Viroids with hammerhead ribozymes: some unique structural and functional aspects with respect to other members of the group.

Viroids, subviral pathogens of plants, are composed of a single-stranded circular RNA of 246-399 nucleotides. Within the 27 viroids sequenced, avocado sunblotch, peach latent mosaic and chrysanthemum chlorotic mottle viroids (ASBVd, PLMVd and CChMVd, respectively) can form hammerhead structures in both of their polarity strands. These ribozymes mediate self-cleavage of the oligomeric RNAs generated in the replication through a rolling circle mechanism, whose two other steps are catalyzed by an RNA polymerase and an RNA ligase. ASBVd, and presumably PLMVd and CChMVd, replicate and accumulate in the chloroplast, whereas typical viroids replicate and accumulate in the nucleus. PLMVd and CChMVd do not adopt a rod-like or quasi rod-like secondary structure as typical viroids do but have a highly branched conformation. A pathogenicity determinant has been mapped in a defined region of the CChMVd molecule.     

The RNA replication enhancer element of tombusviruses contains two interchangeable hairpins that are functional during plus-strand synthesis

Replication of the RNA genomes of tombusviruses, which are small plus-sense RNA viruses of plants, may be regulated by cis-acting elements, including promoters and replication enhancers that are present in the RNA templates. Using a partially purified RNA-dependent RNA polymerase (RdRp) preparation (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), we demonstrate that the minus-strand templates of tombusviruses contain a replication enhancer, which can upregulate RNA synthesis initiating from the minimal plus-strand initiation promoter by 10- to 20-fold in an in vitro assay. Dissection of the sequence of the replication enhancer element revealed that the two stem-loop structures present within the approximately 80-nucleotide-long enhancer region have interchangeable roles in upregulating RNA synthesis. The single-stranded sequence located between the two stem-loops also plays an important role in stimulation of RNA synthesis. We also demonstrate that one of the two hairpins, both of which are similar to the hairpin of the minus-strand initiation promoter, can function as a promoter in vitro in the presence of short cytidylate-containing initiation sites. Overall, the in vitro data presented are consistent with previous in vivo results (D. Ray and K. A. White, Virology 256:162-171, 1999) and they firmly establish the presence of a replication enhancer on the minus-stranded RNA of tombusviruses.     

The sequence of the RNA primer and the DNA template influence the initiation of plus-strand DNA synthesis in hepatitis B virus

For hepadnaviruses, the RNA primer for plus-strand DNA synthesis is generated by the final RNase H cleavage of the pregenomic RNA at an 11 nt sequence called DR1 during the synthesis of minus-strand DNA. This RNA primer initiates synthesis at one of two distinct sites on the minus-strand DNA template, resulting in two different end products; duplex linear DNA or relaxed circular DNA. Duplex linear DNA is made when initiation of synthesis occurs at DR1. Relaxed circular DNA, the major product, is made when the RNA primer translocates to the sequence complementary to DR1, called DR2 before initiation of DNA synthesis. We studied the mechanism that determines the site of the final RNase H cleavage in hepatitis B virus (HBV). We showed that the sites of the final RNase H cleavage are always a fixed number of nucleotides from the 5' end of the pregenomic RNA. This finding is similar to what was found previously for duck hepatitis B virus (DHBV), and suggests that all hepadnaviruses use a similar mechanism. Also, we studied the role of complementarity between the RNA primer and the acceptor site at DR2 in HBV. By increasing the complementarity, we were able to increase the level of priming at DR2 over that seen in the wild-type virus. This finding suggests that the level of initiation of plus-strand DNA synthesis at DR2 is sub-maximal for wild-type HBV. Finally, we studied the role of the sequence at the 5' end of the RNA primer that is outside of the DR sequence. We found that substitutions or insertions in this region affected the level of priming at DR1 and DR2.     

Use of DNA, RNA, and chimeric templates by a viral RNA-dependent RNA polymerase: evolutionary implications for the transition from the RNA to the DNA world.

All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position -11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes.     

A freeze-fracture study of early membrane events during mast cell secretion

The early membrane events taking place during mast cell secretion were followed in transmission and freeze-fracture electron microscopy. In order to slow down exocytosis and capture intermediate stages of membrane fusion, special conditions of incubation and stimulation were used. These were as follows: (a) the use of incubation media with altered ionic composition, and (b) stimulation with a low dosage of polymyxin B sulfate (4 microgram/ml) at low temperature (18 degrees C) for very short incubation times (30-60 s), with or without the presence of formaldehyde (0.8%). Under these conditions, unetchable circular impressions are found on the E face of the plasma membrane, 80-100 nm in diameter, with particles associated with their perimeters. In granule-to-granule fusion, the zone involved is demarcated by one or two rows of particles on the E face. In addition, raised circular areas of varying diameters (43-87 nm) surrounded by similar particles, also found on the E face, may represent potential sites before completion of fusion. Neither the circular impressions on the plasma membrane nor the sites on the granule membrane are permanent, but their appearance coincides with initiation of membrane fusion.     

Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex.

Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribonucleotide. Pol alpha 2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol alpha-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vivo. Pol alpha 2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol alpha-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase alpha affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.     

Characterisation of sequences required for RNA initiation from the PGK promoter of Saccharomyces cerevisiae.

In the phosphoglycerate kinase (PGK) gene of yeast, as in other highly expressed yeast genes, the sequences surrounding the site of RNA initiation have a loosely conserved structure of a CT rich stretch followed by the tetranucleotide CAAG. Using internal deletions and insertions we have identified the elements in the PGK promoter which are required for correct RNA initiation at the CAAG sequence at -39. The results indicate that two different components of the PGK promoter contribute to correct RNA initiation, the TATA homologies, located at -152 and -113, and the sequences at the site of initiation. Both TATA elements can function in RNA initiation. Deletion of the upstream TATA element, TATAI, results in slightly heterogeneous RNA initiation, but the majority of the RNA initiates correctly. Deletion of both the PGK TATA elements results in the majority of the RNA initiating at sites downstream from the wild-type I site, within the structural gene between +40 to +80. The CT rich box is not essential for correct mRNA initiation as shown by deletion analysis. The site of RNA initiation in the PGK promoter appears to be determined by sequences located immediately 5' of the CAAG sequence motif. This short sequence, ACAGATC, when located the correct distance from the TATA elements may be sufficient to determine a discrete initiation site.     

RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae

We identified and mapped RNA-binding sites of yeast Saccharomyces cerevisiae translation initiation factor eIF4G1 and examined their importance for eIF4G1 function in vitro and in vivo. Yeast eIF4G1 binds to single-stranded RNA with three different sites, the regions of amino acids 1-82 (N terminus), 492-539 (middle), and 883-952 (C terminus). The middle and C-terminal RNA-binding sites represent RS (arginine and serine)-rich domains; the N-terminal site is asparagine-, glutamine- and glycine-rich. The three RNA-binding sites have similar affinity for single-stranded RNA, whereas the affinity for single-stranded RNA full-length eIF4G1 is about 100-fold higher (approximate K(d) of 5 x 10(-8) M). Replacement of the arginine residues in the middle RS site by alanine residues abolishes its RNA-binding activity. Deletion of individual RNA-binding sites shows that eIF4G1 molecules lacking one binding site are still active in supporting growth of yeast cells and translation in vitro, whereas eIF4G1 molecules lacking two or all three RNA-binding sites are strongly impaired or inactive. These data suggest that RNA-binding activity is required for eIF4G1 function.     

Resistance of human hepatitis delta virus RNAs to dicer activity

The endonuclease dicer cleaves RNAs that are 100% double stranded and certain RNAs with extensive but <100% pairing to release approximately 21-nucleotide (nt) fragments. Circular 1,679-nt genomic and antigenomic RNAs of human hepatitis delta virus (HDV) can fold into a rod-like structure with 74% pairing. However, during HDV replication in hepatocytes of human, woodchuck, and mouse origin, no approximately 21-nt RNAs were detected. Likewise, in vitro, purified recombinant dicer gave <0.2% cleavage of unit-length HDV RNAs. Similarly, rod-like RNAs of potato spindle tuber viroid (PSTVd) and avocado sunblotch viroid (ASBVd) were only 0.5% cleaved. Furthermore, when a 66-nt hairpin RNA with 79% pairing, the putative precursor to miR-122, which is an abundant liver micro-RNA, replaced one end of HDV genomic RNA, it was poorly cleaved, both in vivo and in vitro. In contrast, this 66-nt hairpin, in the absence of appended HDV sequences, was >80% cleaved in vitro. Other 66-nt hairpins derived from one end of genomic HDV, PSTVd, or ASBVd RNAs were also cleaved. Apparently, for unit-length RNAs of HDV, PSTVd, and ASBVd, it is the extended structure with <100% base pairing that confers significant resistance to dicer action.