Anti-retroviral effects of type I IFN subtypes in vivo.

Type I IFN play a very important role in immunity against viral infections. Murine type I IFN belongs to a multigene family including 14 IFN-alpha subtypes but the biological functions of IFN-alpha subtypes in retroviral infections are unknown. We have used the Friend retrovirus model to determine the anti-viral effects of IFN-alpha subtypes in vitro and in vivo. IFN-alpha subtypes alpha1, alpha4, alpha6 or alpha9 suppressed Friend virus (FV) replication in vitro, but differed greatly in their anti-viral efficacy in vivo. Treatment of FV-infected mice with the IFN-alpha subtypes alpha1, alpha4 or alpha9, but not alpha6 led to a significant reduction in viral loads. Decreased splenic viral load after IFN-alpha1 treatment correlated with an expansion of activated FV-specific CD8(+) T cells and NK cells into the spleen, whereas in IFN-alpha4- and -alpha9-treated mice it exclusively correlated with the activation of NK cells. The results demonstrate the distinct anti-retroviral effects of different IFN-alpha subtypes, which may be relevant for new therapeutic approaches.

In situ localization of globin messenger RNA formation. II. After treatment of Friend virus-transformed mouse cells with dimethyl sulfoxide.

Globin messenger RNA (mRNA) levels in Friend virus-transformed mouse cells have been estimated by in situ hybridization of DNA copy (cDNA) to fixed preparations of cells and by hybridization of cDNA to extracted cytoplasmic RNA in true solution. The results obtained by both methods agree in showing that a low level of globin mRNA can be detected in untreated Friend cells. The levels of hemoglobin and globin mRNA have also been correlated after treatment of Friend cells with dimethyl sulfoxide (DMSO). The results obtained by both experimental approaches show that there is a minimum period of treatment with DMSO required in order that Friend cells may become hemoglobinized, and that this period coincides with the time when globin mRNA accumulates. Moreover, bromodeoxyuridine prevents both hemoglobin and globin mRNA accumulation.

Broad-spectrum antiviral activity of Virazole: 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide.

Virazole is a synthetic nucleoside active in tissue culture against at least 16 DNA and RNA viruses. Applied topically, it inhibits herpetic keratitis in rabbits and tail lesions induced by herpes, vaccinia, and vesicular stomatitis viruses in mice. Injected intraperitoneally into mice, it inhibits splenomegaly and hepatomegaly induced by Friend leukemia virus and respiratory infections caused by influenza A(O), A(2), and B viruses and parainfluenza 1 virus. infections is also effective.

Lack of retrovirus gene expression in somatic cell hybrids of friend cells and teratocarcinoma cells with a teratocarcinoma phenotype.

Two types of hybrids between cells with erythroid phenotype (Friend cells) and teratocarcinoma cells can be distinguished: cell hybrids with an erythroid phenotype, which release or can be induced to release large amounts of Friend spleen focus-forming virus (F-SFFV) on exposure to bromodeoxyuridine and cell hybrids with a teratocarcinoma phenotype, which do not release Friend virus and are not inducible for F-SFFV release. In this paper, we attempted to relate these differences to the expression of F-SFFV and Friend murine leukemia virus (F-MuLV) functions. Teratocarcinoma phenotype hybrids retained F-SFFV-and F-MuLV-related provirus sequences. They did not express F-SFFV- or F-MuLV-related RNA or proteins. The hybrids differentiated to endoderm-like cells on exposure to retinoic acid or hexamethylene-bis -acetamide. These cells, in contrast to the teratocarcinoma phenotype (uninduced) cells expressing SSEA-1-like antigens, did not express SSEA-1-like antigens; they formed typical, prekeratin-staining cytoskeletal structures and could be induced to release mouse interferon. The differentiating cells, but not the uninduced teratocarcinoma hybrids, were infected productively with F-MuLV or the F-MuLV--F-SFFV complex. They, however, did not express endogenous F-SFFV. Endogenous F-SFFV functions could not be rescued by infection with F-MuLV. Induction of teratocarcinoma hybrids with retinoic acid did not activate endogenous F-MuLV or F-SFFV transcription or protein synthesis. These data demonstrated two control mechanisms of Friend virus repression: one which acted trans during formation of the cell hybrids and was maintained only in teratocarcinoma phenotype cells and the other which acted cis and was still operative during induction of endodermal differentiation.

Antiviral effects of amino acid derivatives with the fluorene substituent on murine leukemia viruses.

Two new amino acid derivatives with the fluorene substituent, when administered ip to female inbred ICR-CD1 mice inoculated with Friend murine leukemia virus, significantly inhibited virus-induced splenomegaly, reduced viable virus titers in spleen and plasma, and significantly prolonged survival time. These compounds also inhibited multiplication of the strains of the Friend and Moloney murine leukemia viruses in a cell culture system. The action of these compounds on murine leukemia virus was presumely different from that of tilorone.

Antiviral effect of N-phenylacetoaminomethylene-DL-p-nitrophenylalanine (A-101) on murine leukemia viruses.

N-Phenylacetoaminomethylene-DL-p-nitrophenylalanine (A-101), when administered ip to male DDY mice infected with Friend leukemia virus, significantly inhibited virus-induced splenomegaly, reduced viable virus titers in spleen and plasma, and significantly prolonged survival time. A-101 also inhibited multiplication of the Friend and Moloney viruses in tissue culture systems.

Cellular and serum involvement in protection against Friend leukemia virus.

Treatment of mice with the immunomodulator pyran copolymer inhibited leukemogenesis produced by Friend leukemia virus (FLV) complex, as evidenced by inhibition of the spleen focus-forming virus and lymphatic leukemia virus, as well as by a significant decrease in splenomegaly. In this report we present data suggesting that the protective effect of pyran is mediated by macrophages. Protection was conferred on normal recipient mice when peritoneal exudate cells from pyran-treated mice were transferred to recipient mice infected 24 hr later with FLV. Animals receiving pyran-activated peritoneal cells had a significant reduction of splenomegaly and of titers of spleen focus-forming virus and lymphatic leukemia virus than did control animals. In contrast, when glycogen-elicited peritoneal exudate cells were transferred, the mice were not protected. Pyran-activated peritoneal cells, but not normal peritoneal cells, also inhibited FLV growth in vitro. Serum from pyran-treated, but not glycogen-treated, mice also transferred resistance to FLV-infected mice.

Enhancement of viral gene expression in Friend erythroleukemic cells by 12-O tetradecanoylphorbol-13-acetate.

Tumor-promoting agents are known to inhibit the specific differentiation processes of several animal cell systems in vitro, including the Friend leukemia cell system. We have examined the effect of 12-O tetradecanoylphorbol-13-acetate (TPA) on the latter system and have investigated its action on Friend virus expression. At a concentration of 16.7 nM, TPA inhibits the dimethyl sulfoxide-induced Friend cell terminal differentiation and, at the same time, enhances the expression of the Friend virus genome, as demonstrated by a 2-fold increase in the amount of reverse transcriptase-containing particles released into the culture fluid and in the levels of virus-specific intracytoplasmic RNA. The greatest effect of TPA is evident after 24 hr of treatment. At this time, TPA exerts also its strongest effect upon the induction of the plasminogen activator. Our results indicate that two specific effects of TPA, i.e., block of differentiation and induction of plasminogen activator, correlate well in the Friend cell system with an extracellular and intracellular increase in virus expression.

Protective effect of cholesterol on Friend leukemic cells against photosensitization by hematoporphyrin derivative.

The cytotoxic effects of hematoporphyrin derivative (HPD) on Friend erythroleukemic cells were studied. Upon binding of the porphyrin to the cells, the fluorescence spectra was shifted from 613 to 633 nm regarding the main band and from 676 to 667 nm concerning the secondary band. The kinetics of HPD binding was then determined. Maximum binding already occurred at 60 s after exposure of the cells to HPD. It could be demonstrated that the effect of the photoactivated HPD on cell viability was drug, dose, and light fluorescence dependent. Cellular protein synthesis and Friend virus complex release from the cells were equally inhibited by the photodynamic sensitization of the drug, indicating no specific effect on virus maturation. Since cholesterol affects the fluidity of cell membranes, it was important to study the effect of cholesterol enrichment on the photodynamic sensitization by HPD. It was found that, while a 50% reduction in protein synthesis was monitored following treatment with 20 micrograms of HPD per ml and illumination by a 6-milliwatt white light for 60 s, no inhibition was observed following preenrichment of the cells with 0.5, 1, or 2% of cholesterol hemisuccinate. The same trend of cholesterol protection was demonstrated with longer illumination periods up to 10 min. The protective effect of cholesterol hemisuccinate was also seen using scanning electron microscopy. It is thus concluded that the cholesterol hemisuccinate content of Friend erythroleukemic cell membranes is an important factor in regulating the cytotoxicity of photoactivated HPD.

Protective influence of lactoferrin on mice infected with the polycythemia-inducing strain of Friend virus complex.

Purified iron-saturated human lactoferrin (LF) was assessed in vivo for effects on the survival rates of C57BL X DBA/2 f1 (hereafter called BD2F1) (Fv-2sr) mice and titers of spleen focus-forming viruses (SFFV) in BD2F1 and DBA/2 (Fv-2ss) mice inoculated with the polycythemia-inducing strain of the Friend virus complex (FVC-P). LF prolonged the survival rates and decreased the titers of SFFV in mice given FVC-P. Titers of SFFV, assayed 14 days after administration of FVC-P, were measured by the spleen focus-forming unit assay in secondary mouse recipients. Decreases in titers of SFFV were apparent when LF was given in vivo as a single bolus dose of 200 micrograms within 2 h of the Friend virus complex (FVC), or as a total dosage of 200 micrograms given on days 1, 2, 4, 7, 9, and 11 after FVC-P, and to a lesser degree when LF was given as a total dosage of 200 micrograms on days 3, 4, 7, 9, and 11 after FVC-P. No decreases in titers of SFFV were detected when LF was given up to 3 days before or more than 3 days after FVC-P. LF did not appear to be directly inactivating the viruses as it did not inactivate the SFFV or the Friend murine leukemia helper virus in vitro. The results suggest that the protective effect of LF in vivo is probably due to an action on cells responding to the FVC or to an action on cells which influence the cells responding to the FVC or which influence the virus. It has been shown elsewhere that LF decreases the percentage of marrow and spleen hematopoietic progenitor cells that are in DNA synthesis in vivo and this may be the means by which the protective effect of LF is mediated in mice given the FVC.

Bromodeoxyuridine inhibition of Friend leukemia cell induction. Mechanism of reversal by deoxycytidine.

Deoxycytidine (dC) reverses bromodeoxyuridine (brdUrd) inhibition of induction in Friend leukemia cells only if added within the first 6 h after the addition of brdUrd. dC is shown to reduce the uptake of [3H]brdUrd into both soluble nucleotide pools and DNA, substantially expand the dTTP pool, and result in a lower level of brdUrd substitution in DNA. When the conversion of dC to thymidine nucleotides is prevented in BATH medium (containing brdUrd, aminopterin, thymidine, and hypoxanthine), dC no longer reverses brdUrd inhibition. These results show that dC exerts its primary effect via alterations in thymidine pools and probably through the resultant lower substitution of brdUrd in DNA.

Modulation of Friend virus infectivity in vivo by administration of purified preparations of human lactoferrin and recombinant murine interleukin-3 to mice.

Purified iron-saturated human milk lactoferrin (LF) and purified recombinant murine interleukin-3 (IL-3) were assessed in vivo for their effects on replication of spleen focus forming viruses (SFFV) in spleens of DBA/2 mice injected with the polycythemia-inducing strain of the Friend virus complex. LF and IL-3, inoculated 2 hr prior to the administration of the polycythemia-inducing strain of the Friend virus complex, respectively decreased and increased the replication of SFFV in mice as assessed by the spleen focus forming unit assay in primary and secondary DBA/2 mice. Since virus infectivity is associated with the DNA synthetic phase of the cell cycle and it has been shown elsewhere that LF decreases and IL-3 increases the percent of hematopoietic progenitor cells in S-phase in vivo, the results suggest that the opposing actions of LF and IL-3 on replication of SFFV may reflect the actions of these molecules on cycling of the target cells for SFFV.

Mucosal cytokine therapy: marked antiviral and antitumor activity.

Mucosal administration of the Th1 stimulatory cytokines interleukin-2 (IL-2), IL-12, IL-15, IL-18, or granulocyte-macrophage colony-stimulating factor (GM-CSF) induced antiviral activity in mice challenged systemically with a lethal dose of encephalomyocarditis virus (EMCV) similar to that observed following parenteral administration. In contrast, mucosal administration of the Th2 stimulatory cytokines IL-4, IL-5, IL-10, or IL-13 did not affect significantly the survival of EMCV-infected animals. Mucosal administration of IL-2 or IL-12 also exerted a marked antitumor activity in mice inoculated intravenously with Friend erythroleukemia cells. Recombinant IL-2 and IL-18, but none of the other recombinant cytokines tested, induced low levels of IFN in vitro. Polyclonal antibodies to both mouse and human interferon-alpha/beta (IFN-alpha/beta) abrogated the antiviral activity of IL-2 in vivo, even though the anti-human IFN-alpha/beta antibody did not neutralize mouse IFN-alpha/beta, and neither antibody bound to IL-2. IL-15 did not exhibit antiviral activity in IFN-alpha/beta R-/- mice, which are deficient in natural killer (NK) cell activity. These results suggest that mucosal Th1 cytokine therapy induces a soluble factor or activates a specific cell population in the lymphoid or epithelial tissue of the oropharyngeal cavity, which potentiates elimination of virus-infected or neoplasic cells systemically.

Impeded progression of Friend disease in mice by an inhibitor of retroviral proteases.

The protease of the human immunodeficiency virus type 1 (HIV-1) is essential for the processing of GAG and POL polyproteins and maturation of the virus particles. Using recombinant protease and a truncated GAG polyprotein as substrate, we developed a Western blot assay for the evaluation of inhibitors of the enzyme. Two statine-based inhibitors of the enzyme, KH161 and KH164, were effective in blocking the replication of HIV-1 in acutely infected human T4 lymphoid cells, with potency approaching that of zidovudine (ZDV) when tested in parallel. In chronically infected cells, the production of infectious virus was inhibited by KH161 and KH164, while ZDV was ineffective. Both KH161 and KH164 were also active as antivirals against the replication of murine leukemia virus (MLV) in cultured mouse cells. In an animal model of a murine retroviral disease, KH164 was shown to inhibit in a dose-dependent manner the progression of the disease induced by Friend virus complex (a mixture of Friend MLV and spleen focus-forming virus). The results suggest that the progression of the acquired immune deficiency syndrome (AIDS) may be impeded by inhibitors of HIV-1 protease.

Early-initiated zidovudine therapy prevents disease but not low levels of persistent retrovirus in mice.

An F1 hybrid mouse strain containing the Rfv-3r/s genotype was inoculated with Friend virus complex (FV) and treated with zidovudine (ZDV) intraperitoneally three times daily for 20 days beginning as early as 10 min after initial viral exposure. This strain of mice develops FV-specific neutralizing antibodies that aid in reducing viremia and splenic virus titers but do not prevent splenomegaly and eventual FV-associated death. The virally exposed mice treated with ZDV did not develop splenomegaly or have detectable viremia after the last drug treatment. On day 21, a single animal had demonstrable virus in the spleen as determined by a focal immunoenzyme assay; 57% had detectable virus at 5 weeks, but non displayed splenic virus after 35 weeks. None of the animals died after the 35-week holding period, compared to 38% dying in placebo-treated mice. To detect low levels of the virus, or potentially latent virus, splenocytes were cocultivated with a cell line known to readily propagate FV, and the cells were subsequently passaged four times to amplify replication of the virus. After amplification, a significant increase was seen in the number of mice testing positive for virus. Thus, ZDV treatment initiated early after virus exposure was effective in preventing FV-induced splenomegaly and death, but did not prevent low levels of persistent retrovirus in the mice.

Unsaturated and carbocyclic nucleoside analogues: synthesis, antitumor, and antiviral activity.

A series of unsaturated analogues of nucleosides were prepared and their cytotoxic, antitumor, and antiviral activities were investigated. Alkylation of cytosine with (E)-1,4-dichloro-2-butene gave chloro derivative 2f, which was hydrolyzed to alcohol 2h. Cytosine, adenine, 2-amino-6-chloropurine, thymine, and (Z)-1,4-chloro-2-butene gave compounds 4c-f, which, after hydrolysis, afforded alcohols 4a, 4b, 4g, and 4h. Alkenes 4d and 4e were cyclized to heterocycles 12 and 13. Alkylation of 2,6-diaminopurine with 1,4-dichloro-2-butyne led to chloro derivative 6a, which was hydrolyzed to alcohol 6b. Allenic isomerization of 6b gave compound 5c. Chloro derivatives 2e-g, 4c-f, 5d, and 6c-e as well as pyrimidine oxacyclopentenes 9c and 9d are slow-acting inhibitors of murine leukemia L1210 of IC50 10-100 microM. The most active were analogues 4c, 4d, 4e, and 6e (IC50 10-20 microM). The corresponding hydroxy derivatives were less active of inactive. Inhibition of macromolecular synthesis with compounds 4c, 4d, 6e, 9c, and 9d follows the order: DNA greater than RNA greater than or equal to protein. Cytotoxic effects of 4c, 6e, and 9d are not reversed with any of the four basic ribonucleosides or 2'-deoxyribonucleosides. Inhibitory activity of cytosine derivative 9c is reversed with uridine and 2'-deoxyuridine but not with the corresponding cytosine nucleosides. Zone assays in several tumor cell lines show that active compounds are cytotoxic agents with little selectivity for tumor cells. Analogue 6c showed 16.7% ILS in leukemia P388/o implanted ip in mice at 510 and 1020 mg/kg, respectively. Cytallene (5b) and 6'beta-hydroxyaristeromycin (10) exhibited significant activity against Friend and Rauscher murine leukemia viruses. The rest of the hydroxy derivatives, with the exception of 4a, were moderately effective or inactive as antiviral agents. None of the chloro derivatives or oxacyclopentenes exhibited an antiviral effect at noncytotoxic concentrations. Z-Olefin 4b and 2-aminoadenallene (5c) are substrates for adenosine deaminase.

Accumulation of unintegrated viral DNA in rat glial cells infected with neurotropic Friend murine leukemia virus.

FrC6 virus isolated from Friend murine leukemia virus (FLV) as a neurotropic virus clone induced a high-degree of accumulation of unintegrated viral DNA during infection of rat glial cells by 72 h post-infection. When anti-FLV neutralizing antibody, dextran sulfate, or 3'-azido-3'-deoxythymidine (AZT) was added to the culture within 24 h after infection, the accumulation of unintegrated viral DNA was inhibited. However, after 30-36 h post-infection, addition of anti-FLV antibody or dextran sulfate scarcely inhibited the accumulation of the unintegrated viral DNA, while addition of AZT at 30-36 h post-infection still reduced the amount of unintegrated viral DNA. Our results demonstrate that after 30-36 h post-infection, when second-round infection had already taken place, further superinfection with cell-free virus to glial cells was not required for the accumulation of unintegrated viral DNA. Possible mechanisms are discussed.

Induction of differentiation in Friend erythroleukemia cells with dimethyl sulfoxide, hexamethylene bisacetamide and sodium butyrate is not accompanied by changes in proviral DNA or its expression.

The integration pattern, copy number and DNA expression of the Friend virus genome was examined in Friend erythroleukemia cells induced to differentiate with dimethyl sulfoxide, hexamethylene bisacetamide and sodium butyrate. The integrated proviral DNA in Friend erythroleukemia cells was examined by Southern hybridization with a cloned Friend virus (F-MuLV) probe at days 1 and 4 following inducer treatment, as well as on day 6 at which time the cells had remained for 48 h in inducer free medium. KpnI fragments 9 and 5.7 kb long were observed. The copy number of each fragment remained constant throughout the erythroid differentiation process. EcoRI digestion of DNA isolated from cells at different times following the inducer treatment demonstrated multiple integration sites of the proviral genome, which also remained constant during the differentiation process. Proviral DNA expression was examined at 4 h and 4 days following inducer treatment as well as on day 6 by which time cells remained for 48 h in inducer free medium. Northern blot hybridization to the F-MuLV probe indicated no change in the provirus gene expression independent of the class of inducers. These observations reinforce our conclusions that the viral genome and its transcription product do not play a major role in the differentiation process of Friend erythroleukemic cells.

Antiretroviral activities of hypericin and rose bengal: photodynamic effects on Friend leukemia virus infection of mice.

The ability of hypericin to protect mice from splenomegaly resulting from infection with Friend leukemia virus (FLV) was re-examined in light of recent evidence showing that light is absolutely required for this drug's antiviral activity. FLV-induced splenomegaly was not prevented or ameliorated in mice injected with 100 micrograms hypericin, either mixed with the FLV inoculum or administered 1 day p.i., either under normal laboratory light or in the dark. These results contradict previous findings. Both hypericin and rose bengal, however, inactivated the FLV inoculum at low doses (< 11 micrograms), provided that the mixture was illuminated for 1 h under a normal fluorescent desk lamp. This procedure protected mice completely from FLV-induced splenomegaly, and provided a possible explanation for the discrepancy between our results and those reported previously. We conclude that for FLV, as for other enveloped viruses studied previously, illumination of hypericin with the virus is absolutely required for hypericin's antiviral (virucidal) effects, thus limiting its potential usefulness as an antiretroviral agent.

Factors influencing zidovudine efficacy when administered at early stages of Friend virus infection in mice.

Strategies for zidovudine (AZT) administration in retrovirus infection may greatly influence treatment efficacy, especially in the case of early intervention. Antiretroviral activity of AZT in mice infected with Friend leukemia virus (FLV) has been investigated using various experimental protocols. Mice were inoculated with FLV and treated with AZT either 1 or 4 h after inoculation. A dose/effect relationship of AZT therapy was established for two different loads of virus inoculum. The effects of treatment duration (5 or 14 days) and route of administration (b.i.d. subcutaneous injection or administration in drinking water) were also evaluated. In all cases AZT therapy suppressed or reduced virus-induced splenomegaly and increased survival time. AZT therapy was more effective when started 1 h rather than 4 h after virus inoculation. A mutual influence between the dosage of the antiviral drug and the virus inoculum size was observed. A 5-day therapy was inadequate to suppress infection. AZT therapy led to similar results whether administered subcutaneously or in drinking water. The present results suggest that AZT efficacy declines when the inoculum size is increased, when the initiation of treatment is delayed and when treatment duration is shortened.