Inhibition of hepatitis B virus replication by the interferon-inducible MxA protein.

Human MxA is an alpha/beta interferon-inducible intracytoplasmic protein that mediates antiviral activity against several RNA viruses. We had previously shown that overexpression of the hepatitis B virus (HBV) capsid led to selective downregulation of MxA gene expression, suggesting a mechanism by which the virus escapes from the host defense system (O. Rosmorduc, H. Sirma, P. Soussan, E. Gordien, P. Lebon, M. Horisberger, C. Brechot and D. Kremsdorf, J. Gen. Virol. 80:1253-1262, 1999). In the present study, we investigated the antiviral activity of MxA protein against HBV. MxA-expressing HuH7 clones were established and transiently transfected with HBV, and viral replication was then studied. Viral protein secretion was profoundly reduced in MxA-expressing clones by 80% for HBV surface antigen (HBsAg) and 70% for HBV e antigen (HBeAg). The levels of intracytoplasmic HBsAg and HBeAg were reduced by about 80 and 50% in the two MxA-positive clones tested. A nearly complete disappearance of HBV DNA replicative intermediates was observed in MxA-expressing clones. Although the expression of total viral RNAs was not modified, two- to fourfold reductions in HBV cytoplasmic RNAs were found in MxA-expressing clones. This suggests the inhibition of HBV replication at a posttranscriptional level. Indeed, using the well-characterized posttranscriptional regulation element (PRE) reporter system, we were able to demonstrate a marked reduction (three- to eightfold) in the nucleocytoplasmic export of unspliced RNA in MxA-expressing clones. In addition, MxA protein did not interact with HBV nucleocapsid or interfere with HBV nucleocapsid formation. Our results show an antiviral effect of MxA protein on a DNA virus for the first time. MxA protein acts, at least in part, by inhibiting the nucleocytoplasmic export of viral mRNA via the PRE sequence.

In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA.

Hepatitis B virus (HBV) is a small DNA virus with a compact genomic organization. All HBV proteins identified to date have been encoded by unspliced HBV RNAs. Spliced HBV RNAs have been described, but their functions are unknown. We show here that a singly spliced HBV RNA encodes a novel HBV protein in vivo. This HBV splice-generated protein (HBSP) corresponds to the fusion of a part of the viral polymerase and a new open reading frame that is created by the splicing event. In vivo, HBSP protein was found in HBV-infected liver samples, and anti-HBSP antibodies occurred in one-third of sera samples collected from chronic HBV carriers. In vitro, the ectopic expression of HBSP had no effect on viral DNA replication or transcription but induced cell apoptosis without a cell-cycle block. Overall, our results suggest that HBV has evolved a mechanism that directly modulates virus-cell interaction through RNA splicing.

In vivo selection of a hepatitis B virus mutant with abnormal viral protein expression.

We have investigated the molecular basis for the in vivo selective advantage of a hepatitis B virus (HBV) mutant. We have determined the complete nucleotide sequences of the major HBV forms identified at the beginning (B1-83) and end (B1-89) of a 6 year follow-up of a chronically infected patient. The B1-89 sequence showed marked nucleotide rearrangements (a nucleotide divergence of 11.3 % compared with the adw2 subtype), but sequence comparison showed that both viral molecules were of common origin (62/138 mutations were found on both molecules, compared to adw2). In vitro transfection of Huh7 cells showed important modifications in B1-89 viral protein expression. We observed a decrease in B1-89 envelope protein expression associated with a modification of the migration pattern of the large envelope protein. For the B1-89 capsid protein, an insertion of 36 nucleotides at the 5' end of the C gene resulted in increased expression of a core-specific protein of abnormal size (24 kDa versus 22 kDa). Finally, our data also suggest an increase in the transcomplementation efficiency of the mutated B1-89 polymerase protein. Thus, we were able to demonstrate distinct intrinsic properties of HBV DNA molecules isolated from a chronic carrier with virus multiplication at different times during infection. Modifications of viral protein expression in the mutated form illustrate strategies used by the virus to prevent clearance and to contribute to viral persistence.

Effects of ethanol on hepatitis B virus Pre-S/S gene expression in the human hepatocellular carcinoma derived HEP G2 hepatitis B DNA positive cell line.

BACKGROUND/AIMS: Among the reported interactions between ethanol and hepatitis B virus (HBV), studies of transgenic mice have suggested an effect of ethanol on the secretion of viral envelope proteins. METHODS: We further investigated these interactions in vitro by determining HBs antigen levels and performing northern blots of viral mRNA in human cell culture (HepG2 HBV positive cells) exposed for 3 to 12 days to various concentrations of ethanol. RESULTS: In cultures exposed to 200 mM ethanol, HBs antigen concentrations increased in the medium (p < 0.05) after 3 days as Pre-S1 and Pre-S2 protein concentrations. This increase was not specific, as albumin and ferritin increased in the same proportions. Ethanol also increased the HBs antigen concentration in the cells (p < 0.05), whereas levels of viral mRNA encoding surface proteins were unaffected. CONCLUSIONS: These findings show that short-term ethanol exposure in vitro can induce HBs antigen overexpression via a post-transcriptional mechanism.

Complete nucleotide sequence and viral envelope protein expression of a hepatitis B virus DNA derived from a hepatitis B surface antigen-seronegative patient.

An HBV strain isolated from a patient lacking conventional serological markers of HBV infection was characterized. The complete nucleotide sequence was determined following PCR amplification. Only 22 nucleotide substitutions were found relative to the reported sequence of the ayw subtype. Five of these point mutations in the preS/S and preC/C genes led to amino acid substitutions and, with one exception, were located in regions coding for antigenic determinants of viral envelope or capsid proteins. Eight amino acid substitutions were located in terminal protein and the spacer domain of the polymerase gene product. Despite these amino acid changes, transient expression of the preS2 and S envelope proteins in eucaryotic cells yielded viral proteins detectable in the culture medium with polyclonal and monoclonal anti-preS2 and -S antibodies. These data conclusively demonstrate persistent infection by HBV in subjects without HBV serological markers. The absence of conventional HBV serological markers is probably due to several factors: a low level of viral replication, some genetic modifications, as well as an abnormal immune response to the virus.

Regional mapping to 4q32.1 by in situ hybridization of a DNA domain rearranged in human liver cancer.

Recently, a unique cellular DNA segment, representing the normal allele counterpart of hepatitis B virus integration site, has been isolated. It has allowed the identification of a cellular domain in which rearrangements occur in approximately 10% of primary liver tumours. We here report on the assignment of this probe, D4S112, by in situ hybridization to band 4q32.1.

Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors.

Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. We have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possibly related to HBV integration.

Energy metabolism in adenosine deaminase-inhibited human erythrocytes.

The metabolic changes induced by the deoxycoformycin inhibition of adenosine deaminase were studied in human erythrocytes incubated with nucleosides. 1 Adenosine nucleotide levels and glycolytic rate were increased by adenosine. 2 With deoxyadenosine, the cellular ATP level was reduced when dATP increased and the glycolytic rate was similarly enhanced. 3 The hypoxanthine production was equivalent in both cases. Our data demonstrate that human red cells are able to catabolize adenine deoxynucleotides into hypoxanthine, and the control of energy metabolism is not impaired by adenosine deaminase inhibition when PO identical to 4 and NAD+ are not limiting.

Suppressor cells induced by BCG release non-specific factors in vitro which inhibit DNA synthesis and interleukin-2 production.

Mice injected intravenously with a high dose (5 X 10(7) ) of BCG fail to develop delayed hypersensitivity to BCG and are described as anergic or unresponsive. Spleen cells from these mice release factors on culture which suppress DNA synthesis induced by concanavalin A in vitro. Cell separation experiments showed that both macrophages and T cells produce inhibitory factors. However, the macrophage factor has a molecular weight 10,000-30,000, while the T cell factor has a molecular weight of 50,000-70,000. Further evidence that these two factors are different is provided by the kinetics of their action. The T cell factor only acts when given within 12 hr of stimulation with concanavalin A, while the macrophage factor acts even when given at 48 hr. In the case of the T cell factor, the inhibition of DNA synthesis may be attributed to its ability to block the interleukin-2 production induced by Con A. As similar T cell and macrophage factors are produced in mice responding to simple chemically reactive haptenes (contact sensitizers), it is possible that a similar suppressor circuit is involved in the control of the response to contact sensitizers and in the production of unresponsiveness (anergy) in mice given large doses of BCG.

Isopycnic centrifugation as method for the separation of rat thymocyte subpopulations.

Rat thymocytes were separated into five density layers by isopycnic centrifugation on a discontinuous-density Ficoll gradient and each fraction was tested for metabolic functions at different times during a 96-h culture period in various conditions. Immediately after separation the rate of oxygen consumption per unit of cellular volume is virtually the same in all fractions. However, the capacity to synthesize nucleic acids, proteins, purine and pyrimidine nucleotides by the de novo pathway and the activity of the salvage pathway of purine nucleotides is much greater in the low density fractions (fractions 1 and 2). In in vitro cell culture conditions (1) in the absence of mitogens this metabolic activity (which demonstrates a high spontaneous mitotic capacity of cells in the low-density fractions) decays rapidly to a level which is practically negligible after 24 h of culture; (2) in the presence of mitogens alone: PHA, Con A, PWM or PNA, with or without prior neuraminidase treatment, after 24 h an induction of the synthesis of nucleic acids and protein is observed concomitant to the apparition of blast cells. This blastogenesis peaks at 72 h and occurs in fractions 1 and 2 only; (3) in the presence of Con A and 2-mercaptoethanol the blast transformation corresponding to the response of the thymocytes to TCGF is observed in all of the thymocyte fractions and extends beyond 72 h of culture. It remains much more intense in the low-density fractions 1 and 2.

Study of rat lymphocytes by use of peanut agglutinin.

Peanut agglutinin (PNA) was used to identify rat lymphocyte subpopulations. The binding of PNA on the surface of lymphocytes was assessed by fluorescence microscopy using fluoresceinated PNA. It was shown that most of the thymocytes and bone marrow cells have receptors for PNA; in spleen and lymph nodes the percentages of PNA-positive cells are around 40%. PNA was also used as a potentially mitogenic lectin. It was ascertained that lymphocytes incubated with PNA are stimulated only if they are pretreated with neuraminidase, and that only 'immunocompetent' thymocytes exhibit a very weak response. The fact that PNA slightly inhibits the [3H]uridine uptake of resting thymocytes might explain the weak mitogenicity of PNA.

Consequences of adenosine deaminase deficiency on thymocyte metabolism.

Using 2'-deoxycoformycin inhibition of adenosine deaminase as a model of adenosine deaminase deficiency, the effects of 10 microM 2'-deoxyadenosine (dAdo) on the metabolism of concanavalin A (Con A)-stimulated rat thymocytes were studied. When dAdo and Con A were added simultaneously, a strong inhibition of the incorporation of [3H]thymidine (84%); [3H]uridine (98%) and L-[3H] leucine (46%) in the acid-insoluble fraction, and of [14C]formate (78%) and H14CO-3 (43%) uptake is observed after 48 h of incubation. When dAdo is added after 12 h of Con A stimulation, no such inhibition is observed, but when added after 24 h of stimulation, there is an enhancement of blastogenesis as measured by nucleic acid, protein, and purine and pyrimidine base synthesis. More detailed studies of thymocytes stimulated by Con A for 0-72 h, followed by short-term incubation periods with dAdo (1-5 h), revealed that thymocyte metabolism becomes progressively less sensitive to dAdo-mediated inhibition during the course of blastogenesis. These results suggest that (a) the inhibition of ribonucleotide reductase is not the only mechanism involved in the inhibition of blastogenesis by dAdo and that (b) such inhibition of thymocyte metabolism is essentially dependent upon the activation state of the cell.