APOBEC-mediated viral restriction: not simply editing?

The APOBEC family of cytidine deaminases inhibit the mobility of diverse retroviruses, retrotransposons and other viruses. Initial reports proposed that these effects were due to the DNA editing capabilities of these enzymes; however, many recent studies have provided evidence suggesting that APOBEC proteins can inhibit these elements by several mechanisms, including editing-dependent and editing-independent processes. Investigating these modes of action and the potential contribution that each one makes to the antiviral activities of various APOBEC proteins is vital if we are to understand how APOBEC proteins protect host genomes from invading nucleic acids.

Cutting edge: increased NK cell activity in HIV-1-exposed but uninfected Vietnamese intravascular drug users.

We addressed the role of innate immunity in the protection against HIV-1 infection by studying NK cell function in 37 Vietnamese intravascular drug users (IDUs), who appeared to remain HIV-1 uninfected despite many years of high-risk exposure (exposed uninfected, EU), 10 IDUs who underwent seroconversion and 28 unexposed blood donors. Main results were: NK cell lytic activities against both the NK-susceptible K562 cell line and the NK-resistant Daudi cell line were significantly augmented in EU IDUs compared with either controls or seroconverters before or after seroconversion; NK cells producing the cytokines IFN-gamma and TNF-alpha and the beta chemokines CCL3, CCL4, and CCL5 were also increased in the EU IDUs, either after in vitro activation or without stimulation. The finding of an enhanced NK cell function in EU IDUs, especially compared with IDUs who became HIV-1 infected, supports the hypothesis that NK cells contribute to the protection against HIV-1 infection.

Antiviral agents with activity against human retroviruses.

The ability of various known anti-HIV antivirals to inhibit four different strains of human immunodeficiency virus type 1 (HIV-1), a strain of type 2 (HIV-2), and a human T-cell lymphotropic virus type I (HTLV-I) was tested. The tested substances included two sulfated polysaccharides (lentinan sulfate and dextran sulfate) and a nonsulfated polysaccharide PSK, E-P-LEM, glycyrrhizin sulfate, and nucleoside analogues (AZT and DHT). The effects of the substances were measured quantitatively with two different assays: (i) inhibition of cell-free viral infection and (ii) inhibition of the fusion reaction induced by cell-to-cell infection. The results showed that cell-free infection of HIV-1 and HIV-2 was almost completely blocked in the presence of all of the substances tested. However, cell-to-cell infection by HIV-1, HIV-2, and HTLV-I was inhibited only by the polysaccharides, E-P-LEM, and glycyrrhizin sulfate but not by the two nucleoside analogues. Moreover, the extent of inhibition of the fusion reaction by the substances varied significantly from strain to strain in HIV-1.

The effects of tumor promoters on HTLV-1 expression in a low-virus producer and a non-virus producer cell line.

Chemical agents including 5-iodo-2'-deoxyuridine (IUdR), 4-O-methyl 12-O-tetradecanoyl phorbol-13-acetate (TPA), phorbol, and the tumor promoters teleocidin and TPA were tested for their ability to induce Human T-cell leukemia-lymphoma virus Type I (HTLV-I) expression in the low-virus producer MT-1 cell line and in the non-virus producer C63/CRII-2 cell line, which contains at least one integrated HTLV-I genome equivalent per cell [26]. Viral antigen expression increased from 0.3% to 8.7% in the MT-1 cell line after treatment with IUdR, while TPA and teleocidin were marginally or non-effective as inducers. Chemicals did not induce HTLV-I antigen expression in the C63/CI(II-2) cell line. No enhancement of reverse transcriptase activity or alteration of proviral organization was observed after chemical treatment of MT-1 cells.

Close association between interleukin 2 receptor mRNA expression and human T cell leukemia/lymphoma virus type I viral RNA expression in short-term cultured leukemic cells from adult T cell leukemia patients.

Human T cell leukemia/lymphoma (T-lymphotropic) virus type I (HTLV-I) infection has been considered to be closely associated with the leukemogenesis of adult T cell leukemia (ATL), in which interleukin 2 (IL-2) receptors are abnormally expressed. In this study, however, Southern blot analysis revealed no gross rearrangement or obvious amplification of the IL-2 receptor gene in ATL leukemic cells, indicating that abnormal IL-2 receptor expression in ATL is not due to the structural change of its gene. Hence, we studied the expression of the IL-2 receptor and HTLV-I at the RNA level during short-term cultures of leukemic cells from 9 ATL patients. Cytoplasmic dot hybridization and Northern hybridization revealed that fresh leukemic cells from seven of nine patients expressed a small amount of IL-2 receptor mRNA but HTLV-I RNA was undetectable in all cases. After cultures for up to 7 d, both IL-2 receptor mRNA and HTLV-I RNA (including pX message) expression concomitantly increased, whereas the amounts of other cellular genes, except for beta-actin, did not. The increases in their RNA expression were inhibited by early addition (within 12 h after the beginning of the culture) of cycloheximide, indicating that these increases are mediated by newly synthesized protein(s). These results strongly suggested that IL-2 receptor expression is closely associated with HTLV-I expression in leukemic cells from ATL patients.

A biological response modifier, PSK, inhibits human immunodeficiency virus infection in vitro.

PSK, a biological response modifier (BRM), was studied to determine its anti-viral activity on human immunodeficiency virus (HIV) in vitro. Either a novel infection system using human T-cell lymphotropic virus type I (HTLV-I)-carrying MT-4 cells or a coculture system using MOLT-4 cells and its virus-producing cells MOLT-4/HIVHTLV-IIIB which induces multinucleated giant cells very efficiently was used. PSK almost completely blocked the cytopathic effect such as giant cell formation and HIV-specific antigen expression both in MT-4 cells and MOLT-4 cells at a concentration of 0.4 and 0.8 mg/ml, respectively. Pretreatment of the virus with PSK may specifically interfere with early stages of HIV infection by modifying the viral receptor.

Pharmacological inhibition of in vitro infectivity of human T lymphotropic virus type I.

Human T lymphotropic virus type I (HTLV-I) is an exogenous RNA tumor virus etiologically linked to adult T cell leukemia and related diseases. In this paper, we describe that two 2',3'-dideoxynucleoside analogues, erythro 3'-azido-2',3'-dideoxythymidine (also called azidothymidine) and 2',3'-dideoxycytidine can inhibit the infectivity of HTLV-I against helper/inducer T cells in vitro. Both 2',3'-dideoxynucleoside analogues inhibited the overgrowth of target T cells, which was a consequence of virally mediated transformation, when they were exposed to the virus and cultured with the compounds. A profound decrease in the expression of HTLV-I gag-proteins was also observed. Moreover, we observed that the amount of proviral DNA detected in cellular DNA from the target T cells was substantially reduced when the cells were protected by the compounds against the virus and that at certain concentrations of the compounds the synthesis of viral DNA was completely suppressed. These results may be of value in developing a new pharmacological strategy for preventing the replication and possibly blocking the transmission of HTLV-I and related retroviruses in human beings.

CD4 receptor binding peptides that block HIV infectivity cause human monocyte chemotaxis. Relationship to vasoactive intestinal polypeptide.

The octapeptide Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr (peptide T) and two structural analogs are potent agonists of human monocyte chemotaxis, evincing identical rank potency orders as was previously shown for their inhibition of human immunodeficiency virus (HIV) envelope binding and T cell infectivity. Chemotactic activity could be inhibited by anti-CD4 monoclonal antibodies (Mabs), but not other mononuclear cell Mabs. The core peptide required for chemotactic activity is a pentapeptide related to the sequence Thr-Thr-Asn-Tyr-Thr. Homologous pentapeptides, identified by computer search, were detected in several other non-HIV-related viruses as well as the neuropeptide vasoactive intestinal polypeptide (VIP). The CD4 molecule, therefore, appears to be a recognition molecule for a small signal peptide ligand whose active sequence is a homolog of peptide T and which may be the neuropeptide VIP.

Substances affecting the infection and replication of human immunodeficiency virus (HIV).

The effects of various compounds were studied quantitatively on the growth of human immunodeficiency virus (HIV). For this we used a human T-cell leukemia virus type I carrying cell line, MT-4 which is most permissive for HIV infection. The results are summarized as follows: 1) Prostaglandin E2 and 12-0-tetradecanoylphorbol-13-acetate enhanced the production of HIV significantly in MT-4 cells as well as a continuous HIV producer Molt-4/HTLV-III cells. 2) Interferon gamma, retinoic acid and 3'-azido-3'-deoxythymidine inhibited the replication of HIV at the concentrations which were not cytotoxic. Mechanism of action of these compounds and its clinical implications are discussed.

Recombinant human interferon gamma suppresses HTLV-III replication in vitro.

Effect of human interferon gamma (rINF gamma) on HTLV-III replication was evaluated quantitatively via a novel infection system using HTLV-I-carrying MT-4 cells. Treatment of HTLV-III-infected MT-4 cells with different concentrations (I-1,000 U/ml) of rINF gamma, which did not affect the growth or viability of uninfected cells, significantly blocked the appearance of immunofluorescent antigens of HTLV-III and the virus-induced cytopathic effect in a dose-dependent manner. A plaque assay was applied to measure the exact amount of viral particles released from HTLV-III-infected MT-4 cultures either untreated or treated with rINF gamma after infection. The number of plaques per dish decreased with increasing drug concentrations. About 50% and 80% of HTLV-III replication were inhibited by the addition of 100 and 1,000 U/ml of rINF gamma, respectively. The effects of INF were observed by day 5 of incubation with the chemical. However, longer treatment of cells with rINF gamma permitted a gradual increase in viral replication. Re-addition of fresh INF into cultures did not change this pattern significantly.

Synthesis and antiviral activity of certain nucleoside 5'-phosphonoformate derivatives.

(Ethoxycarbonyl)phosphonic dichloride (3) was synthesized by chlorination of bis(trimethylsilyl) (ethoxycarbonyl)phosphonate with thionyl chloride. Adenosine 5'-(ethoxycarbonyl)phosphonate (4), guanosine 5'-(ethoxycarbonyl)phosphonate (5), 2'-deoxyadenosine 5'-(ethoxycarbonyl)phosphonate (18) and 2'-deoxyguanosine 5'-(ethoxycarbonyl)phosphonate (19) were synthesized by coupling of compound 3 with adenosine, guanosine, 2'-deoxyadenosine, and 2'-deoxyguanosine, respectively. Alkaline treatment of 4, 5, 18, and 19 gave the corresponding adenosine 5'-(hydroxycarbonyl)phosphonate (14), guanosine 5'-(hydroxycarbonyl) phosphonate (15), 2'-deoxyadenosine 5'-(hydroxycarbonyl)phosphonate (20), and 2'-deoxyguanosine 5'-(hydroxycarbonyl) phosphonate (21). Treatment of 4 and 5 with methanolic ammonia resulted in the production of adenosine 5'-(aminocarbonyl)phosphonate (12) and guanosine 5'-(aminocarbonyl)phosphonate (13), respectively. The nucleotide analogue 20 exhibited the most potent antiviral activity of this group of nucleotide tested in vitro and was most active against herpes viruses especially HSV-2. The nucleotide analogue 21 had lower, but significant, activity against HSV-2. All of the compounds tested were nontoxic to confluent Vero cells at concentrations as high as 5000 microM.

Synergistic inhibition of human T-cell lymphotropic virus type III replication in vitro by phosphonoformate and recombinant alpha-A interferon.

Phosphonoformate and recombinant alpha-A interferon synergistically inhibited the replication of human T-cell lymphotropic virus type III in cultured peripheral blood lymphocytes. T-cell proliferative capability was maintained by this combination, and toxicity was minimal.

Neutralization of HTLV-III/LAV replication by antiserum to thymosin alpha 1.

An antiserum prepared against thymosin alpha 1, a hormone secreted by the thymus gland, effectively neutralized the AIDS-associated virus [HTLV-III/LAV (clone BH-10)] and blocked its replication in H9 cells. Reverse transcriptase activity and expression of the HTLV-III/LAV antigens p15 and p24 were inhibited by purified immunoglobulin G preparations of antisera to thymosin alpha 1. The antiviral activity of the antiserum was found to be due to a region of homology between thymosin alpha 1 and p17, a product of the gag gene of HTLV-III/LAV. Comparison of the primary sequences of thymosin alpha 1 and the gag protein revealed a 44% to 50% homology in an 18-amino acid region, between positions 11 and 28 on thymosin alpha 1 and 92 and 109 on the gag protein. The effectiveness of the thymosin alpha 1 antiserum and of immunoglobulin G-enriched preparations in blocking replication of HTLV-III(BH-10) in H9 cells suggests a novel approach to the development of an AIDS vaccine. A vaccine directed against the gag protein might overcome the problem of genetic drift in the envelope region of the virus and be useful against all genetic variants of HTLV-III/LAV.