Efficient hepatitis delta virus RNA replication in avian cells requires a permissive factor(s) from mammalian cells.

Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian cells, avian cells do not support efficient HDV RNA replication and that this defect cannot be rescued by provision of HDV gene products in trans. Contrary to earlier assertions, this defect is not due to enhanced apoptosis triggered in avian cells by HDV. Fusion of avian cells to mammalian cells rescues HDV replication in avian nuclei, indicating that the nonpermissive phenotype of avian cells is not due to the presence of dominantly acting inhibitors of replication. Rather, avian cells lack one or more essential permissive factors present in mammalian cells. These results set the stage for the identification of such factors and also explain the failure of earlier efforts to transmit HDV infection to avian hosts harboring indigenous hepadnaviruses.

Hepatitis delta virus replication generates complexes of large hepatitis delta antigen and antigenomic RNA that affiliate with and alter nuclear domain 10.

Hepatitis delta virus (HDV), a single-stranded RNA virus, bears a single coding region whose product, the hepatitis delta antigen (HDAg), is expressed in two isoforms, small (S-HDAg) and large (L-HDAg). S-HDAg is required for replication of HDV, while L-HDAg inhibits viral replication and is required for the envelopment of the HDV genomic RNA by hepatitis B virus proteins. Here we have examined the spatial distribution of HDV RNA and proteins in infected nuclei, with particular reference to specific nuclear domains. We found that L-HDAg was aggregated in specific nuclear domains and that over half of these domains were localized beside nuclear domain 10 (ND10). At later times, ND10-associated proteins like PML were found in larger HDAg complexes that had developed into apparently hollow spheres. In these larger complexes, PML was found chiefly in the rims of the spheres, while the known ND10 components Sp100, Daxx, and NDP55 were found in the centers of the spheres. Thus, ND10 proteins that normally are closely linked separate within HDAg-associated complexes. Viral RNA of antigenomic polarity, whether expressed from genomic RNA or directly from introduced plasmids, colocalizes with L-HDAg and the transcriptional repressor PML. In contrast, HDV genomic RNA was distributed more uniformly throughout the nucleus. These results suggest that different host protein complexes may assemble on viral RNA strands of different polarities, and they also suggest that this RNA virus, like DNA viruses, can alter the distribution of ND10-associated proteins. The fact that viral components specifically linked to repression of replication can associate with one of the ND10-associated proteins (PML) raises the possibility that this host protein may play a role in the regulation of HDV RNA synthesis.

Long-range interactions at the HO promoter.

The SWI5 gene encodes a zinc finger DNA-binding protein required for the transcriptional activation of the yeast HO gene. There are two Swi5p binding sites in the HO promoter, site A at -1800 and site B at -1300. Swi5p binding at site B has been investigated in some detail, and we have shown that Swi5p binds site B in a mutually cooperative fashion with Pho2p, a homeodomain protein. In this report, we demonstrate that Swi5p and Pho2p bind cooperatively to both sites A and B but that there are differences in binding to these two promoter sites. It has been shown previously that point mutations in either Swi5p binding site only modestly reduce HO expression in a PHO2 strain. We show that these mutant promoters are completely inactive in a pho2 mutant. We have created stronger point mutations at the two Swi5p binding sites within the HO promoter, and we show that the two binding sites, separated by 500 bp, are both absolutely required for HO expression, independent of PHO2. These results create an apparent dilemma, as the strong mutations at the Swi5p binding sites show that both binding sites are required for HO expression, but the earlier binding site mutations allow Swi5p to activate HO, but only in the presence of Pho2p. To explain these results, a model is proposed in which physical interaction between Swi5p proteins bound to these two sites separated by 500 bp is required for activation of the HO promoter. Experimental evidence is presented that supports the model. In addition, through deletion analysis we have identified a region near the amino terminus of Swi5p that is required for PHO2-independent activation of HO, suggesting that this region mediates the long-range interactions between Swi5p molecules bound at the distant sites.

A cellular homolog of hepatitis delta antigen: implications for viral replication and evolution.

Hepatitis delta virus (HDV) is a pathogenic human virus whose RNA genome and replication cycle resemble those of plant viroids. However, viroid genomes contain no open reading frames, whereas HDV RNA encodes a single protein, hepatitis delta antigen (HDAg), which is required for viral replication. A cellular gene whose product interacts with HDAg has now been identified, and this interaction was found to affect viral genomic replication in intact cells. DNA sequence analysis revealed that this protein, termed delta-interacting protein A (DIPA), is a cellular homolog of HDAg. These observations demonstrate that a host gene product can modulate HDV replication and suggest that HDV may have evolved from a primitive viroidlike RNA through capture of a cellular transcript.

Determining the requirements for cooperative DNA binding by Swi5p and Pho2p (Grf10p/Bas2p) at the HO promoter.

SW15 encodes a zinc finger DNA binding protein required for the transcription of the Saccharomyces cerevisiae HO gene, and PHO2 encodes a homeodomain DNA binding protein. In vitro biochemical studies using purified Swi5p and Pho2p proteins have demonstrated that Swi5p and Pho2p bind cooperatively to the HO promoter. In this report we investigate the regions of the Swi5p and Pho2p proteins required for cooperative DNA binding. The analysis of each protein gives a similar result: the zinc finger or homeodomain DNA binding domains are each sufficient for in vitro DNA binding, but additional regions of each protein are required for cooperative DNA binding. In vitro and in vivo experiments were conducted with promoters with altered spacing between the Pho2p and Swi5p binding sites. Mutations that increased the distance between the two binding sites had minimal effects on either in vitro cooperative DNA binding or in vivo upstream activating sequence activity. These observations suggest that the interaction domains of Swi5p and Pho2p are flexible and can tolerate an increase in distance between the two binding sites. The mechanism of the cooperative DNA binding by Swi5p and Pho2p is discussed.

The Swi5 zinc-finger and Grf10 homeodomain proteins bind DNA cooperatively at the yeast HO promoter.

SWI5 encodes a zinc-finger protein required for expression of the yeast HO gene. Using Swi5 protein that was purified from a bacterial expression system, we previously isolated a yeast factor that stimulates binding of Swi5 to the HO promoter. N-terminal amino acid sequence analysis identified the Swi5 stimulatory factor as the product of the GRF10 gene, which encodes a yeast homeodomain protein. GRF10, also known as PHO2 and BAS2, is a transcriptional activator of the PHO5 acid phosphatase gene and the HIS4 histidine biosynthesis gene. Grf10 protein purified from a bacterial expression system binds DNA cooperatively with Swi5 in vitro. Analysis of disassociation rates indicates that the Grf10-Swi5-DNA complex has a longer half-life than protein-DNA complexes that contain only Swi5 or Grf10. Finally, we show that HO expression is reduced in yeast strains containing grf10 null mutations and that full expression of a heterologous promoter containing a SWI5-dependent HO upstream activation sequence element requires GRF10.

Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein.

The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large excess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts. SWI5 and GRF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.

Transcriptional organization of the rfaGBIJ locus of Salmonella typhimurium.

The transcriptional organization of the rfaGBIJ gene cluster of Salmonella typhimurium was studied by using lacZ and cat transcriptional probes. The results indicated that the leftward end of the gene cluster (rfaG-rfaB-rfaI) is an operon that is transcribed from one or more promoters that lie upstream of rfaG. The results further indicated that the product of the rfaH (sfrB) gene acts as a positive regulator of transcription of the entire rfaGBIJ cluster. At least one site required for the RfaH-mediated transcriptional regulation lies within or very close to the upstream promoter.

Suppression of the abnormal phenotype of Salmonella typhimurium rfaH mutants by mutations in the gene for transcription termination factor Rho.

Mutations in the rfaH gene have previously been shown to cause premature termination of transcription of the traYZ operon of the F factor and also to prevent expression of the rfaGBIJ gene cluster of Salmonella typhimurium. In the present study, mutants were selected for their ability to restore the normal pattern of rfaGBIJ function. On the basis of this initial section, several classes of extragenic suppressor mutants were isolated that completely or partially corrected the Tra- and Rfa- phenotypes of the prototype rfaH mutant. The suppressor mutations included mutations in rho and mutations that mapped in or close to rpoBC. Other suppressor mutations were located elsewhere on the chromosome, presumably identifying other genes that play a role in the RfaH-mediated transcriptional regulation.