Review

HIV and hepatitis delta virus: evolution takes different paths to relieveblocks in transcriptional elongation

Yuki Yamaguchia,c, Sophie Deléhouzéea, Hiroshi Handab,*a Graduate School of Bioscience and Biotechnology, 4259 Nagatsuta, Yokohama 226-8503, Japan

b Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8503, Japanc PRESTO, Japan Science and Technology Corporation, 4259 Nagatsuta, Yokohama 226-8503, Japan

Abstract

The elongation step of transcription by RNA polymerase II (RNAPII) is controlled both positively and negatively by over a dozen cellularproteins. Recent findings suggest that two distinct viruses, human immunodeficiency virus type 1 and hepatitis delta virus, encode proteinsthat facilitate viral replication and transcription by targeting the same cellular transcription elongation machinery. © 2002 Éditionsscientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Hepatitis delta antigen; Hepatitis delta virus; Human immunodeficiency virus; RNA polymerase II; Tat; Transcriptional elongation

1. Introduction

RNA polymerase II (RNAPII), the DNA-dependent RNApolymerase responsible for cellular mRNA synthesis, alsoplays important roles in the viral life cycle. RNAPII isinvolved in gene expression and/or genome replication ofmost DNA viruses and some RNA viruses. These virusesoften encode proteins that interact with the RNAPII tran-scription machinery, such as the adenovirus E1A protein,the human papillomavirus E7 protein, the human T-cellleukemia virus Tax protein, and the HIV-1 Tat protein.These proteins facilitate viral transcription/replication andcan also affect cellular gene expression to the benefit of viralproliferation and survival. Thus, the elucidation of this typeof host–virus interaction is central to our understanding of awide variety of pathogenic viruses and to the developmentof antiviral therapies.

In this review, we focus on HIV-1 and hepatitis deltavirus (HDV), two RNA viruses with quite different lifecycles. Recent studies have suggested that these viruses areunexpectedly similar in the way they control viraltranscription/replication. Tat and hepatitis delta antigen(HDAg), the proteins encoded by HIV-1 and HDV, respec-tively, likely target the same RNAPII transcription machin-ery that controls the transcriptional elongation process. We

will first briefly overview the RNAPII elongation processbefore going into the details of the mechanism of viraltranscriptional regulation. For readers who are not familiarwith HDV, we will also give an outline of its life cycle.

2. Regulation of RNAPII elongation

Synthesis of mRNA by eukaryotic RNAPII is a complexprocess that can be divided into at least four steps, i.e.preinitiation or the assembly of transcription complexes ona promoter, initiation, elongation, and termination[1,2]. Intheory, each of these steps can be regulated, resulting ineither overall stimulation or repression of transcription.While it is widely accepted that the preinitiation step playsa critical role in transcriptional regulation, over the pastseveral years, increasing attention has focused on the role ofthe elongation step. An obvious reason for the importance ofthe elongation process is the size of protein-coding genes inhigher eukaryotes: many genes span regions of hundreds ofkilobases and take several hours to be completely tran-scribed[3]. Therefore, a lack of regulation of the elongationstep would mean that the expression of some genes wouldbe uncontrolled for a long period of time. The control ofRNAPII elongation would allow a rapid response to envi-ronmental changes or to extracellular stimuli, and in addi-tion, would allow a more sophisticated control of gene

* Corresponding author. Tel.: +81-45-924-5872; fax: +81-45-924-5145.E-mail address: hhanda@bio.titech.ac.jp (H. Handa).

Microbes and Infection 4 (2002) 1169–1175

www.elsevier.com/locate/micinf

© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.PII: S 1 2 8 6 - 4 5 7 9 ( 0 2 ) 0 1 6 4 1 - 6

expression when combined with regulation of the preinitia-tion step.

Transcriptional elongation by RNAPII is controlled by anumber of trans-acting factors called transcription elonga-tion factors [2,4]. During mRNA synthesis, RNAPII fre-quently encounters blocks to elongation; a primary cause ofpause or arrest is thought to be the secondary structureformed by nascent transcripts. Transcription elongationfactors such as TFIIF, elongin, ELL, and TFIIS interact withRNAPII and thereby prevent it from pausing or reactivate itfrom arrest.

The recent discovery of a new class of transcriptionelongation factors, which include DRB-sensitivity inducingfactor (DSIF), negative elongation factor (NELF), andpositive transcription elongation factor b (P-TEFb), hasshed new light on the control of RNAPII elongation [4–6].Shortly after the initiation of transcription, RNAPII is

subjected to both negative and positive control by thesefactors. DSIF and NELF cause transcriptional pausing byphysically associating with RNAPII (Fig. 1A) [7–10].Conversely, P-TEFb allows RNAPII to enter a productiveelongation phase by preventing DSIF and NELF fromacting [8,9,11,12]. P-TEFb is a protein kinase that stronglyphosphorylates the C-terminal domain (CTD) of RNAPIIand the Spt5 subunit of DSIF [11,13]. Several, but not all,lines of evidence suggest that P-TEFb-dependent phospho-rylation of the CTD facilitates the release of DSIF andNELF from RNAPII, thereby counteracting transcriptionalrepression (Fig. 1A) [9,11,12,14]. Thus, DSIF, NELF, andP-TEFb appear to constitute a critical rate-limiting step oftranscription during the early phases of RNAPII elongation.

Interestingly, DSIF is also capable of activating tran-scriptional elongation. Although the mechanism is largelyunknown, available data are consistent with the model that

Fig. 1. Hypothetical models for transcriptional elongation control by cellular and viral proteins. (A) During transcription of cellular genes, DSIF and NELFblock mRNA chain elongation through direct association with RNAPII (left). P-TEFb alleviates this block by phosphorylating the RNAPII CTD and DSIF(center). A multiprotein complex, which is not well characterized but possibly contains P-TEFb and DSIF [15], interacts with RNAPII to further increasethe processivity of transcriptional elongation (right). (B) During transcription of the HIV-1 proviral genome, RNAPII prematurely stops at ∼ +60 in theabsence of the viral protein Tat. For this premature termination, several mechanisms have been proposed, including one involving DSIF and NELF (left)[20–22]. Tat binds to the TAR RNA structure located near the 5’ end of the HIV-1 transcript, and thereby recruits P-TEFb (center) as well as the multiproteincomplex (right), which together strongly activate RNAPII elongation in a manner similar to that described in (A). (C) During transcription of cellular genesand transcription/replication of the HDV genome RNA, DSIF and NELF block RNA chain elongation through direct association with RNAPII (left). Theviral protein HDAg-S binds to RNAPII and alleviates the block by displacing NELF and moreover, further activates RNAPII elongation (right) in a mannersimilar to that of such conventional transcription elongation factors as Elongin and ELL [47]. Transcription/replication of the HDV genome likely proceedsthrough a rolling-circle mechanism, i.e. newly made RNA remains associated with the template RNA. P’s indicate phosphorylation of proteins. Nascenttranscripts are shown as pink lines.

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DSIF, but not NELF, participates in a large protein complexthat binds to RNAPII and enhances the processivity oftranscriptional elongation (Fig. 1A) [15,16].

3. HIV-1

HIV-1, the etiological agent for the acquired immunode-ficiency syndrome, is a retrovirus with a linear single-stranded RNA genome of ∼ 9 kb [17,18]. The RNA genomeis converted to double-stranded DNA by a viral reversetranscriptase in infected cells and then integrated into thehost genome. RNA synthesis of the proviral DNA ismediated by host RNAPII. Either a terminal sequence of theproviral DNA or the long terminal repeat functions as apromoter for RNAPII and directs synthesis of the entireviral RNA. Some RNA copies are encapsulated into newvirions, while others are processed to form mRNAs for allthe known viral proteins. Thus, RNAPII-mediated RNAsynthesis serves in viral genome replication as well as inviral gene expression.

Successful transcription of the HIV-1 proviral DNArequires the viral transactivator Tat [18–20]. In its absence,RNAPII encounters a strong block to elongation ataround +60 from the transcriptional start site (Fig. 1B). Ithas been proposed that a downstream DNA element (calledinducer of short transcripts), the secondary structure of thenascent transcripts, and the host factors DSIF and NELF areresponsible for the negative control of HIV-1 transcription[20–22]. Tat strongly stimulates the synthesis of full-lengthHIV-1 transcripts through its interaction with the trans-activation response element (TAR) RNA located near the 5’end of the nascent transcript [23,24]. Through this interac-tion, Tat recruits P-TEFb to the arrested polymerase [25].P-TEFb has been shown to be essential for Tat-dependenttranscriptional activation, and it functions, at least in part,by overcoming the transcriptional block imposed by DSIF

and NELF (Fig. 1B) [26,27]. Tat also recruits multiplecellular cofactors other than P-TEFb, including the DSIF-containing positive transcription elongation factor complexmentioned above, which collectively further promote tran-scriptional elongation (Fig. 1B) [15].

Tat is a small nuclear RNA-binding protein of 101 aminoacids (Fig. 2A) [28] with an arginine-rich motif (ARM)-typeRNA-binding domain in the central region. The ARM isfound in a variety of proteins involved in RNA metabolism[29], including hepatitis delta antigen (HDAg) (see below)and the bacteriophage λ antiterminator protein N [30]. TheARM of Tat also functions as a nuclear localization signal.Another important function is encoded by the N-terminal 48amino acids, which include multiple conserved cysteineresidues and constitute a minimal transcriptional activationdomain. Little is known about the roles of the C-terminalregion of Tat. Some viral strains have premature terminationcodons in the Tat gene, leading to the production of aC-terminally truncated protein [28]. Thus, the C-terminalregion of Tat appears to be dispensable for the HIV-1 lifecycle under certain conditions.

Tat binds to the TAR RNA stem-loop structure throughthe ARM RNA-binding domain [31]. Tat also binds to thecyclin T1 subunit of P-TEFb through the transcriptionalactivation domain, and in addition, cyclin T1 recognizes theTAR substructure and increases the specificity and stabilityof the Tat-TAR interaction [25]. Overall, Tat recruitsP-TEFb through multiple protein–protein and protein–RNAinteractions to the arrested polymerase.

4. HDV

The first report on HDV cases was made in 1977 byRizzetto and colleagues [32], with the identification of anew antigen, termed HDAg, in liver cells and serum ofhepatitis B patients. HDAg was initially thought to be

Fig. 2. Schematic structures of HIV-1 Tat and HDV HDAg. (A) Tat is a 101-amino acid (aa) protein with an ARM-type RNA-binding domain. The N-terminal48-amino acid segment including a conserved cysteine (Cys)-rich region functions as a minimal transcriptional activation domain. (B) HDAg-S and HDAg-Lare 195- and 214-amino acid proteins that are identical except for the C-terminal 19 amino acids unique to HDAg-L. Several distinct domains and functionsof HDAg have been assigned, including the N-terminal coiled-coil region involved in homo- and hetero-oligomerization, two ARM-type RNA-bindingdomains, and the NES located within the L-specific peptide. ‘S’ on HDAg-L indicates the position of the cysteine residue that is farnesylated in infected cells.

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derived from hepatitis B virus (HBV), but later found tooriginate from a new and different virus termed HDV[33,34]. HDV is unable to complete a viral life cycle on itsown. Instead, HDV requires HBV to supply its envelopecomponent or hepatitis B surface antigen (HBsAg), whichenables the formation of new infectious HDV particles.HDV is, thus, a satellite virus of HBV, capable of producingprogeny viruses only in HBV-infected cells [33,34].

Compared to HBV-infected patients, those infected withboth HBV and HDV tend to develop more severe clinicalsymptoms [35,36]. HDV infection can occur either as acoinfection with HBV or as a superinfection of patients withchronic HBV infection. Superinfection with HDV of HBVcarriers leads to more progressive chronic liver disease, withhigher incidence of cirrhosis and hepatocellular carcinoma.In contrast, individuals with HBV-HDV coinfection havemore severe acute disease and a higher risk of fulminanthepatitis compared to those infected with HBV alone.

4.1. The structure and the life cycle of HDV

HDV is a spherical virus of approximately 36 nm indiameter [37]. The envelope contains HBsAg, which isderived from coinfecting HBV (Fig. 3A). The nucleocapsidis a ribonucleoprotein complex of about 19 nm in diameter,which is composed of the genomic RNA and an average of70 HDV-encoded HDAg proteins per RNA molecule. Twospecies of HDAg (HDAg-L and HDAg-S) are found in thevirion with varying ratios [37].

The HDV genome is a 1.7-kb single-stranded circularRNA (Fig. 3B) [38,39]. Due to a high level of sequencecomplementarity, 70% of the genome forms intramolecularbase pairs. Indeed, HDV RNAs appear as unbranched,rod-shaped structures when examined by electron micros-copy under non-denaturing conditions [40]. This circularRNA contains a single ORF encoding HDAg on theantigenomic strand and a sequence with ribozyme activityon both the genomic and antigenomic strands, which causessite-specific cleavage of the HDV RNA (Fig. 3B).

HDV progresses in its viral life cycle through RNA-dependent RNA synthesis [33,34]. Once inside the cell, the1.7-kb RNA genome is used as a template to produce a1.7-kb antigenomic RNA intermediate and a 0.8-kb poly-adenylated HDAg mRNA. The antigenome is then used asa template to produce RNA genomes, which are subse-quently packaged into new HDV particles or used as atemplate for the next round of replication. The replication ofboth the genomic and antigenomic strands is considered tooccur through a rolling-circle mechanism [33,34]. As repli-cation proceeds on the circular templates, multimeric repli-cation products appear that are subsequently cleaved intomonomers through the action of the ribozymes. The unit-length RNAs are self-ligated to form circles by an as yetunknown mechanism [41].

Both genomic RNA replication and transcription involveRNA-dependent RNA synthesis without DNA intermediatesand are considered to occur through a similar mechanism.HDV does not encode its own polymerase and, therefore,must use the host’s machinery. Most previous studies havepointed to the host RNAPII as being the enzyme responsiblefor RNA-dependent RNA synthesis of the HDV genome[42–45]. The mechanism by which a DNA-dependent RNApolymerase is recruited to transcribe an RNA template hasnot been fully deciphered; however, the viral protein HDAgmay be involved in an unconventional use of the hostpolymerase, as discussed below.

4.2. The multiple functions of HDAg

Two HDAg protein forms, HDAg-S (195 amino acids)and HDAg-L (214 amino acids), are translated from viralmRNA through a process known as RNA editing [46].During replication, site-specific mutations occur in the HDVgenome through the action of a cellular enzyme, double-stranded RNA-specific adenosine deaminase. This convertsthe UGA termination codon of the HDAg mRNA to a UGGtryptophan codon. As a result, the coding region is extendedto the next termination codon, and the translated protein

Fig. 3. Schematic structures of HDV virion and genome. (A) HDV are spherical viruses of ∼ 36 nm in diameter [37]. The envelope contains HBsAg, whichis derived from coinfecting HBV. The nucleocapsid is a ribonucleoprotein complex composed of the genomic RNA and HDV-encoded HDAg. (B) HDV hasa 1.7-kb single-stranded circular RNA genome with an unbranched, rod-like structure [38–40]. Two RNA species are generated from the genome: a 1.7-kbantigenomic RNA intermediate and a 0.8-kb polyadenylated mRNA for HDAg. Short segments of both genomic and antigenomic strands of the HDV RNAshow ribozyme activities that cause site-specific cleavage of the HDV RNA.

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increases in size from that of the original HDAg-S to that ofHDAg-L, which is 19 amino acids longer (Fig. 2B).

The two HDAg forms show opposing biological activi-ties [33,34]. HDAg-S, synthesized early in infection, isessential for viral transcription and replication: disruption ofthe HDAg-S ORF abolishes viral transcription and replica-tion in host cells. On the other hand, HDAg-L is synthesizedlater in infection and acts as a dominant-negative inhibitorof HDAg-S, thereby preventing further viraltranscription/replication. In addition, HDAg-L is consideredto promote the assembly of viral particles through nuclearexport of viral ribonucleoprotein complexes and interactionwith HBsAg molecules at the cellular membrane.

A recent report has defined the role of HDAg-S inRNAPII transcription. The transcription elongation factorNELF is composed of five polypeptides, and one of thesesubunits, termed NELF-A, shows limited sequence similar-ity to HDAg [47]. This similarity has stimulated subsequentstudy and led to the important finding that HDAg-S is theviral transcription elongation factor. The direct associationof HDAg with RNAPII competes with the NELF–RNAPIIinteraction, and the regions of similarity shared by HDAgand NELF-A may form a conserved structure that recog-nizes a common surface on RNAPII. HDAg-S is proposedto stimulate transcriptional elongation by two differentmechanisms: (i) HDAg-S reverses the negative effect ofDSIF and NELF by displacing NELF from RNAPII, and (ii)the HDAg-S–RNAPII interaction by itself further activatestranscriptional elongation (Fig. 1C) [47]. In support of thesecond model, HDAg-S strongly stimulates elongation ofpurified RNAPII on a modified DNA template in theabsence of any other protein factors [47]. Proteins with suchan activity have been considered to be transcription elon-gation factors [48,49]. In this sense, HDAg-S is the firstviral transcription elongation factor to be identified.

4.3. The modular structure of HDAg

The similarities and differences between the functions ofthe two HDAgs can be attributed to the protein domains thateach possesses [33,34]. Both HDAg-S and HDAg-L have aleucine-zipper motif at the N-terminus that enables homo-and hetero-oligomerization (Fig. 2B). HDAg-S andHDAg-L also share two ARMs, both of which are necessaryfor specific binding to HDV RNA [50]. HDAgs bind toHDV genomic and antigenomic RNA with similar affinity,and seem to recognize the overall rod-shaped structureinstead of specific RNA sequences. HDAg-S and HDAg-Lalso encode a typical nuclear localization signal whichenables the bound HDV RNA to be imported to the nucleus,where HDV transcription and replication occur.

The differences between the functions of HDAg-S andHDAg-L lie in the C-terminus. HDAg-S binds to RNAPIIthrough its CTD (Fig. 2B) [47]. HDAg-L, with an additional19 amino acids at the C-terminus, however, has only a weakRNAPII-binding activity [47]. The L-specific peptide is

farnesylated at the 211th cysteine residue (Fig. 2B) [51,52].This farnesylation reportedly causes a conformationalchange of a region spanning the RNAPII-binding domain ofHDAg [53]. In addition, the L-specific peptide functions asa nuclear export signal (NES) [54], interacts with HBsAg[55], and is necessary for copackaging with HBsAg intonew HDV particles [51]. Taken together, the L-specificpeptide may inhibit the HDAg–RNAPII interaction bycausing a conformational change of the RNAPII-bindingdomain of HDAg or by redirecting HDAg to the cytoplasm.The inhibitory effect of the L-specific peptide may extend toHDAg-S through hetero-oligomerization between HDAg-Land HDAg-S.

4.4. Future directions

HDAg stimulates transcriptional elongation from bothDNA and HDV RNA templates in vitro [47]. While HDAgis known to stimulate transcription/replication of HDVRNA strongly in vivo, it remains to be determined whetherHDAg has any effect on the transcription of protein-codinggenes from the host genome. Considering the enhanceddisease severity observed during dual infection by HBV andHDV, including the higher incidence of hepatocellularcarcinoma, it is an interesting, testable idea that HDAg maydirectly cause cytopathic effects by disturbing host genomeexpression.

It is largely unknown how HDV RNA-dependent RNAsynthesis proceeds. Sequences near the ends of the rod-likestructure of HDV RNA appear to function as an RNAPIIpromoter, directing the synthesis of a complementary RNAstrand [44,56]. Little is known about the cellular factor(s)responsible for ‘promoter’ recognition and the RNAPIIrecruitment steps. These issues which need to be addressedin the future. Possibly, besides its roles as a transcriptionelongation factor, HDAg-S may be involved, through itsinteractions with HDV RNA and RNAPII, in the recruit-ment of RNAPII onto non-physiological templates.

A few laboratories have successfully reconstituted thetranscription initiation reaction using crude cell extracts andgenomic fragments of HDV [43,44,56]. One of these studies[44] demonstrated that the RNA template somehow under-goes an endonucleolytic cleavage at a unique site, and thatthe free 3’ end is used as a primer for the synthesis of thecomplementary strand. Interestingly, in the absence ofHDAg-S, transcription ceases after 41 nucleotides of thetemplate are copied, and the addition of HDAg-S allowsRNA synthesis to resume. Thus, transcription/replication ofHDV, as well as that of HIV-1, may be controlled at an earlyphase of transcriptional elongation.

5. Comparison of HIV and HDV

HIV-1 and HDV are two viruses with entirely differentstructures, life cycles, and host ranges. They are similar,

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however, in the way they rely on the host RNAPII for viraltranscription/replication and proliferation. In both cases,viral transcription encounters a block at an early stage of theelongation process, and the block is relieved by a virallyencoded protein. HIV-1 Tat and HDV HDAg may differstructurally and functionally: whereas Tat targets P-TEFband stimulates a positive elongation factor of transcription,HDAg targets NELF and displaces a NELF. Both are,however, small nuclear proteins containing an ARM RNA-binding domain, and both function overall in a similar wayto facilitate viral transcription and promote successful viralproliferation. An interesting similarity can be seen in somebacteriophage proteins. The phage λ-encoded N protein isan ARM-containing RNA-binding protein that is respon-sible for the antitermination of viral transcription [30]. Nprotein binds to the bacterial RNA polymerase and causesread-through at terminator sites on λ operons, which resultsin the production of longer transcripts that are required forλ replication.

Transcriptional pausing has been found to occur duringtranscription of many viral genomes, including those ofHIV, HDV, adenovirus, polyomavirus, simian virus 40, andminute virus of mice [3]. A transcriptional elongation block,thus, seems to play a general role in the prevention of viralproliferation. Although HIV and HDV are so far the onlyanimal viruses known to encode proteins that relieve thetranscriptional block, other viruses may also have evolvedin one way or another to either counteract such regulation oruse it for their survival.

Acknowledgements

Supported in part by a Grant for Research and Develop-ment Projects in Cooperation with Academic Institutionsfrom the New Energy and Industrial Technology Develop-ment Organization to H.H.

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