Induction of interferon-alpha by glycoprotein D of herpes simplex virus: a possible role of chemokine receptors.

The induction of type I interferons by most RNA viruses is initiated by virus-derived double-stranded (ds)RNA. However, retro- and DNA-viruses, which do not synthesize dsRNA, must rely on different mechanisms of induction. For human immunodeficiency virus type 1 (HIV-1), recombinant glycoproteins 120 or 160 suffice to induce interferon (IFN)-alpha in blood-derived lymphocytes [H. Ankel, M. R. Capobianchi, C. Castilletti, and F. Dianzani (1994). Virology 205, 34-43]. Here we show that for herpes simplex virus type 1 (HSV-1) recombinant glycoprotein, gD is the major inducer, whereas gB, gC, gE, gG, gI, and the complex of gH and gL are poor inducers. The recombinant extramembrane fragment of gD was sufficient to induce IFN-alpha levels comparable to that of intact virus. Like with HIV-1, induction was inhibited by a monoclonal antibody that recognizes cerebrosides and sulfatides. Furthermore, monoclonal antibodies specific for the chemokine receptors CCR3 and CXCR4 also blocked induction. We conclude that HSV-1 induces IFN-alpha by interaction of its glycoprotein gD with appropriate receptors on IFN-producing cells. Based on the known receptor roles of galactosyl cerebrosides and chemokine receptors in HIV infection, such structures on IFN-producing cells could also participate in the induction of IFN-alpha by HSV-1.

Assessment of antiviral activity, efficacy, and toxicity of prostaglandin A2 in a rabbit model of herpetic keratitis.

Prostaglandin A2 (PGA2) inhibited the replication of herpes simplex virus type 1 in rabbit and human cornea stromal cells at concentrations of 1 to 5 microM while causing significant toxicity at 55 to 150 microM. Despite favorable therapeutic indices in cultured cells, PGA2 was not effective as a therapeutic agent in the treatment of herpetic keratitis in a rabbit model. The sequelae of disease appeared more severe in animals receiving PGA2 than in untreated or placebo-treated controls. The recovery of virus from tissues of latently infected rabbits was not affected by therapy. PGA2 therapy alone induced breakdown of the blood-aqueous barrier, indicating that pharmacologically active concentrations of drug were achieved in the eye. Thus, PGA2 had antiviral activity, but its proinflammatory effects appeared to be more detrimental than beneficial in the treatment of herpetic keratitis.

Interferon induction by HIV-1-infected cells: a possible role of sulfatides or related glycolipids.

We have investigated the mechanism of interferon (IFN) induction in peripheral blood mononuclear cells by HIV-1(IIIB)-infected H9 cells or by recombinant gp120. A monoclonal antibody specific for the galactosylsphingosinyl moiety in galactocerebrosides and sulfatides inhibited IFN induction in a dose-dependent manner. Furthermore, exogenous sulfatides inhibited with an ID50 of approximately 1 microM, whereas galactocerebrosides were not inhibitory at 40 times higher concentrations. These studies suggest that sulfate containing galactolipids such as sulfatides on responder cells may be part of the gp120-membrane complex that initiates the induction of IFN. A partial homology of an epitope on the V3 loop of gp 120 with a previously suggested binding domain for sulfated glycoconjugates supports this conclusion.

Antiviral effect of cyclopentenone prostaglandins on vesicular stomatitis virus replication.

Prostaglandins are potentially useful antiviral agents, however their mechanism of action is unclear. Recent evidence suggests that RNA transcription of vesicular stomatitis virus (VSV) is inhibited by prostaglandins (Bader and Ankel, J. Gen. Virol. 71, 2823-2832, 1990). Prostaglandins are known to have multiple effects on cells which may or may not be related to their antiviral action. We examined the effects of prostaglandins on cells and on VSV RNA polymerase in vitro to seek the mechanism of antiviral action. Actinomycin D inhibited cellular RNA synthesis but failed to block the antiviral activity of prostaglandins on VSV. Thus induction of host cell RNA transcription is not involved in the antiviral action. Neither modulation of the cellular glutathione level by prostaglandins nor formation of prostaglandin-glutathione conjugates was required for the antiviral action. The relative inhibition of VSV RNA polymerase in vitro by prostaglandins with different structures correlated to inhibition of VSV replication in infected cells. This result indicates that the same step in VSV replication is inhibited by prostaglandins both in the in vitro RNA polymerase assay and in the infected cell.

Interferon induction by HIV glycoprotein 120: role of the V3 loop.

We have studied the mechanism of IFN induction by HIV-1 in peripheral blood mononuclear cells (PBMC), using recombinant viral membrane glycoproteins as potential inducers. Whereas 8 nM HIVIIIB-derived gp120 resulted in IFN levels between 80 and 2000 IU/ml with PBMC from different donors, gp120 from the MN strain was not an inducer. Preincubation of HIVIIIB-gp120 with a monoclonal antibody (mAB) to its CD4 binding domain or of PBMC with a mAB to the gp120 binding domain of CD4 abolished IFN induction. Antibodies against the third extracellular domain of CD4 which did not block binding of gp120, however, were also inhibitory. Furthermore, several mABs to the third variable loop (V3) of HIVIIIB-gp120 also blocked IFN induction, suggesting an important role of V3 in this process. This was further supported by the inhibitory action of peptides homologous to complete or partial sequences of V3. We conclude that after binding of gp120 to its CD4 receptor the V3 loop can be positioned close to the membrane of the responder cells by bending of gp120-occupied CD4 at its hinge region between extracellular domains 2 and 3. As a result V3 is able to interact with a V3-specific "secondary receptor" on the membranes of these cells. We suggest that it is the latter interaction which triggers IFN induction.

Acid lability is not an intrinsic property of interferon-alpha induced by HIV-infected cells.

Human immunodeficiency virus (HIV)-infected cells induce acid-labile interferon-alpha (al-IFN-alpha) in cultures of mononuclear cells from peripheral human blood. We have investigated the physiochemical properties of such preparations to elucidate the reasons for acid-lability of this IFN. Al-IFN-alpha is a mixture of both glycosylated and unglycosylated molecules as shown by separation on Concanavalin-A Sepharose. Acid-lability is associated only with glycosylated molecules. Upon chromatography of the glycosylated fraction on Sepharose coupled to IFN-alpha-specific antibody, the portion of the IFN that is retained and eluted with guanidine-HCl is acid-stable, whereas the excluded antiviral activity is acid-labile, and is partially neutralized by antibodies to either IFN-alpha or IFN-gamma. Also, upon further purification of the unglycosylated fraction on the same antibody column, all antiviral activity remains indistinguishable from conventional IFN-alpha. Reconstitution experiments showed that glycosylated material excluded from the anti-IFN-alpha column potentiates antiviral activity of the IFN that is specifically retained by the column. This potentiation is abolished by acid treatment. Similar results are obtained with al-IFN-alpha from the serum of acquired immunodeficiency syndrome (AIDS) patients, indicating that its acid-lability is also the consequence of an acid-labile component that is capable of enhancing the antiviral activity. The potentiation of antiviral activity obtained by combining recombinant preparations of IFN-alpha and IFN-gamma suggests that the cooperating molecule is IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant glycoprotein 120 of human immunodeficiency virus is a potent interferon inducer.

Cells infected with human immunodeficiency virus (HIV) induce antiviral activity in peripheral blood mononuclear cells (PBMC) from healthy donors. This activity is neutralized by anti-interferon-alpha antibody and partially destroyed at pH 2. Previous studies with enriched cell populations and monoclonal antibodies suggest that B lymphocytes are the main IFN-producing cells, and that both CD4 and HLA class II antigens are essential for IFN induction. Since the initial event of HIV infection of CD4+ cells is the interaction of the virus coat glycoprotein gp120 with CD4 molecule, we investigated whether gp120 is responsible for IFN induction. Using PBMC and recombinant gp120 obtained from a baculovirus expression system, dose-dependent induction of antiviral activity was observed with titers approaching 10(3) IU/ml. This induction was blocked in the presence of antibody to gp120. The antiviral activity was characterized as IFN-alpha by neutralization with IFN alpha-specific antibody. Preincubation of PBMC with anti-CD4 or the presence of soluble CD4 during incubation inhibited IFN induction, indicating that interaction of gp120 with cell-associated CD4 is responsible for this induction. Neither lymphoproliferation nor interleukin-2 (IL-2) production was observed during IFN induction. However, class G immunoglobulin secretion was enhanced by gp120, indicating that B cells are direct or indirect targets of gp120 stimulation in this experimental system. Since gp120 is shed from HIV-infected cells and occurs in the serum of acquired immunodeficiency syndrome (AIDS) patients, our data suggest that this glycoprotein is responsible for the induction of endogenous IFN and the polyclonal activation of B cells both of which are observed in AIDS patients.

Formation of a prostaglandin A2-glutathione conjugate in L1210 mouse leukemia cells.

Prostaglandins containing a cyclopentenone moiety are potent antiviral and antigrowth compounds. Some evidence indicates that these prostaglandins are conjugated to glutathione by cells. However, the metabolism of one group, the prostaglandins of the A type, is unclear due to conflicting reports. We studied the uptake and metabolism of prostaglandin A2 (PGA2) in mouse L1210 leukemia and L929 fibroblast cell lines in which this prostaglandin has antiviral and antigrowth effects. Both cell types took up the PGA2 and then metabolized it to a more polar compound. Inside L1210 cells, PGA2 was initially conjugated to glutathione and then reduced at the 9-keto position to form 9-OH-PGA2-GSH. The 9-OH-PGA2-GSH was then secreted from the cells and apparently degraded to form the CysGly and Cys derivatives. Intracellular glutathione was decreased markedly by the addition of the PGA2 in L1210 and L929 cells. This result confirms that conjugation of PGA2 to glutathione occurs in both cell types. Formation of the 9-OH-PGA2-GSH and other glutathione-related conjugates was prevented when glutathione was depleted by growth in buthionine sulfoximine. The glutathione-depleted cells were insensitive to the cytotoxicity of the PGA2, suggesting that one of the glutathione-related conjugates may be involved in the cytotoxicity of PGA2. These results end the controversy over the metabolism of PGA2 and suggest mechanisms for its antiviral and antigrowth actions.

Antiviral activity of the prostanoid clavulone II against vesicular stomatitis virus.

Prostaglandins of the A series exhibit the most pronounced antiviral activity in cells infected with RNA or DNA viruses as compared to other prostaglandins. Clavulone is a prostaglandin A analog found in the soft coral Clavularia viridis. Using vesicular stomatitis virus in mouse L929 fibroblasts as a model system, 50% inhibition of viral yield was seen at a concentration of 1-1.5 microM, whereas 50% cytotoxicity required 50-70 times higher inhibitor concentrations. For a further elucidation of the antiviral mechanism a temperature-sensitive mutant, tsG 41, was used, which is replication-negative at the restrictive temperature. Results obtained with this mutant suggest that inhibition of VSV replication occurs at the level of transcription.

Prostaglandin A inhibits replication of human immunodeficiency virus during acute infection.

An antiviral effect of prostaglandins (PGs) of the A series on the replication of human immunodeficiency virus (HIV) has been determined. In the T cell line C8166 under single growth cycle conditions, PGA1 reduced the number of infectious progeny 1000-fold in the absence of cytotoxicity. Thus, inhibition of HIV replication by PGA1 represents a true antiviral phenomenon. The number and size of virus-induced syncytia, and the amount of viral antigen were also drastically reduced. The effect was specific for PGAs because PGA2 was also inhibitory, whereas PGB1, PGE1 and PGE2 were inactive. Virus adsorption and penetration do not appear to be targets of antiviral action because PGA1 substantially reduced virus replication, even when added 5 h post-infection. PGA1 did not inhibit viral reverse transcriptase, as determined by in vitro assays, suggesting that its antiviral action is not the consequence of a direct inhibitory effect on this enzyme. PGA1 also inhibited the replication of HIV-1 in CEM x 174 cells, but with less potency. Previously, intravenous infusion of PGA1 into human volunteers has shown no significant deleterious side-effects and thus these observations suggest that PGAs might have potential as antiviral agents in humans.

Role of glycosilation in the susceptibility of "acid labile" interferon alpha to acid treatment.

Mononuclear cells from blood of healthy donors produce acid-labile interferon (IFN) alpha when stimulated with HIV-infected cells. A large proportion of this IFN appears to be glycosilated, as treatment with neuraminidase causes a shift of the isoelectric point (IP) from pH = 5.2-5.4 to pH = 5.8-6.2. To assess the role of glycosilation in determining the instability of antiviral activity after exposure to acid (pH lower than 4) peripheral blood mononuclear cells (PBMC) were induced to produce IFN with HIV-infected cells in the presence of tunicamycin, an inhibitor of glycosilation. The IFN produced under such experimental conditions (tu-IFN) was acid-stable. Tu-IFN was compared to a standard acid-labile IFN by affinity chromatography on Con A-sepharose. The elution pattern showed that tu-IFN does not bind to the gel, whereas the acid-labile IFN is eluted in two fractions, one unbound, which is stable at pH2, and one bound, which retains the initial acid-lability. These results suggest that acid-labile IFN alpha is largely glycosilated, and that the presence of glycosilated molecules contribute to render the IFN molecule unstable at acidic pH. It is to be determined whether some glycosilated molecule present in the IFN preparation, or glycosilation of the IFN molecule per se, is responsible for acid-lability of the antiviral activity.

Inhibition of primary transcription of vesicular stomatitis virus by prostaglandin A1.

Prostaglandin A (PGA) exhibits antiviral activity against RNA and DNA viruses. The effect of PGA1 on vesicular stomatitis virus (VSV) was investigated. When VSV-infected L-1210 cells were kept in the presence of PGA1 the amount of all five viral proteins and their respective mRNAs was dose-dependently decreased. To determine whether the effect was on viral transcription or translation, the temperature-sensitive VSV mutant tsG 41 was employed. This is a good model system for the investigation of primary transcription; at the restrictive temperature of 39 degrees C, tsG 41 is unable to replicate but can transcribe viral mRNA. Mutant mRNA synthesis was strongly inhibited by PGA1 at this temperature, indicating that the major effect is on primary transcription. This conclusion is supported by data demonstrating that in vitro transcription of viral genomic RNA was also inhibited by PGA1.

Tumour necrosis factor enhances induction by beta-interferon of a ubiquitin cross-reactive protein.

Tumour necrosis factor alpha (TNF-alpha) elicited an antiviral response in some cell lines (MG-63 and HEp-2) but not in others (MDBK). Cell lines that generated an antiviral response to TNF-alpha also showed induction of a 15K protein which shared sequence homology with ubiquitin and reacted with an antibody to ubiquitin. This ubiquitin cross-reactive protein (UCRP) had been demonstrated previously to be induced by interferon. The TNF-alpha induction of UCRP occurred at the level of transcription. TNF-alpha induction of both the antiviral state and the 15K protein was blocked by either monoclonal or polyclonal anti-beta-interferon (IFN-beta) antibody. However no measurable increase in the mRNA specific for IFN-beta was detected after TNF-alpha treatment. Nonetheless, in supernatants from cell cultures, the presence of an antiviral activity inhibitable by anti-IFN-beta antibody indicates that these cells are making IFN-beta already. We conclude that the TNF-alpha induction of antiviral activity and UCRP in cells is dependent upon the presence of constitutive low levels of IFN-beta in the responding cells. Furthermore TNF functions to enhance the existing IFN-beta activity.

Natural killer cells discriminate between high mannose- and complex-type asparagine-linked oligosaccharides.

Chinese hamster ovary cell lines with different types of N-linked oligosaccharides were tested as targets for control and lymphokine treated natural killer (NK) cells. The targets tested were parent cells, Lec1 mutants and Lec4 mutants. Due to an apparent defect in GlcNAc transferase V, Lec4 cells produce complex-type N-linked oligosaccharides devoid of GlcNAc beta(1-6) linked branches. Lec1 cells form only high mannose-type N-linked oligosaccharides because they lack GlcNAc transferase I activity. Lec1 cells are very sensitive to lysis by beta-interferon treated human NK cells, but both parent and Lec4 cells are resistant to NK lysis. The ability to discriminate between parent and Lec1 targets was demonstrated with untreated control effectors as well as those which were pretreated with either beta-interferon, gamma-interferon or interleukin-2. Both control and lymphokine-boosted NK cells exhibit much greater lytic activity against targets having only high mannose-type N-linked oligosaccharides. Five oligosaccharide structures resembling those found on N-linked glycoproteins were tested for their ability to block NK lysis of Lec1 targets. Only the high mannose-type glycopeptide from 7S soybean glycoprotein was inhibitory in the mu molar range. At the same concentration, none of the complex-type oligosaccharides had any effect on lytic activity. The results suggest that a high mannose-type N-linked oligosaccharides is recognized at some step in NK cell-mediated lysis.

Interferon induces a 15-kilodalton protein exhibiting marked homology to ubiquitin.

Immunochemical methods were used to examine the effect of viral infection on the dynamics of intracellular ubiquitin pools. Infection of either the human lung carcinoma line A-549 or the mouse fibroblast line L929 with encephalomyocarditis virus had little effect on either the distribution or fractional level of intracellular ubiquitin conjugates. In contrast, viral infection resulted in a significant decline in the steady state content of the mono-ubiquitin conjugate to histone 2A (uH2A). Prior treatment with interferons protected against this decrease of uH2A. Furthermore, interferons induced the de novo synthesis of a 15-kDa protein immunologically related to ubiquitin. The ubiquitin cross-reactive protein (UCRP) was not constitutively present in control cells but was significantly induced in various cells sensitive to the biological effects of interferons. Induction of UCRP with respect to both time and interferon concentration dependence closely paralleled the appearance of resistance to viral infection and could be blocked by low levels of actinomycin D. Subsequent studies demonstrated that UCRP was identical to an interferon-induced 15-kDa protein whose sequence has recently been reported (Blomstrom, D. C., Fahey, D., Kutny, R., Korant, B. D., and Knight, E. (1986) J. Biol. Chem. 261, 8811-8816). An authentic sample of the 15-kDa protein was found to co-migrate with UCRP and to cross-react with two different anti-ubiquitin antibodies. Using the authentic 15-kDa protein as a standard, UCRP accumulated to 6.2 +/- 0.5 pmol/10(6) cells and 34 +/- 2 pmol/10(6) cells in interferon-treated A-549 and L929 cultures, respectively. Comparison of the primary sequence of the 15-kDa protein to that of ubiquitin indicated that the former is composed of two domains, each of which bears striking homology to ubiquitin. These observations suggest that the 15-kDa protein may represent one example of a functionally distinct family of ubiquitin-like proteins.

The role of asparagine-linked carbohydrate in natural killer cell-mediated cytolysis.

Chinese hamster ovary cell lines with specific lesions in the formation of glycoconjugates were tested for their sensitivity to lysis by interferon-boosted human natural killer cells. We report here that the type of asparagine-linked carbohydrate present on target cell glycoproteins determines their susceptibility to natural killer lysis. The targets tested were Chinese hamster ovary parent cells and Lec1, Lec2, and Lec8 mutants. Lec8 and Lec2 cells show an overall reduction of galactose and/or sialic acid in their glycoconjugates due to defects in the translocation of UDP-galactose and CMP-sialic acid, respectively. Due to a specific block in N-linked carbohydrate processing, Lec1 cells produce only high mannose-type oligosaccharides, but their glycolipids are identical to those of the parent. Both Lec2 and Lec8 mutants are more sensitive to natural killer lysis than the parent cells. This is consistent with their extensive reduction in cell surface sialic acid. Furthermore, Lec1 mutants are more susceptible to natural killer lysis than the parent cells. To confirm that the increased natural killer sensitivity of Lec1 cells was due to the modification of N-linked carbohydrate, parent cells were treated with swainsonine, a specific inhibitor of N-linked oligosaccharide processing. Swainsonine-treated parent cells are nearly as sensitive to natural killer lysis as the Lec1 mutants.

Differential effects of beta- and gamma-interferons on natural killer cell-mediated lysis of lung carcinoma cells.

Peripheral blood lymphocytes from healthy donors (PBL) poorly lyse lung carcinoma cell lines A-549, A-427 and SK- MES-1 when tested in a short-term chromium release assay. When PBL are preincubated with human beta-interferon (IFN-beta), these cell lines are lysed with an efficacy comparable to that of erythroleukemia K-562 cells, the standard targets used in natural killer cell assays. However, when PBL are preincubated with gamma-interferon (IFN-gamma) instead, lysis of the lung carcinoma lines is little augmented. Unlabeled lung carcinoma A-549 cells block chromium release from labeled K-562 cells with non-boosted and IFN-gamma or IFN-beta-boosted effector cells. Also with the IFN-beta treated effectors, chromium release from A-549 targets is inhibited by unlabeled K-562 cells. Therefore, cells that lyse K-562 cells must be able to recognize A-549 cells, and, in the case of IFN-beta pretreated effectors, cause the killing of these cells as well. Data obtained with effector cells separated on discontinuous Percoll gradients also indicate that the same cells that lyse A-549 cells are responsible for lysis of K-562 cells. We conclude that in response to IFN-beta, effector cells previously able to lyse K-562, but unable to lyse A-549 targets, mature into fully competent killer cells capable of lysing tumor cells from lymphoid as well as from lung cancer origin. This effect is not elicited by IFN-gamma, indicating that killer cells respond differently to both interferon types.

Antiviral activity of prostaglandin A on encephalomyocarditis virus-infected cells: a unique effect unrelated to interferon.

Antiviral effects of prostaglandins of the A series (PGAs) on Sendai, vaccinia and vesicular stomatitis viruses have previously been reported and a relationship between the antiviral actions of PGAs and interferons has been suggested. We have investigated the antiviral activity of PGAs on encephalomyocarditis (EMC) virus. Using single-cycle assays of virus replication our results indicate that PGAs only inhibit when present in the culture medium after the cells are infected, and that they are most effective during incubation periods including from 3 to 5 h post-infection. Furthermore, viral RNA synthesis is blocked in infected cells treated with PGA and, as a result, viral antigens are greatly reduced in the cytoplasm of the cells 5 h post-infection. Since the antiviral effect of PGAs is unperturbed by actinomycin D, when cellular RNA synthesis is greatly reduced, it appears unlikely that induction of new cellular proteins is the reason for the antiviral activity of PGAs. In separate experiments we were unable to demonstrate directly the induction of interferon, or of the two dsRNA-dependent enzymes, 2',5'-oligoadenylate synthetase and protein kinase, which are greatly increased in interferon-treated cells. Thus, we conclude that the antiviral activity of PGAs is unrelated to the antiviral action of interferons and involves a unique mechanism independent of cellular protein synthesis.

Sensitivity to alpha/beta-interferon is independent of N-linked complex-type oligosaccharides on cell-surface-membrane glycoproteins.

The extent of involvement of carbohydrate structures in the mechanism of action of alpha and beta-interferon (IFN-alpha, IFN-beta) is undefined. In this report we examine the role of complex-type N-linked oligosaccharides in the response to these interferons. The response of mouse leukemia L 1210S cells, grown in the presence of swainsonine, an inhibitor of Golgi mannosidase II [Tulsiani, D. R. P., Harris, T. M. and Touster, O. (1982) J. Biol. Chem. 257, 7936-7939; Elbein A. D., Solf, R., Dorling, P. R. and Vosbeck, K. (1981) Proc. Natl Acad. Sci. USA 78, 7393-7397], to mouse IFN-alpha/beta, both with respect to antiviral and antigrowth effects, remains intact in spite of the total absence of complex-type N-linked oligosaccharides. Also, there is no difference in the response to human IFN-beta of a parental Chinese hamster ovary cell line and a mutant lacking beta-N-acetylglucosaminyltransferase I and therefore unable to synthesize complex-type N-linked oligosaccharides [Stanley, P., Callibot, V. and Siminovitch, L. (1975) Cell 6, 121-128]. These results are significant in permitting the conclusion that the carbohydrate-specific binding of IFN-alpha and IFN-beta to gangliosides cannot be due to a similarity of the ganglioside carbohydrate to that of a glycoprotein containing a complex-type N-liked oligosaccharide.

Binding of interleukin 2 to gangliosides.

Exogenous gangliosides inhibit interleukin 2 (IL2)-dependent growth of a T cell line, AKIL -1.E8. IL2 activity is retained by columns of ganglioside covalently linked to poly(L-lysine)-agarose and is not eluted with ethylene glycol but is completely recovered by elution with 1% SDS. The ability of gangliosides to inhibit IL2 activity is directly related to the complexity of their carbohydrate portion, and related ceramide derivatives at similar concentrations do not inhibit IL2 activity. We conclude that IL2 bound to exogenous gangliosides is inactive and that the carbohydrate portion of the ganglioside is crucial to its interaction with IL2.