Chronic hepatitis B virus (HBV) infection is a major cause of liver disease. Only interferon-alpha and the nucleosidic inhibitors of the viral polymerase, 3TC and adefovir, are approved for therapy. However, these therapies are limited by the side effects of interferon and the substantial resistance of the virus to nucleosidic inhibitors. Potent new antiviral compounds suitable for monotherapy or combination therapy are highly desired. We describe non-nucleosidic inhibitors of HBV nucleocapsid maturation that possess in vitro and in vivo antiviral activity. These inhibitors have potential for future therapeutic regimens to combat chronic HBV infection.

Virus-infected cells can be eliminated by cytotoxic T lymphocytes (CTL), which recognize virus-derived peptides bound to major histocompatibility complex (MHC) class I molecules on the cell surface. Until now, this notion has relied on overwhelming but indirect evidence, as the existence of naturally processed viral peptides has not been previously reported. Here we show that such peptides can be extracted from virus-infected cells by acid elution. Both the naturally processed H-2-Db-restricted and H-2-Kd-restricted peptides from influenza nucleoprotein are smaller than the corresponding synthetic peptides, which have first been used to determine the respective CTL epitopes. As with minor histocompatibility antigens, occurrence of viral peptides seems to be heavily dependent on MHC class I molecules, because infected H-2d cells do not contain the H-2-Db-restricted peptide, and infected H-2b cells do not contain the H-2-Kd-restricted peptide. Our data provide direct experimental proof for the above notion on MHC-associated viral peptides on virus-infected cells.

Cytotoxic T lymphocytes (CTL) constitute an essential part of the immune response against viral infections. Such CTL recognize peptides derived from viral proteins together with major histocompatibility complex (MHC) class I molecules on the surface of infected cells, and usually require in vivo priming with infectious virus. Here we report that synthetic viral peptides covalently linked to tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can efficiently prime influenza-virus-specific CTL in vivo. These lipopeptides are able to induce the same high-affinity CTL as does the infectious virus. Our data are not only relevant to vaccine development, but also have a bearing on basic immune processes leading to the transition of virgin T cells to activated effector cells in vivo, and to antigen presentation by MHC class I molecules.

The carboxy-terminal sequence of the hepatitis B virus (HBV) core protein constitutes a nucleic acid binding domain that is rich in arginine residues and contains three serine phosphorylation sites. While dispensable for capsid assembly, this domain is involved in viral replication, as demonstrated by the effects of mutations on RNA packaging and/or reverse transcription; however, the underlying mechanisms are poorly understood. Here we tested a series of core protein mutants in which the three serine phosphorylation sites were replaced by glutamic acid, in parallel with a previously described deletion variant lacking the 19 C-terminal amino acid residues, for their ability to support viral replication in transfected hepatoma cells. Replacement of all serines and the deletion gave rise to nucleocapsids containing a smaller than wild-type DNA genome. Rather than a single-stranded DNA intermediate, as previously thought, this was a 2.0-kbp double-stranded DNA molecule derived from spliced pregenomic RNA (pgRNA). Interestingly, full-length pgRNA was associated with nucleocapsids but was found to be sensitive to nuclease digestion, while encapsidated spliced RNA and 3 truncated RNA species were nuclease resistant. These findings suggest that HBV pgRNA encapsidation is directional and that a packaging limit is determined by the C-terminal portion of the core protein.Hepatitis B virus (HBV) is an important human pathogen accounting for about one million deaths each year (13). The viral genome, as present in infectious virions, is a 3.2-kbp circular, partially double-stranded DNA (dsDNA) molecule that contains overlapping reading frames encoding the core protein, a reverse transcriptase (P), three surface proteins, and the X protein (20,25).Viral replication involves reverse transcription of a pregenomic RNA (pgRNA) intermediate inside nucleocapsids, which are formed by 180 or 240 core protein subunits (4-6, 30). Specific encapsidation of pgRNA occurs via binding of P protein to a 5Ј-proximal RNA stem-loop structure, termed ε. Subgenomic RNAs lack the 5Ј ε and are excluded from encapsidation. Notably, however, spliced RNA species containing all sequence elements necessary for packaging and for reverse transcription have been observed in liver tissue and in transfected cells (9,23,(26)(27)(28)(29). Viral DNA synthesis is a highly complex process involving three translocation events of P to priming sites located at the respective 5Ј and 3Ј ends of the template (17). Thus, both ends must be accessible to the encapsidated P protein. The final product is a partially doublestranded, relaxed circular DNA (rcDNA). Occasionally, one of the translocation steps fails (in situ priming), resulting in a double-stranded linear molecule (17,19,24,25).The C-terminal domain of the core protein plays a crucial role in viral replication. It is rich in arginine residues and contains three serine phosphorylation sites. Though dispensable for particle assembly, it is required for pgRNA packaging (2,3,7,10). Further, the phenotypes of core pr...

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SummaryVirus-specific cytotoxic T lymphocytes (CTL) recognize virus-derived peptides presented by major histocompatibility complex (MHC) class I molecules on virus-infected cells. Such peptides have been isolated from infected cells and were compared to synthetic peptides . We found previously the Kd-or Db-restricted natural influenza nucleoprotein peptides to coelute on reversed phase high performance liquid chromatography columns with certain peptidic by-products present in synthetic peptide preparations . Here we show by extensive biochemical and immunological comparison that the natural peptides in all respects behave as the surmised synthetic nonapeptides, and thus, must be identical to them. The absolute amounts of these natural peptides contained in infected cells could be determined to be between 220 and 540 copies by comparing with defined amounts of pure synthetic nonapeptides. The comparison of the natural Kd-restricted peptide with published synthetic peptides known to contain other Kd-restricted CTL epitopes suggested a new MHC allele-specific T cell epitope forecast method, based on the defined length of nine amino acid residues and on critical amino acid residues at the second and the last position .

Synthetic peptides and a novel type of lipopeptide vaccine, both containing T cell epitopes recognized by Kd-restricted, influenza virus-specific cytotoxic T cells (CTL) were compared in their efficiency to induce virus-specific CTL in vivo. All attempts to induce virus-specific CTL with synthetic peptide (in the absence of adjuvants) failed. However, a latent immunization was observed, resulting in an increased response to the injected peptide seen only after boosting the recipients with immunogenic virus. In contrast, priming with synthetic lipopeptide vaccine (tripalmitoyl-S-glycerylcysteinyl-seryl-serine [P3CSS] coupled to peptide) was successful under most conditions, and matched the priming efficiency seen with infectious virus. The requirements for in vivo priming of virus-specific CTL using lipopeptide suggest that attachment of the lipopeptide to a hydrophobic entity, such as the cell membrane, is responsible for its efficiency.

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