Analogues of 2',5'-oligoadenylates (2-5A), the cordycepin (3'-deoxyadenosine) core trimer (Co3) and its 5'-monophosphate derivative (pCo3), were shown to display pronounced anti-human immunodeficiency virus type 1 (HIV-1) activity in vitro. Treatment of HIV-1 infected H9 cells with 1 microM Co3 or pCo3 resulted in an almost 100% inhibition of virus production. The compounds were encapsulated in liposomes targeted by antibodies specific for the T-cell receptor molecule CD3. Substitution of one or two cordycepin units in Co3 or pCo3 decreased the antiviral activity of the compounds. pCo3 did not stimulate 2-5A-dependent ribonuclease L activity and displayed no effect on the amount of cellular RNA and protein. At a concentration of 10 microM the cellular DNA polymerases alpha, beta, and gamma were almost insensitive toward Co3 or pCo3. In contrast, these compounds reduced the activity of HIV-1 reverse transcriptase (RT) by 90% at a concentration of 10 microM if the viral RNA genome and the cellular tRNALys.3 was used as template/primer system; if the synthetic poly(A).(dT)10 was used as template/primer, no marked inhibition was observed. Dot-blot, gel-retardation, and cross-linking assays showed that Co3 or pCo3 interfere with the binding site of tRNALys.3 to RT. These results indicate that inhibition of RT at the level of initiation of the enzymic reaction is a novel approach to inhibit HIV-1 replication.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of % 1 x 10" M-l. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 1251-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPasedriven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclearenvelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.

Sulphoevernan is a sulphated ~-1 --, 3, 1 -, 4 polyglucan (Mr 20000) with a helical structure. This compound effectively inhibits both human immunodeficiency virus type 1 (HIV-1) and type 2 infection of cells in vitro at concentrations around 0-5 ~tg/ml. Moreover, the compound completely inhibits HIV-l-induced syncytium formation at a concentration of 1 ~tg/ml. Competition experiments with 35S-labelled sulphoevernan revealed that the mannose-specific lectin from Narcissus pseudonarcissus prevented binding of sulphoevernan to HIV-1, whereas the antibody OKT4A did not reduce the amount of sulphoevernan bound to MT-2 cells. These data indicate that the non-cytotoxic polymer sulphoevernan binds to the virus rather than to the host cell. In vivo studies, using Rauscher leukaemia virus in NMRI mice, revealed that, at a daily dose of 20 mg/kg, the animals were protected against virus-induced increases in spleen weight. From these in vitro and in vivo data we conclude that sulphoevernan has potential in the treatment of acquired immunodeficiency syndrome.

2',3'-Dideoxy-3'-fluorothymidine (FddThd), 2',3'-didehydro-2',3'-dideoxythymidine (ddeThd), and 3'-fluoro-5-methyl-deoxycytidine (FddMeCyt) are, in their triphosphate forms, selective inhibitors of human immunodeficiency virus type 1 reverse transcriptase. We report that 0.3 ,uM FddThd, FddMeCyt, or ddeThd as well as 3'-chloro-5-methyl-deoxycytidine (ClddMeCyt) or 3'-amino-5-methyl-deoxycytidine (AddMeCyt) almost completely blocked production of hepatitis B virus (HBV) particles by HBV DNA-transfected cell line 2.2.15 in vitro. Only at an at least 10-fold-higher concentration was a cytotoxic effect observed. These results indicate that FddThd, FddMeCyt, ClddMeCyt, AddMeCyt, and ddeThd are potent anti-HBV agents in vitro.Hepatitis B is a disease with continuously increasing occurrence and is caused by the hepatitis B virus (HBV) (11). Vaccination against hepatitis B is one way of effectively preventing HBV infection (2). Two lines of treatment of hepatitis B infection have been followed: (i) treatment with cytokines (8) to substitute the impaired production of peripheral blood mononuclear cells (7, 32) and (ii) treatment with antiviral agents, e.g., adenine arabinoside (18,21,29). The rationale for a chemotherapeutic treatment for hepatitis B is the inhibition of the viral DNA polymerase (4).Recently, we found that the triphosphate of 2',3'-dideoxy-3'-fluorothymidine (FddThd) is a potent inhibitor of reverse transcriptase of human immunodeficiency virus, a weak inhibitor of cellular DNA polymerase ,, and not effective at all against DNA polymerase a (12). In addition, we established that FddThd is well phosphorylated to the 5'-triphosphate in relevant cell lines (13). Moreover, it was found that the triphosphate of FddThd and the triphosphates of 2',3'-didehydro-2',3'-dideoxythymidine (ddeThd) and 3'-fluoro-5-methyl-deoxycytidine (FddMeCyt) FddThd, FddMeCyt, ClddMeCyt, AddMeCyt, and ddeThd were synthesized as described previously (6, 9, 30; E. Matthes, C. Lehmann, D. Scholz, M. von Janta-Lipinski, K. Gaertner, P. Langen, and H. A. Rosenthal, European Patent 0254268A2, 1987).In vitro assay for antiviral activity. The human hepatoblastoma cell line HepG2 was transfected with pDolTHBV-1, a vector which contains HBV (27). The clonal line of cells was designated 2.2.15 and was found to secrete both hepatitis B surface antigen (HBsAg) and HBV DNA (27). The 2.2.15 cells were kept in RPMI 1640 medium supplemented with 2 mM glutamine and 10% (vol/vol) fetal bovine serum. The cultures were incubated at 37°C in 5% CO2 in air.Cells were planted at a density of 5 x 105/ml in plastic flasks. One hour later the compounds were added and incubation was continued for 4 days; the cultures reached confluence on day 5. On day 2 fresh serum was added to the culture medium and the respective compound concentration was renewed.Cell growth was determined by two methods. First, after the 4-day incubation period, the cultures were incubated for an additional 24 h with 3 pXCi of [3H]dAdo per ml. In order to avoid direct interference of th...

A new lectin has been isolated from the sponge Chondrilla nucula. The purified CN lectin is a protein composed of four polypeptide chains with a molecular weight (MW) of 15600. The isoelectric point is 4.5 and the amino acid composition is rich in aspartic and glutamic acid. The lectin precipitates erythrocytes from humans (A, B, O) with a titre between 25 and 210. The CN lectin is d-galactose-specific and displays a moderate mitogenic effect on spleen lymphocytes from mice and on CD4-positive human H9 cells. An interesting feature of this lectin is its ability to stimulate the (2′-5′)oligoriboadenylate [(2′-5′)A] metabolic pathway in non-infected and human immunodeficiency virus (HIV-1)-infected H9 cells. Moreover, HIV-1-infected H9 cells show an increase in the time period of HIV-1 release in response to CN lectin treatment. These data suggest that CN lectin causes a retardation of HIV-1 release due to further increase of the elevated intracellular level of (2′-5′)A.