Weidong Zhong

Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative because it does not directly affect nuclear HBV closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target cccDNA regulation would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report, to our knowledge, the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes, and from HBV-infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome and, surprisingly, very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection.

The most abundantly expressed latent transcripts encoded by the Kaposi's sarcoma (KS)-associated herpesvirus derive from the genomic region surrounding open reading frame (ORF) K12 (kaposin A). Here we show that these transcripts, initially described as limited to ORF K12 itself, more frequently encompass upstream sequences spanning two sets of 23-nucleotide GC-rich direct repeats (DRs) (DR1 and DR2). Although the DRs lack AUG codons and were previously presumed to be noncoding, a monoclonal antibody raised to infected cells detected multiple polypeptides encoded by this region. These proteins are expressed during latency and upon induction of lytic viral replication in both primary effusion lymphoma (PEL) cell lines and KS tumors. Biochemical and genetic analyses reveal that these proteins are derived from variant translational initiation at CUG codons. The predominant translation product in the PEL cell line BCBL-1 derives from the 5'-most CUG codon in the transcript, resulting in a protein (termed kaposin B) which is encoded largely by the repeats themselves and which does not include K12 sequences. Other non-AUG codons in alternate reading frames are also used at lower efficiency, including one that initiates translation of a DR-K12 fusion protein (kaposin C) that is predicted to sort to a different subcellular locale than kaposin B. Thus, the products of the K12 region, which is the most abundantly transcribed region in latency, are surprisingly complex and may encompass multiple biological functions.

RNA-dependent RNA polymerase (RdRp) encoded by positive-strand RNA viruses is critical to the replication of viral RNA genome. Like other positive-strand RNA viruses, replication of hepatitis C virus (HCV) RNA is mediated through a negative-strand intermediate, which is generated through copying the positive-strand genomic RNA. Although it has been demonstrated that HCV NS5B alone can direct RNA replication through a copy-back primer at the 3' end, de novo initiation of RNA synthesis is likely to be the mode of RNA replication in infected cells. In this study, we demonstrate that a recombinant HCV NS5B protein has the ability to initiate de novo RNA synthesis in vitro. The NS5B used HCV 3' X-tail RNA (98 nucleotides) as the template to synthesize an RNA product of monomer size, which can be labeled by ¿gamma-(32)Pnucleoside triphosphate. The de novo initiation activity was further confirmed by using small synthetic RNAs ending with dideoxynucleotides at the 3' termini. In addition, HCV NS5B preferred GTP as the initiation nucleotide. The optimal conditions for the de novo initiation activity have been determined. Identification and characterization of the de novo priming or initiation activity by HCV NS5B provides an opportunity to screen for inhibitors that specifically target the initiation step.