiation factor eIF-2 alpha correlated with the decay of the antiviral state. The decay rates of the antiviral state and protein kinase observed in mouse L929 cells that had been treated with natural mouse IFN synthesized in Newcastle disease virus-induced L929 cells were comparable to the decay rates observed in L929 cells that had been treated with recombinant human IFN-alpha A/D synthesized in Escherichia coli. The induction and decay of the antiviral state and protein kinase following treatment with a single dose of IFN did not significantly affect the sensitivity of the cell population to a subsequent treatment with a single dose of IFN. However, continuous treatment of L929 cells with natural mouse IFN or recombinant human IFN prevented the decay of both the antiviral state and protein kinase but also ultimately lead to cell death. The results suggest that protein phosphorylation may play an important role in the mechanism of IFN action in mouse L929 fibroblasts.     

Sensitivity of rabies virus to type I interferon is determined by the phosphoprotein gene

The growth of a virulent strain of fixed rabies virus, Nishigahara, in mouse neuroblastoma NA cells treated with type I interferon (IFN) was compared with that of a derivative avirulent strain, Ni-CE. Nishigahara strain was slightly sensitive to IFN treatment but still grew more efficiently than did Ni-CE strain in IFN-treated NA cells. Furthermore, a virulent chimeric virus with the phosphoprotein gene from Nishigahara strain in the Ni-CE genome was less sensitive to IFN treatment than was Ni-CE strain, indicating that the IFN sensitivity is determined by the phosphoprotein gene of the virus.     

Phospholipase C and phospholipase A2 are involved in the antiviral activity of human interferon-alpha

Treatment of human amniotic cells (UAC) with human interferon-alpha (Hu-IFN alpha) or phorbol myristate acetate (PMA) resulted in translocation of protein kinase C (PK-C) activity from the cytosol fraction to that of the membranes. Analysis of 32P incorporation into phospholipid fractions and studies of alterations in fatty acid content for the major phospholipids of IFN-treated cells suggest that phospholipases C and A2 are activated by Hu-IFN alpha. Addition of neomycin (an inhibitor of phospholipase C), as well as mepacrine (an inhibitor of phospholipase A2) to IFN-treated cells inhibited the antiviral activity of Hu-IFN alpha in the vesicular stomatitis virus (VSV)-UAC system used. These observations indicate that (i) activation of PK-C and (ii) diacylglycerol formation, arachidonic acid and/or lysophosphatidylcholine release are important steps in the mechanism of action of IFN.     

Mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion U cells by cloned human leukocyte interferon. I. Effect on early and late stages of the viral multiplication cycle

The kinetics of induction in human amnion U cells of the antiviral activity against vesicular stomatitis virus (VSV) produced by a single molecularly cloned subspecies of human leukocyte interferon (IFN-alpha A) were examined. IFN-alpha A-induced inhibition was found to be biphasic over a period of 24 h with the major extent of VSV inhibition occurring within the first 6 h of IFN treatment. The relationship of this major phase of inhibition to the early and late events of the VSV multiplication cycle was investigated. IFN-alpha A treatment had no detectable effect on the adsorption and penetration of VSV virions or on their uncoating to yield viral nucleocapsids. The polypeptides of adsorbed or uncoated VSV particles were neither preferentially degraded nor detectably altered in IFN-treated cells, as compared to untreated cells. Progeny virions released from IFN-treated cells, although greatly reduced in number, were found to be equally as infectious as those released from untreated cells. Progeny virions from IFN-treated cells also had a normal complement of VSV proteins in the same ratios as were seen in virions from untreated cells; specifically, IFN treatment produced no reduction in the incorporation of G or M protein into assembled virions. These results suggest that conditions of IFN treatment sufficient to reduce the yield of infectious VSV progeny greater than 99% do not detectably affect either the early or the late stages of the VSV multiplication cycle.     

Suppression of bacterial cell wall-induced polyarthritis by recombinant gamma interferon

Group A streptococcal cell wall fragments (SCW) induce erosive polyarthritis, characterized by synovial cell hyperplasia and intense mononuclear cell infiltration, in susceptible rats. Because of the known antiproliferative and immunomodulatory effects of interferon (IFN), we evaluated the effect of systemically administered alpha, beta and gamma IFN on the evolution of these destructive lesions. Treatment with gamma IFN not only reduced the acute response, but had an even greater suppressive effect on the chronic mononuclear cell-mediated destructive phase of the disease (articular index 10.2 +/- 1.2 for SCW only versus 3.8 +/- 0.7 for SCW + gamma IFN; p less than 0.01). Treatment with gamma IFN was more effective in the suppression of the arthritis than alpha, beta IFN. Histopathologic evaluation of the joints demonstrated that gamma IFN-treated animals had significantly fewer inflammatory cells, and less synovial hyperplasia and erosions than the SCW controls. gamma IFN suppression of mononuclear cell prostaglandin synthesis and synovial fibroblast proliferation was consistent with its anti-arthritic effects. These data indicate that the pathophysiology of SCW-induced erosive polyarthritis is subject to regulatory control by gamma IFN and that the mechanisms of suppression may be relevant in the treatment of rheumatoid arthritis.     

Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus

Mx+ mice are much more resistant to influenza virus than Mx- strains. The resistance is mediated by interferon (IFN) alpha/beta. After IFN treatment, Mx+ but not Mx- cells accumulate Mx protein and become specifically resistant to orthomyxoviruses. cDNA encoding Mx protein was cloned and sequenced. Southern analyses indicate that Mx- alleles derive from their Mx+ counterpart by deletions. IFN-treated Mx+ cells contained a 3.5 kb Mx mRNA, while Mx- cells showed only traces of shorter Mx RNA. Mx- cells transformed with Mx cDNA expressed Mx protein constitutively to varying extents; resistance of individual cells to influenza virus correlated with Mx protein expression. Thus, specific resistance to influenza virus in vivo may be attributed to Mx protein expression and is independent of other IFN-mediated effects.     

Mechanism of interferon action. Increased phosphorylation of protein synthesis initiation factor eIF-2 alpha in interferon-treated, reovirus-infected mouse L929 fibroblasts in vitro and in vivo

The effect of interferon (IFN) treatment and virus infection on the phosphorylation both in vitro and in vivo of the alpha subunit of protein synthesis initiation factor eIF-2 (eIF-2 alpha) was examined in mouse fibroblast L929 cells. The [gamma-32P]ATP-mediated in vitro phosphorylation of eIF-2 alpha catalyzed by cell-free extracts prepared from IFN-treated, uninfected cells was dependent upon exogenously added double-stranded RNA (dsRNA). However, the dsRNA requirement for eIF-2 alpha phosphorylation in vitro was eliminated by prior infection of cells with reovirus Dearing strain virions but not with defective top component particles. The enhanced phosphorylation in vitro of eIF-2 alpha and ribosome-associated protein P1 depended in a similar manner upon the multiplicity of virus infection. The extent of phosphorylation in vivo of eIF-2 alpha prepared from L929 cells was also examined by utilizing two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting techniques. About 5-10% of the eIF-2 alpha was typically phosphorylated in vivo in untreated, mock-infected cells, whereas 25-30% was phosphorylated in IFN-treated, reovirus-infected cells. An intermediate extent of eIF-2 alpha phosphorylation, routinely between 15 and 20%, was observed with either IFN treatment or reovirus infection alone. The integrity of eIF-4A and eIF-4B was also examined by two-dimensional electrophoresis and immunoblotting, and no significant alterations in molecular size or charge heterogeneity were detected when these factors were prepared from IFN-treated, reovirus-infected cells as compared to untreated, uninfected cells.     

Interferon-alpha down-regulates insulin receptors in lymphoblastoid (Daudi) cells. Relationship to inhibition of cell proliferation

The Daudi line of human lymphoblastoid cells requires insulin and transferrin for growth in serum-free medium and is highly sensitive to the inhibitory effect of human leukocyte interferon (IFN-alpha) on cell proliferation. A variant subline of Daudi cells, which is resistant to the antiproliferative action of IFN-alpha, also has been grown in serum-free medium containing insulin and transferrin. The proliferation of IFN-sensitive and -resistant Daudi cells is dependent on the occupancy of insulin receptors, with optimal cell proliferation observed at high receptor occupancy (nearly 100%). No evidence was found for receptors for insulin-like growth factor I on Daudi cells. IFN treatment of IFN-sensitive cells decreased the capacity of the cells to bind 125I-insulin. The altered binding capacity was due to diminished specific, lower affinity insulin binding, as detected at high 125I-insulin concentrations. Higher affinity insulin binding was not altered by IFN. Insulin binding was also reduced in detergent-solubilized extracts from IFN-treated sensitive Daudi cells and the magnitude of the effect was comparable to that observed in intact cells. This indicates that the total number of insulin binding sites (surface + internal) is decreased in IFN-treated sensitive cells. Insulin binding to IFN-sensitive cells decreased linearly with time between 6 and 48 h from the addition of IFN. The effect on lower affinity insulin binding developed more rapidly than the inhibitory effect of IFN on cell proliferation. The insulin-binding capacity of Daudi cells resistant to the antiproliferative effect of IFN was unaffected by IFN, despite the fact that these cells contain as many cell surface IFN receptors as sensitive cells. These observations raise the possibility that lower affinity insulin binding is important in the growth-promoting actions of insulin.     

The effect of human alpha leukocyte interferon (IFN) on the frequency of sister chromatid exchanges (SCE) in the KB cell line

Concentrations of 50, 500 and 5,000 iu/ml of natural human alpha leukocyte interferon (IFN) were added into the culture medium of KB cells, 4 h after serial passage, in the presence of 5-bromo-desoxyuridine (Brdu) at 10 micrograms/ml. Similar cultures without IFN were set up as controls. After 72 h of incubation, the harlequin technique (differential staining of sister chromatids) was applied in order to discriminate among the metaphases of different generations and to appreciate the frequency of sister chromatid exchanges (SCE) in the second generation cells. The incidence of sister chromatid exchanges was slightly increased following IFN treatment but no dose-effect relationships were observed. At the same time, cell cycle kinetics estimated as replication index (RI) and average generation time (AGT) was not modified in IFN-treated cells as against the controls.     

Alpha interferon inhibits early stages of the human immunodeficiency virus type 1 replication cycle.

In this study, we have analyzed the effect of human alpha interferon (IFN-alpha) on a single replication cycle of human immunodeficiency virus type 1 (HIV-1) infection in the lymphocytic cell line CEM-174, which is highly sensitive to the antiviral effects of IFN. Pretreatment of cells with 50 to 500 U of recombinant human IFN-alpha per ml resulted in a marked reduction in viral RNA and protein synthesis. The effect of IFN-alpha was dose dependent and was amplified in multiple infection cycles. IFN-induced inhibition of viral protein synthesis could be detected only when cells were treated with IFN-alpha prior to infection or when IFN-alpha was added up to 10 h postinfection, but not if IFN-alpha was added at the later stages of HIV-1 replication cycle or after the HIV-1 infection was already established. Analysis of the integrated HIV-1 provirus showed a marked decrease in the levels of proviral DNA in IFN-treated cells. Thus, in contrast to the previous studies on established HIV-1 infection in T cells, in which the IFN block appeared to be at the posttranslational level, during de novo infection, IFN-alpha interferes with an early step of HIV-1 replication cycle that occurs prior to the integration of the proviral DNA. These results indicate that the early IFN block of HIV-1 replication, which has been previously observed only in primary marcophages, can also be detected in the IFN-sensitive T cells, indicating that the early IFN block is not limited to macrophages.