Effect of oxetanocin G, a novel nucleoside analog, on DNA synthesis by hepatitis B virus virions.

The novel nucleoside oxetanocin G, 9-(2-deoxy-2-hydroxymethyl-beta-D-erythro-oxetanosyl)guanine (OXT-G), that is a derivative of oxetanocin A, was studied in relation to its action on the synthesis of hepatitis B virus (HBV) DNA and cellular DNA in an HBV-producing cell line, HB611 (T. Tsurimoto, A. Fujiyama, and K. Matsubara, Proc. Natl. Acad. Sci. USA 84:444-448, 1987). The median effective concentration of OXT-G against HBV replication was 1.5 microM, and the median cytotoxic concentration was more than 1,000 microM. At the same concentration, OXT-G did not inhibit cellular DNA synthesis or viral RNA synthesis. Chemically synthesized OXT-GTP inhibited the HBV endogenous DNA polymerase reaction and was incorporated into HBV DNA strands at a low efficiency compared with the incorporation of dGTP. A synthetic primer-template study revealed that OXT-GTP was incorporated into DNA strands at a low efficiency and that further extension of the DNA strand by using the 2' position of the incorporated OXT-G could take place.     

Inhibition of infectivity of human immunodeficiency virus by a novel nucleoside, oxetanocin, and related compounds.

Oxetanocin is a novel nucleoside containing a 4-membered sugar, oxetanosyl-N-glycoside, and adenine. The effects of oxetanocin and related compounds on the infectivity of human immunodeficiency virus (HIV) were examined. They inhibited HIV infectivity in vitro. Allopurinol and mycophenolic acid produced additive anti-HIV effects when added with these compounds.     

Mechanism of selective inhibition of human cytomegalovirus replication by 1-beta-D-arabinofuranosyl-5-fluorouracil.

Four kinds of 1-beta-D-arabinofuranosyl-5-halogenouracil were examined for inhibition of human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) replication. 1-beta-D-Arabinofuranosyl-5-fluorouracil (ara-FU) was the most effective against HCMV, whereas 1-beta-D-arabinofuranosyl-5-bromouracil was the most effective against HSV-1 and HSV-2. The mechanism of action of ara-FU on HCMV replication was also studied. The dTTP pool size in human embryonic fibroblasts was increased 33-fold by HCMV infection. However, treatment with ara-FU decreased the size of the dTTP pool by approximately 50%. On the other hand, 1-beta-D-arabinofuranosyl-5-fluorouracil-5'-triphosphate inhibited HCMV DNA polymerase activity competitively with dTTP. These results suggest that ara-FU acts as a bifunctional inhibitor of HCMV replication. Ara-FU is phosphorylated by cellular thymidine kinase to 1-beta-D-arabinofuranosyl-5-fluorouracil-5'-monophosphate, which inhibits cellular thymidylate synthetase, which in turn decreases the dTTP pool size in infected cells. As the dTTP pool size is reduced, inhibition of viral DNA polymerase by 1-beta-D-arabinofuranosyl-5-fluorouracil-5'-triphosphate becomes more efficient.     

Effects of the nucleoside analog 2''-nor-2''-deoxyguanosine on human cytomegalovirus replication.

The nucleoside analog 2'-nor-2'-deoxyguanosine (2'NDG) effectively inhibits the replication of several laboratory and clinical isolates of human cytomegalovirus. These isolates included viruses obtained from congenitally infected infants and patients suffering from acquired immune deficiency syndrome. The dose of 2'NDG that inhibited cytomegalovirus plaque formation ranged from 0.1 to 1.6 micrograms/ml. At 10 micrograms/ml, 2'NDG completely blocked the production of virus progeny but not the expression of immediate early and early virus gene functions. Cytomegalovirus DNA was not detectable in 2'NDG-treated virus-infected human embryo lung cells when assayed by CsCl density gradient centrifugation. In contrast, the guanosine analog acyclovir at 100 micrograms/ml did not inhibit the production of virus or the synthesis of cytomegalovirus DNA. In virus-infected cells, 2'NDG and acyclovir at 10 and 100 micrograms/ml, respectively, inhibited the incorporation of [3H]thymidine and 32Pi into cellular DNA by ca. 50%. Uninfected human embryo lung cells grown in these concentrations of acyclovir or 2'NDG exhibited a slightly transient lag phase but, overall, cell growth was not retarded, and there was no decrease in cell viability. The extended lag in cell division was not due to inactivation or breakdown of the antiviral compounds but may be due in part to a temporary decrease in cellular DNA synthesis.     

Potent and selective inhibition of human cytomegalovirus replication by 1263W94, a benzimidazole L-riboside with a unique mode of action

Benzimidazole nucleosides have been shown to be potent inhibitors of human cytomegalovirus (HCMV) replication in vitro. As part of the exploration of structure-activity relationships within this series, we synthesized the 2-isopropylamino derivative (3322W93) of 1H-beta-D-ribofuranoside-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and the biologically unnatural L-sugars corresponding to both compounds. One of the L derivatives, 1H-beta-L-ribofuranoside-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94), showed significant antiviral potency in vitro against both laboratory HCMV strains and clinical HCMV isolates, including those resistant to ganciclovir (GCV), foscarnet, and BDCRB. 1263W94 inhibited viral replication in a dose-dependent manner, with a mean 50% inhibitory concentration (IC(50)) of 0.12 +/- 0.01 microM compared to a mean IC(50) for GCV of 0.53 +/- 0.04 microM, as measured by a multicycle DNA hybridization assay. In a single replication cycle, 1263W94 treatment reduced viral DNA synthesis, as well as overall virus yield. HCMV mutants resistant to 1263W94 were isolated, establishing that the target of 1263W94 was a viral gene product. The resistance mutation was mapped to the UL97 open reading frame. The pUL97 protein kinase was strongly inhibited by 1263W94, with 50% inhibition occurring at 3 nM. Although HCMV DNA synthesis was inhibited by 1263W94, the inhibition was not mediated by the inhibition of viral DNA polymerase. The parent benzimidazole D-riboside BDCRB inhibits viral DNA maturation and processing, whereas 1263W94 does not. The mechanism of the antiviral effect of L-riboside 1263W94 is thus distinct from those of GCV and of BDCRB. In summary, 1263W94 inhibits viral replication by a novel mechanism that is not yet completely understood.     

The dynamics of human cytomegalovirus replication in vivo.

Cytomegalovirus (CMV) is generally described as a slowly replicating virus. During studies of immunocompromised patients, we observed rapid changes in the quantity of CMV DNA present in serial blood samples by quantitative-competitive polymerase chain reaction commensurate with a doubling time of <2 d. To further investigate the dynamics of replication in vivo, patients in three distinct situations were studied in detail: (a) those receiving intravenous ganciclovir; (b) those in whom ganciclovir-resistant strains appeared during long-term therapy; and (c) those in whom ganciclovir-resistant strains disappeared with alternative drug therapy. In all cases, it was possible to provide accurate estimates of the doubling time of CMV and its half-life of disappearance after antiviral chemotherapy. The results from all three approaches demonstrated that the doubling time/half-life of CMV in blood is approximately 1 d when frequent samples are collected. These results show that CMV DNA replication in vivo is a highly dynamic process. We conclude that the reputation of CMV as a slowly replicating virus based on the time taken to produce cytopathic effects in vitro is unwarranted. These findings have implications for the potency, dose, and duration of antiviral chemotherapy needed for the effective treatment of this important human pathogen.     

Interactions of 1263W94 with other antiviral agents in inhibition of human cytomegalovirus replication

Inhibition of human cytomegalovirus (HCMV) by 1263W94 was additive dosewise in combination with ganciclovir, acyclovir, and foscarnet. None of the commonly used anti-human immunodeficiency virus agents antagonized the inhibition of HCMV by 1263W94. The data were analyzed by a modified isobologram procedure that measures the strength and statistical significance of drug interactions.     

Phosphorothioate-modified oligodeoxynucleotides inhibit human cytomegalovirus replication by blocking virus entry

Studies in animal models have provided evidence that Toll-like receptor 9 (TLR9) agonists, such as synthetic oligodeoxynucleotides (ODNs) that contain immunostimulatory deoxycytidyl-deoxyguanosine (CpG) motifs (CpG ODNs), protect against a wide range of viral pathogens. This antiviral activity has been suggested to be indirect and secondary to CpG-induced cytokines and inflammatory responses triggered through TLR9 activation. However, few studies have addressed the potential of CpG ODNs as direct antiviral agents. Here, we report on the ability of some CpG ODNs to directly suppress, almost completely, human cytomegalovirus (HCMV) replication in both primary fibroblasts and endothelial cells. Murine CMV replication was inhibited as well, whereas no inhibition was observed for herpes simplex virus type 1, adenovirus, or vesicular stomatitis virus. The antiviral activity of these ODNs was significantly reduced when they were added after virus adsorption, indicating that their action may be primarily targeted to the very early phases of the HCMV cycle. In fact, the B-class prototype CpG ODN 2006 effectively prevented the nuclear localization of pp65 and input viral DNA, which suggests that it inhibits HCMV entry. Moreover, a CpG 2006 control, ODN 2137 without CpG motifs, also showed a potent inhibitory activity on the HCMV entry phase, indicating that the anticytomegaloviral activity is independent of the CpG motif. In contrast, a phosphodiester version of CpG 2006 showed reduced antiviral activity, indicating that the inhibitory activity is dependent on the phosphorothioate backbone of the ODN. These results suggest that this yet-unrecognized activity of CpG ODNs may be of interest in the development of novel anticytomegaloviral molecules.     

Selective inhibition of human diploid fibroblast collagen synthesis by interferons.

The effects of alpha- and gamma-interferons (IFNs) on collagen production by confluent human diploid fibroblasts in culture were examined. It was found that partially purified alpha-IFNs and affinity purified gamma-IFNs caused greater than 50% inhibition of collagen synthesis by these cells independently of their effect on cell proliferation. Recombinant alpha-IFNs showed a similar effect (38.8% inhibition), indicating that collagen synthesis inhibition was a constitutive property of IFNs. Collagen synthesis inhibition by IFNs was concentration dependent. Gel filtration chromatography of the newly synthesized proteins from the media of fibroblasts incubated with partially purified alpha-IFNs demonstrated a selective depression of molecules eluting in the region of procollagen. No detectable increase in collagen degradation products or underhydroxylation of procollagen was observed. Short-term kinetic studies further demonstrated that the major effect of IFNs was due to a net decrease in fibroblast collagen production rather than to impairment of secretion or increased extracellular degradation of the newly synthesized molecules. These results indicate that alpha- and gamma-IFNs are potent inhibitors of human fibroblast collagen production and suggest that they may play an important role in the regulation of normal and pathologic fibrogenesis.     

Selective inhibition of human immunodeficiency virus type 1 replication by the (-) but not the (+) enantiomer of gossypol.

The (-) enantiomer of gossypol but not the (+) enantiomer had good antiviral activity in peripheral blood mononuclear cells against human immunodeficiency virus type 1 at a concentration more than 20-fold lower than that required for cytotoxicity; however, in H9 cells the (-) enantiomer, although more potent as an antiviral agent, was more cytotoxic.     

Inhibition of human cytomegalovirus replication by benzimidazole nucleosides involves three distinct mechanisms

The benzimidazole nucleosides 2-bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB) and 2-isopropylamino-5,6-dichloro-1-(beta-l-ribofuranosyl)benzimidazole (1263W94, or maribavir) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. These inhibitors act by two different mechanisms: BDCRB blocks the processing and maturation of viral DNA, whereas maribavir prevents viral DNA synthesis and capsid nuclear egress. In order to determine by which of these two mechanisms other benzimidazole nucleosides acted, we performed time-of-addition studies and other experiments with selected new analogs. We found that the erythrofuranosyl analog and the alpha-lyxofuranosyl analog acted late in the viral replication cycle, similar to BDCRB. In marked contrast, the alpha-5'-deoxylyxofuranosyl analog of 2,5,6-trichloro-1-(beta-d-ribofuranosyl)benzimidazole (compound UMJD1311) acted early in the replication cycle, too early to be consistent with either mechanism. Similar to other reports on early acting inhibitors of herpesviruses, compound 1311 was multiplicity of infection dependent, an observation that could not be reproduced with UV-inactivated virus. HCMV isolates resistant to BDCRB and maribavir were sensitive to compound 1311, as were viruses resistant to ganciclovir, cidofovir, and foscarnet. The preincubation of host cells with compound 1311 and removal prior to the addition of HCMV did not produce an antiviral cellular response. We conclude that this newly discovered early mode of action occurs at a stage of viral replication after entry to cells but prior to viral DNA synthesis, thereby strongly suggesting that the trisubstituted benzimidazole nucleoside series possesses three distinct biochemical modes of action for inhibition of HCMV replication.     

Inhibition of ganciclovir-resistant human cytomegalovirus replication by Kampo (Japanese herbal medicine)

We examined the effect of Kampo on the replication of ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) in the human embryonic fibroblast cell line MRC-5. Treatment of HCMV-infected cells with Sho-seiryu-to (SST; Xiao-Qing-Long-Tang in Chinese) resulted in the inhibition of viral replication without affecting the cell growth. SST treatment decreased the synthesis of viral DNA, but had no virucidal effect on cell-free HCMV. However, the inhibitory effect of SST on HCMV replication was ablated by anti-interferon-beta (IFN-beta) antibody suggesting that SST inhibits the replication of GCV-resistant HCMV through the induction of IFN-beta. These results suggest that SST is a novel compund with potential as an anti-HCMV.     

Inhibition of human cytomegalovirus by trifluorothymidine.

The antiviral activity of trifluorothymidine (TFT) singly and in combination with other antiviral agents against human cytomegalovirus (HCMV) was evaluated by using an infectious center plaque reduction assay. The 50% inhibitory dose of TFT against six different patient HCMV strains was 0.57 (+/- 0.24, standard deviation) microM and ranged from 0.32 to 0.97 microM. The 50% inhibitory dose for the laboratory-adapted HCMV strain, AD-169, was 2.1 microM. When TFT (0.17 microM) was combined with human fibroblast interferon (25 U/ml), the combination was additive against all four HCMV isolates evaluated. Synergism was observed when TFT (0.17 microM) was combined with phosphonoformic acid (25 microM) for all strains studied or with acyclovir (20 microM) for three of the four clinical HCMV strains tested. Each of the three antiviral agents, when combined with TFT, exhibited additive effects against strain AD-169. TFT at concentrations of 0.5, 1.7, and 3.5 microM had an increasing inhibitory effect on uninfected human embryonic lung fibroblast (HEL) cell growth over 72 h, with 16% growth inhibition at 3.5 microM after 3 days. There was no increased toxicity to growing HEL cells when the paired antiviral agent combinations were evaluated. These findings suggest that TFT may be useful singly or in combination with other antiviral agents in treating HCMV infections.     

Cyclobut-A and cyclobut-G, carbocyclic oxetanocin analogs that inhibit the replication of human immunodeficiency virus in T cells and monocytes and macrophages in vitro.

Two newly synthesized carbocyclic oxetanocin analogs, (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine (cyclobut-A) and (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine (cyclobut-G) were tested for activity against the infectivity of human immunodeficiency virus (HIV) in vitro. A number of other carbocyclic oxetanocin analogs failed to exert good antiretroviral effects. Both cyclobut-A and cyclobut-G protected CD4+ ATH8 cells against the infectivity and cytopathic effect of HIV type 1 (HIV-1) and suppressed proviral DNA synthesis in ATH8 cells exposed to HIV-1 in vitro at concentrations of 50 to 100 microM. These compounds also inhibited the in vitro infectivity of another human pathogenic retrovirus, HIV-2. Furthermore, both compounds completely suppressed the replication of a monocytotropic strain of HIV-1 in monocytes and macrophages at concentrations as low as 0.5 microM, as assessed by inhibition of HIV-1 p24 gag protein production. We also found that 2'-deoxyguanosine readily reversed the antiretroviral activity of cyclobut-G in our system, whereas the activity of cyclobut-A was hardly reversed by 2'-deoxyadenosine or 2'-deoxycytidine. We noted, however, that these compounds inhibited the proliferation of peripheral blood mononuclear cells at concentrations of greater than or equal to 100 microM in vitro. Although both cyclobut-A and cyclobut-G appear to have a certain level of in vitro toxicity, our observations may have theoretical and clinical implications in understanding the structure-activity relationships of antiretroviral agents active against HIV.