The honeybee hive product, propolis, is a folk medicine employed for treating various ailments. Many important pharmaceutical properties have been ascribed to propolis, including anti-inflammatory, antiviral, immunostimulatory and carcinostatic activities. Propolis extracts have provided an active component identified as caffeic acid phenethyl ester (CAPE), which was readily prepared in one step. Differential cytotoxicity has been observed in normal rat/human versus transformed rat/human melanoma and breast carcinoma cell lines in the presence of CAPE.

Chloroquine and its analogue hydroxychloroquine (HCQ) have been shown to inhibit a variety of viral infections including influenza and adenovirus through blockade of viral entry via inhibition of endosomal acidification. We have extended these observations to human immunodeficiency virus type 1 (HIV-1) infection utilizing primary T cells and monocytes, a T cell line (CEM), and a monocytic cell line (U-937). HCQ inhibited HIV-1 replication (> 75%), as measured by reverse transcriptase activity, in the primary T cells and monocytes as well as the T cell and monocytic cell lines. HCQ itself had no anti-reverse transcriptase activity and was not toxic to the cells at concentrations inhibitory to viral replication. Intracytoplasmic staining with an anti-p24 antibody, 24 h after infection, revealed the presence of intracytoplasmic virus, suggesting that the drug does not block viral entry. The production of steady-state HIV-1 mRNA was not affected by HCQ in that comparable levels of HIV-1 mRNA could be detected by Northern blot analysis and by in situ hybridization in both the HCQ-treated and untreated cells. However, HCQ does appear to affect production of infectious HIV-1 virions because viral isolates from HCQ-treated cells could not infect target CEM cells. These data suggest that HCQ may be useful adjunctive therapy in the treatment of HIV-1 infection.

We have investigated the role of liver-specific trans-acting factor(s) in the regulation of hepatitis B virus (HBV) gene expression. A recorder plasmid (pEcoAluCAT; HBV nucleotides 1 through 1878) was constructed containing the HBV enhancer and the promoter region of the pregenomic RNA, which was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene. Upon transfecting this plasmid into various cell lines, the CAT gene was expressed only in cells of liver origin. Moreover, competition cotransfections with pEcoAluCAT and plasmids containing HBV enhancer sequences in human hepatoblastoma-derived HepG2 cells indicated the presence of titratable trans-acting factor(s) in these cells. Gel mobility shift assays using HBV enhancer and core promoter domains confirmed the existence of sequence-specific DNA-binding proteins in liver cell nuclear extract which bound to these regions. These binding sites encompass 17-and 12-nucleotide palindromes in the HBV enhancer and core promoter domains, respectively, when mapped by the methylation interference assay.The hepatitis B virus (HBV), a small partially doublestranded DNA virus which belongs to the Hepadnaviridae family of animal viruses (38), is a significant worldwide cause of serious illness and mortality (40). The virus is responsible for causing acute and chronic hepatitis, and chronic carriers of HBV are at a highly increased risk of developing hepatocellular carcinoma (3). Although there is some evidence for replication of this virus in nonliver tissues (12,22), the principal site of clinical pathology is the liver, and HBV actively replicates in human hepatocytes. Evidence for the hepatocyte restriction of HBV replication comes from clinical data and studies on HBV mRNA transcription. During productive infection, two major viral RNAs (2.1 and 3.5 kilobases [kb] in length) are transcribed in relatively equal abundance (6, 46). The 3.5-kb RNA, which is greater than the length of the HBV genome, serves as the template for reverse transcription of the HBV genome (31, 39) and thus is integral to viral replication.Recently, many studies on the control of eucaryotic transcription have focused on the regulatory role of trans-acting, sequence-specific, DNA-binding proteins (for reviews, see references 10, 11, and 24). The binding of these regulatory proteins to their respective sites in the DNA has been implicated in mediating the activities of cis-acting viral and cellular DNA elements in various systems (1,5,15,33,37).An enhancer element (ENH), putatively mapped between nucleotides (nt) 1050 and 1250 in the HBV genome (35, 41), has been found to be active selectively in cell lines derived from human hepatocytes (19,42). These reports also suggest that the HBV ENH could be responsible for the hepatocytespecific replication of HBV via hepatocyte-restricted expression of the 3.5-kb RNA replication intermediate.We have investigated the role of sequence-specific DNAbinding proteins on 3.5-kb RNA expression. By using human hepatoblastoma-derived HepG2 cells (21), w...

The carbocyclic analog of deoxyguanosine inhibits hepatitis B virus replication by greater than 95% in the hepatitis B virus-producing cell line (2.2.15) as monitored by decreases of secreted hepatitis B virus DNA, hepatitis B virus polymerase activity and intracellular episomal hepatitis B virus DNA. Transcription of hepatitis B virus RNA from chromosomally integrated hepatitis B virus DNA was unaffected. Radioactive carbocyclic 2'-deoxyguanosine was directly phosphorylated within the 2.2.15 cells and was incorporated exclusively into DNA. In contrast, radioactive deoxyguanosine was presumably metabolized through the "salvage" pathway in which the guanine was primarily incorporated into cellular RNAs. The rate of incorporation of carbocyclic 2'-deoxyguanosine in 2.2.15 cells was similar to that in the parental cell line (HepG2), which does not contain hepatitis B virus sequences. Greater than 90% of the analog was present at internal sites within the DNA, indicating that the analog did not function as a DNA chain terminator. Kinetic analysis of the Km and Ki of dGTP and carbocyclic 2'-deoxyguanosine 5'-triphosphate, respectively, using both hepatitis B virus polymerase and DNA polymerase delta indicated that the analog is a competitive inhibitor for dGTP. Although both polymerases had similar Km's for dGTP, the Ki for carbocyclic 2'-deoxyguanosine 5'-triphosphate was about 6 times lower using the hepatitis B virus polymerase. This would indicate that, at low concentrations of intracellular carbocyclic 2'-deoxyguanosine 5'-triphosphate, the hepatitis B virus polymerase would be preferentially inhibited. We propose this to be the mechanism acting to inhibit preferentially hepatitis B virus replication in the tissue culture cells.

Using human macrophage hybridomas infected with HIV-1, we investigated monocyte function over a 5-week period after HIV-1 infection. Two clones, 63 and 30, were infected with HIV-1IIIB. Infection was documented by RT activity (15 x 10(6) cpm/ml), intracytoplasmic staining with an anti-p24 antibody, in situ hybridization with an HIV-1-specific riboprobe, and electron microscopy showing intracytoplasmic virus. Two weeks after infection, clones 63 and 30 lost expression of all class II antigens (DR, 81.7 vs. 0%; DQ, 15.6 vs. 0%; and DP, 76.9 vs. 0%) while retaining expression of class I (87.4 vs. 84.1%), LFA-1 (82.4 vs. 83.1%), and LFA-3 (79.1 vs. 74.7%) antigens when compared to uninfected cells. When tested for functional integrity, infected but not uninfected clone 63 cells failed to stimulate a tetanus-specific MHC-restricted T cell proliferative response 2 weeks after infection. Cytokine secretion and antigen processing were also perturbed as production of IL-1 was abolished 2 weeks after infection (although IL-6 secretion was augmented) and infected clone 63 cells failed to process exogenous antigen. Last, the viability of T cells cocultured with infected clone 63 was dramatically decreased 35 days after infection (85 vs. 15%). There was no evidence of transmission of HIV-1 to T cells, suggesting a toxic effect of infected clone 63. Taken together, these data suggest that altered macrophage function in our system occurs at multiple levels, which may account for the early immunological defects described in HIV-1 infection.