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Hepatitis Delta Antigen and RNA Polymerase II

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Replication and transcription of HDV proceed via RNA-dependent RNA synthesis. These reactions are thought to be catalyzed at least in part by host RNA polymerase II (RNAPII). Hepatitis delta antigen (HDAg), which is critical for these processes, was recently proposed to function as a transcription elongation factor for RNAPII. The involvement of a DNA-dependent RNA polymerase in RNA-dependent RNA synthesis is itself intriguing and poses fundamental questions as to how RNA synthesis initiates, elongates, and terminates on an unusual HDV RNA template. In addition, the presence of a "viral" transcription elongation factor is unprecedented in eukaryotes, whereas a few are known to exist in prokaryotes. Thus, the study of HDV replication and transcription should provide tremendous insight into the basic mechanism underlying RNAPII transcription.

Introduction

Three types of RNA-dependent RNA synthesis occur during the HDV life cycle: (i) antigenomic RNA synthesis from genomic RNA, (ii) genomic RNA synthesis from antigenomic RNA, and (iii) HDAg mRNA synthesis from genomic RNA (see Chapter 4 for details). The first and second types of reactions are steps in replication that are thought to proceed by a "rolling cycle" mechanism. This mechanism is analogous to DNA replication of many plasmids and filamentous bacteriophages. As for the third type of reaction, based on the analysis of the mRNA's 5' end, it is assumed that the transcription is initiated from a position that is very close to an end of the rod-like structure of the HDV genome. By extension, the first type of reaction, which also utilizes genomic RNA as a template, may be initiated from the same position of the HDV genome. In this chapter, we refer to the three types of reactions simply as _transcription_.

Several lines of evidence suggest that RNAPII is involved in HDV RNA transcription. First, viroid RNAs, infectious agents in plants that show structural similarity to HDV RNA, are reportedly transcribed by RNAPII in cell-free extracts.1 Second, as reported by a few laboratories, HDV RNA can also be transcribed by RNAPII in vitro.2-4It should be noted, however, that the studies completed thus far have been unable to synthesize full-length complementary RNAs. In one report, for example, RNAPII in the nuclear extract of human HeLa cells directed a genomic strand synthesis of up to ~40 nt using an antigenomic fragment of HDV RNA as a template.3Third, HDAg, the sole HDV protein, directly binds to RNAPII and remarkably stimulates DNA-directed and HDV RNA-directed transcription in vitro.5-7The second half of this chapter deals with this topic. Fourth, HDAg mRNA is capped and polyadenylated at its 5apos; and 3' ends, respectively.4These processing events are tightly coupled to RNAPII transcription and occur in all the known mRNA species synthesized by RNAPII, with the exception of histone mRNA. Conversely, essentially no RNA species synthesized by other RNA polymerases are capped or polyadenylated. Fifth, in intact cells and in isolated nuclei, transcription of the HDV genome is reportedly sensitive to the mushroom toxin αmanitin at concentrations low enough to selectively inhibit RNAPII.8,9One may need to view this with caution, however, because opposing results have been presented by another laboratory10,11(see Chapter 4 for more discussion). With these findings taken together, it should be reasonable to conclude that RNAPII is responsible at least in part for HDV RNA transcription.

Variation on a Theme: Initiation, Elongation, and Termination of HDV RNA Transcription

The idea that RNAPII, a DNA-dependent RNA polymerase, directs RNA-dependent RNA synthesis poses several interesting questions as to how RNA synthesis initiates, elongates, and terminates on an unusual HDV RNA template. From a mechanistic point of view, such an RNA-directed transcription seems quite a challenge to RNAPII, as discussed below. Elucidation of this mechanism may lead to the identification of new molecular targets to prevent the pathogenic virus. Furthermore, such knowledge should add insightful information on the basic mechanism of RNAPII transcription.

Before moving on to the central issue, we first overview the process of DNA-directed transcription by RNAPII. The transcription process comprises several distinct steps, including: (i) preinitiation complex assembly, (ii) promoter opening, (iii) transcription initiation, (iv) promoter escape, (v) transcription elongation, and (vi) transcription termination (Fig. 1). The first four steps occur around transcription initiation sites. RNAPII alone is unable to initiate transcription. Instead it forms a preinitiation complex together with general transcription factors, including transcription factor (TF) IIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, on a promoter.12Core promoter elements, such as TATA boxes and Inr elements, are important for the assembly. TFIIH then facilitates the conversion of a closed-to-open complex by its DNA helicase activity in an ATP-dependent manner (promoter opening).12 Next, RNAPII starts to synthesize nascent RNA but immediately encounters a transcriptional block when it reaches 9~12 bp downstream. TFIIH helicase suppresses the block and facilitates the transition to the elongation phase (promoter escape).13This step is equated with the dissociation of RNAPII from promoter-bound transcription factors. During transcription elongation, RNAPII forms a ternary complex together with template DNA and nascent RNA. Within the "transcription elongation complex", 12-15 bp of DNA are unwound to form a "transcription bubble". In addition, 8-9 nt of RNA in the 3'-end are contained by forming a hybrid with the template stand of DNA, with the growing 3'-end usually maintained at the active site of RNAPII.14 Termination of premRNA synthesis is tightly but not entirely coupled to 3'-end processing.15 The processing event, composed of transcript cleavage and polyadenylation, takes place 23 or 24 nt downstream of the AAUAAA sequence. Transcription termination seems to occur rather randomly between 200-2000 bp downstream of the poly(A) signal, triggered by preceding transcript cleavage and polyadenylation.

Figure 1. Comparison of DNA-directed (A) and HDV RNA-directed (B) transcription by RNAPII.

Figure 1

Comparison of DNA-directed (A) and HDV RNA-directed (B) transcription by RNAPII. The termination step is omitted in the illustration. DNA/RNA templates and nascent RNA transcripts are drawn in black and blue, respectively. Numerals indicate positions (more...)

How is transcription initiated on the HDV RNA template? A few laboratories investigated the requirement for cis-acting elements in HDV transcription in vitro. According to these studies, small bulges close to the ends of the rod-like genome, where transcription is considered to be initiated, are important for efficient transcription.2,3These studies, however, were unable to establish the necessary and sufficient conditions for transcription initiation in sufficient detail. Moreover, virtually nothing is known about trans-acting factors involved in the initiation process. One can postulate several possibilities. First, RNAPII recruitment and transcription initiation may require some or all of the general transcription factors, and both processes proceed in manners analogous to those of DNA-directed transcription. Of the general transcription factors, at least TFIID and TFIIB bind to DNA with sequence specificity. It is not known whether they can also bind to RNA. Second, protein factors other than the general transcription factors, such as cellular RNA-binding proteins and the viral HDAg, may be involved in the initiation process (Fig. 1). Third, RNAPII alone may be sufficient to initiate transcription on an unusual template.

Perhaps, data obtained using special DNA templates may provide clues to the solution. On a DNA template containing a bubble or nonbase-paired region of ~10 nt at a promoter, RNAPII initiates transcription without some of the general transcription factors, namely TFIIE and TFIIH16(Fig. 2). On the other hand, on a linear DNA template carrying a 3'-overhang of oligo(dC), RNAPII alone can initiate transcription from its end efficiently17 (Fig. 2). Apparently, these DNA templates show some degree of structural similarity to the HDV genome. Even if general transcription factors are involved in the HDV RNA-directed transcription, we suspect that the factor requirement may be quite different from that of DNA-directed transcription.

Figure 2. Comparison of the rod-like HDV genome and DNA templates used in in vitro study.

Figure 2

Comparison of the rod-like HDV genome and DNA templates used in in vitro study. Normal double-stranded (ds) DNA template, premelted template, and dC-tailed template show different factor requirements in transcription initiation. Also shown at the bottom (more...)

How does a nascent RNA chain elongate on the HDV RNA template? As transcription proceeds by the rolling circle mechanism, a nascent transcript forms a long double-stranded region with the RNA template (Fig. 1). By comparison, during accurately initiated transcription on a DNA template, a nascent transcript is separated from DNA within the transcription elongation complex, and only 8-9 bases of RNA form a hybrid with the template strand of DNA (Fig. 1). RNAPII seems to be intrinsically capable of both types of RNA synthesis. During transcription of a dC-tailed template, sometimes a significant fraction of the nascent transcripts displace the nontemplate strand of DNA to form long DNA-RNA hybrids17 (Fig. 2). The efficiency of this unusual synthesis pathway seems affected in part by the G/C content of the initially transcribed sequence.17

Another consideration concerns structural differences between DNA and RNA, which may pose a considerable problem for the progression of RNAPII. HDV RNA's differences from DNA include 2'-OH versus 2'-H, double helices in an A form versus a B form, and the presence versus absence of bulges and single-stranded regions that interrupt double helices. Data obtained with DNA templates have shown that a small change in the nucleic acid structure often causes a strong transcriptional block.18,19Thus, positive transcription elongation factors that suppress the transcriptional block and enhance the efficiency of elongation may play important roles in HDV RNA-directed transcription, as discussed later.

How does transcription terminate on the HDV RNA template? In the case of the synthesis of HDAg mRNA, it is very likely that the termination process is coupled with 3'-end processing. As evidence, the HDV genome encodes canonical poly(A) signals, and the viral mRNA is polyadenylated.4By contrast, little is known as to where and how the syntheses of the multimeric forms of the HDV genome and antigenome terminate.

HDAg As a Viral Transcription Elongation Factor

RNAPII elongation on a DNA template is controlled by over a dozen transcription elongation factors.20Strictly defined, a transcription elongation factor is a protein that directly associates with RNA polymerase and thereby regulates its elongation activity.

A class of transcription elongation factors, including TFIIF, TFIIS, Elongin, and ELL, interacts with RNAPII and accelerates elongation (TFIIF plays dual roles in transcription initiation and elongation) (Fig. 3). This class of factors probably does so in part by preventing RNAPII from frequent pauses and arrests caused by inhibitory structures of nascent RNA, low concentrations of NTP, and misincorporation of NMP. In this context, a pause is a transient inactive state, whereas an arrest is a more persistent state that eventually leads to termination without reactivation by positive transcription elongation factors.

Figure 3. Two modes of elongation control.

Figure 3

Two modes of elongation control. (A) A class of transcription elongation factors, including TFIIF, TFIIS, Elongin, and ELL, interacts with RNAPII and accelerates elongation by preventing RNAPII from frequent pauses and arrests caused by inhibitory structures (more...)

Another class of transcription elongation factors includes DRB sensitivity-inducing factor (DSIF), negative elongation factor (NELF), and positive transcription elongation factor b (P-TEFb), which together constitute a critical rate-limiting step of transcription20,21(Fig. 3). Shortly after the initiation of transcription, RNAPII is subjected to both negative and positive control by these factors. DSIF and NELF together cause a transcriptional block by binding to RNAPII. Conversely, P-TEFb allows RNAPII to enter a productive elongation phase by preventing DSIF and NELF from acting. P-TEFb is a protein kinase that strongly phosphorylates the C-terminal domain of RNAPII and the Spt5 subunit of DSIF22(Fig. 4). Several, but not all, lines of evidence suggest that P-TEFb-dependent phosphorylation of the CTD facilitates the release of NELF from RNAPII, thereby overcoming the transcriptional block21,23(Fig. 4).

Figure 4. HDAg is a viral transcription elongation factor.

Figure 4

HDAg is a viral transcription elongation factor. (A) DSIF and NELF bind to RNAPII and repress its elongation. P-TEFb phosphorylates RNAPII and DSIF to reverse the repression. (B) According to a proposal,5 HDAg reverses the negative effect of DSIF and (more...)

Recent papers have presented biochemical evidence for the role of HDAg in RNAPII elongation.5-7NELF is composed of five polypeptides, and one of these subunits, called NELF-A, was found to show limited sequence similarity to HDAg.5This similarity stimulated subsequent study and led to the following findings. First, HDAg directly binds to RNAPII in a manner competitive to NELF5(Fig. 4). The regions of similarity shared between HDAg and NELF-A may form a conserved structure that recognizes a common surface on RNAPII. This idea is supported by the results of a more recent deletion analysis of NELF-A.24Second, HDAg activates RNAPII elongation on an HDV RNA template as well as on a DNA template in vitro5-7(Fig. 4). When a dC-tailed template and pure RNAPII are used, for example, HDAg remarkably enhances the rate of elongation regardless of the presence of DSIF and NELF. Thus, the activation mechanism appears two-fold: HDAg reverses the negative effect of DSIF and NELF by displacing NELF from RNAPII, and the HDAg-RNAPII interaction by itself further activates transcription elongation. HDAg can be judged on several criteria as a new member, and in fact it is the first viral member of the transcription elongation factors for RNAPII.

HDAg is thought to be a multifunctional protein playing important roles in nucleocytoplasmic transport of HDV RNA, transcriptional regulation, and viral assembly, among others (see Chapters 4 and 5). Therefore, dissection of the protein motifs responsible for each function is important in order to understand the relative significance of these functions in the cellular context. Deletion and mutagenic analyses of HDAg have found the following: Two-thirds of HDAg protein, containing the oligomerization domain, the nuclear localization signal, and the Arg-rich RNA-binding motif, are dispensable for RNAPII-binding and elongation stimulation activities in vitro (5 and our unpublished data). On the other hand, the C-terminal region of HDAg, which is well conserved among different HDV genotypes and among clinical isolates of HDV, is involved in HDAg's interaction with RNAPII.5 Point mutations of the conserved amino acid residues within this region strongly reduce the affinity of the HDAg-RNAPII interaction in vitro (our unpublished data). The same set of mutations also impairs HDAg's ability to support HDV replication in cell culture (our unpublished data), suggesting that RNAPII binding and activation by HDAg is important for HDV replication in vivo.

Based on the available data, we present a hypothetical model as to how HDAg activates HDV RNA transcription. In vitro transcription systems that use a genomic fragment of HDV RNA and HeLa cell nuclear extracts have been often criticized for their low efficiency and their inability to synthesize full-length complementary RNAs.4 These defects are most likely attributable to the lack of HDAg. In one report, RNAPII stopped transcription after synthesizing a ~40 nt complementary RNA in the absence of HDAg.5 On a DNA template, DSIF and NELF are known to repress an early step of elongation, primarily around 30~50 nt downstream of the transcription initiation site.21 The close correspondence of the stalled positions suggests that HDAg stimulates HDV RNA transcription by overcoming the repression imposed by DSIF and NELF. At further downstream sites, HDAg may also stimulate elongation by suppressing pauses or arrests that are induced by unfavorable structures of the RNA template or transcript.

As for the two isoforms of HDAg, it is generally thought that HDAg-S activates HDV transcription, whereas HDAg-L represses HDV transcription, possibly through its dominant-negative effect on HDAg-S, and directs viral assembly. In vitro transcription assays using bacterially expressed recombinant HDAg proteins have shown that HDAg-L is capable of activating transcription, even though it is several times less active than HDAg-S.5 Naturally, that experiment does not consider post-translational modifications such as phosphorylation and prenylation. Especially, prenylation is known to occur within the L-specific peptide.25 Such modifications in vivo might make HDAg-L transcriptionally inactive either directly, by preventing its interaction with RNAPII, or indirectly, by sequestering it to the cytoplasm, and thereby allow HDAg-L to inhibit HDV transcription in a dominant-negative manner.

Molecular Analysis of Elongation Control by HDAg

The small size and robust effect of HDAg make it an ideal model for understanding the molecular mechanism underlying elongation control. In recent papers, the elongation process on immobilized DNA templates has been analyzed in detail at nucleotide resolutions of millisecond time resolution.6,7At least four chemical steps are required for each nucleotide addition: NTP binding to the RNAPII active site, forward translocation of RNAPII, phosphodiester bond formation, and pyrophosphate release. Kinetic analysis shows that there is a rate-limiting step during each nucleotide addition, and that this step is activated by HDAg. Circumstantial evidence suggests that this step is equal to translocation. HDAg may thus facilitate the otherwise slow translocation step to accelerate RNAPII elongation.

The structural basis of the RNAPII-HDAg interaction is now being investigated (our unpublished data). RNAPII is a half megadalton, twelve-subunit protein complex. Since the crystal structure of yeast RNAPII was solved in 2001,26,27much attention has been focused on the interactions between RNAPII and accessory proteins. Given the situation that crystallization of human RNAPII is still a long way off, an alternative approach is site-specific photocrosslinking combined with proteolytic mapping. This method requires site-specific introduction of a photoactivatable crosslinker to a protein of interest, usually through a unique cysteine residue. Unlike the known "cellular" transcription elongation factors, HDAg is a small protein consisting of only 195 amino acids and with no cysteine. Therefore, the crosslinker can be easily introduced at a desired position by cysteine substitution. Initial data suggest that the C-terminal region of HDAg contacts with the RNAPII Rpb1 and Rpb2 subunits, which consist of a "clamp". The clamp is a mobile structure that grips DNA and RNA during elongation. An interesting hypothesis is that HDAg may bind and loosen the clamp not only to facilitate RNAPII translocation, as mentioned above, but also to influence template recognition. During HDV RNA transcription, RNAPII is converted from a DNA-dependent RNA polymerase to a dual-specificity RNA polymerase. This loss of template selectivity may be accounted for by HDAg loosening the clamp.

Targeting Transcription Elongation: A General Strategy for Viruses?

In the last section, we pointed out an interesting functional similarity between HDAg and other viral proteins in terms of elongation control. The Tat protein encoded by human immunodeficiency virus (HIV) is one of such examples. HIV establishes latent infection following provirus integration into the host genome. Tat plays a critical role in activating HIV transcription by host RNAPII and thereby regulates the switch from latent to productive infection. Extensive studies have shown that Tat binds to TAR, an RNA element located on the nascent transcript, and recruits P-TEFb to the promoter-proximal region. P-TEFb then facilitates the synthesis of full-length HIV transcripts, in part by counteracting the repression imposed by DSIF and NELF (Fig. 4). Thus, HDAg and Tat exert their functions by targeting the same transcriptional regulatory machinery in different ways. The N protein encoded by bacteriophage λs another example. N plays an important role in switching from the lysogenic to the lytic cycle through elongation control, better known as termination and antitermination in prokaryotes. N binds to nut, which are RNA elements located on nascent transcripts of λ operons. N also interacts with cellular transcription factors and RNA polymerase, prevents termination at multiple downstream sites, and allows the production of full-length transcripts encoding the "late" genes. Thus, controlling transcription elongation may be viruses' general strategy for successfully completing their life cycle.

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