Hepadnaviridae and Deltavirus

Properties of Hepadnaviridae 359

Clinical Features of Hepatitis B 362

Pathogenesis and Immunity..____364

Laboratory Diagnosis 366

Epidemiology 369

Control 369

Deltavirus (Hepatitis D) 373

Further Reading 379

In 1963 Blumberg, a geneticist investigating hereditary factors in the sera of isolated racial groups, discovered an antigen in the serum of an Australian aborigine that reacted with sera from multiply transfused American hemophiliacs. In due course the antigen was demonstrated to be present on the surface of particles with three different morphologic forms (Fig. 22-1) and to be associated with the disease serum hepatitis, now known as hepatitis B. The 22 nm particles of "Australia antigen," subsequently renamed HBsAg, for hepatitis B surface antigen, were found to be noninfectious, but the 42 nm particles were shown to be infectious virions capable of transmitting hepatitis to chimpanzees. The unique characteristics of these viruses led to their classification within a new family, named Hepadmi'iridae to reflect the association with hepatitis and the DNA genome. The very small genome replicates via a unique mechanism.

Hepatitis B is one of the world's major unconquered diseases. Some 300 million people are chronic carriers of the virus, and a significant minority go on to develop cirrhosis or cancer of the liver from which over 1 million die every year. Although hepatitis B virus (HBV) has yet to be cultivated repro-ducibly nt vitro, reliable diagnostic procedures and a much-needed vaccine are available.

Fig. 22-1 Hepadnavtridac. Negatively stained preparation of hepatitis B virions (large arrow) and accompanying HBsAg particles (small arrows) Bar, 100 nm. (Courtesy of Dr. I D. Gust and J. Marshall.)

Properties of Hepadnaviridae

The family Hepadnaviridae contains hepatitis viruses specific for humans, woodchucks, ground squirrels, ducks, and herons. We are concerned here only with the agent of human hepatitis B (Table 22-1).

The virion possesses two shells (Fig. 22-1), a 27 nm icosahedral nucleocap-sid (core) constructed from 180 capsomers, surrounded by a closely fitting envelope. The virion is relatively heat-stable but labile to acid and to lipid solvents. The genome consists of a 3.2 kbp molecule of circular dsDNA of most unusual structure (Fig. 22-2). The plus strand is incomplete, leaving 15-50% of the molecule single-stranded; the minus strand is complete but contains a discontinuity ("nick") at a unique site. The 5' termini of the plus and minus strands overlap by about 240 nucleotides and include short direct repeats, DR1 and DR2, producing "cohesive" ends that base-pair to maintain the chromosome in a relaxed circular configuration. A "terminal protein" is

Table 22-1

Properties of Hepatitis B Virus

Spherical enveloped virion, 42 nin, enclosing inner icosahedral 27 nm nucleocapsid (core) Envelope contains glycoprotein, HBsAg; core contains phosphoprotem, HBcAg, plus polymerase with three enzyme activities' reverse transcriptase, DNA polymerase, RNase H Circular dsDNA genome, 3 2 kb, cohesive 5' ends; minus strand nicked, 5' terminal protein, plus strand incomplete, 5' RNA primer Four overlapping open reading frames S, C, P, and X

Genome converted to supercoiled covalently closed circular form and transcribed in nucleus to produce full-length pregenome RNA and subgenomic mRNAs RNA pregenome in cytoplasmic core particles reverse transcribed to dsDNA, some return to nucleus to augment pool of viral supercoiled DNA Cores bud through endoplasmic reticulum, acquiring lipid membrane containing HBsAg, non-cytocidal

Subtypes differ in allelic pairs of HBsAg determinants (d or y, r or re)

covalently attached to the 5' end of the minus strand, whereas a 5'-capped oligoribonucleotide primer is attached to the 5' end of the plus strand.

The minus strand contains four open reading frames: pre-S/S, pre-C/C, P (or POL), and X (Fig. 22-2). The P gene, which compromises 80% of the genome and overlaps all the other genes, encodes a polymerase with three distinct enzymatic functions (DNA polymerase, reverse transcriptase, and RNase H) and also encodes the terminal protein primer. Gene X, spanning the cohesive ends of the genome, encodes a transactivating protein that up-regulates transcription from all the viral and some cellular promoters. The C gene has two initiation sites that divide it into a prc-C and a C region, producing two distinct proteins, HBeAg and HBcAg, respectively. The pre-S/S gene encodes the envelope protein, S, which occurs in three forms: a large (L) protein, translated from the first of the three in-phase initiation codons, is a single polypeptide encoded by the pre-Sl, pre-S2, plus S regions of the genome and occurs in the envelope of infectious virions; a middle-sized (M) protein comprises the product of pre-S2 plus S; and finally, the most abundant product is the S protein, the basic constituent of noninfectious HBsAg particles, comprising only the product of the 5 ORF. All three forms are glycosylated, and the pre-Sl product is myristylated.

Fig. 22-2 HBV genome Wavy lines denote viral transcripts; boxes, viral open reading frames (ORFs); arrows, direction of transcription and translation; innermost circles, structure of virion DNA, DR1, DR2, positions of direct repeat sequences involved in the priming steps in viral DNA synthesis (From D Ganern and H. E Varmus, Awni Rci> Riochem 56,651(1987).)

Fig. 22-2 HBV genome Wavy lines denote viral transcripts; boxes, viral open reading frames (ORFs); arrows, direction of transcription and translation; innermost circles, structure of virion DNA, DR1, DR2, positions of direct repeat sequences involved in the priming steps in viral DNA synthesis (From D Ganern and H. E Varmus, Awni Rci> Riochem 56,651(1987).)

There are a number of subtypes of hepatitis B virus, defined by various combinations of antigenic determinants present on the HBsAg. All have the same group-specific determinant, a, but there are four major subtype-specific determinants, certain pairs of which (d and if; r and iv) tend to behave as alleles, that is, as mutually exclusive alternatives. The w determinant displays considerable heterogeneity. Hence we have subtypes designated at/w, ayw2, ayiv3, ayw4, ayr, adw4, adr, and so on. There are also some more unusual combinations, and additional determinants such as q and j or g have been described, as well as some variants with mutations in any of the determinants. Different subtypes tend to show characteristic geographical distributions, though they often overlap.

Viral Replication

In the absence of a conventional cell culture system for HBV much of our current understanding of the replication of hepadnaviruses comes from studies of woodchuck, ground squirrel, or duck hepatitis viruses, or from the growth of HBV in human hepatocytes either in vivo or in primary culture, or following transfection of hepatoma cell lines by various HBV DNA constructs.

The HBV genome is remarkably compact and makes use of overlapping reading frames to produce seven primary translation products from only four ORFs ("genes"): S, C, P, and X. Transcription and translation are tightly regulated via the four separate promoters and at least two enhancers plus a glucocorticoid-responsive element. Transcription occurs in the nucleus, whereas replication of the genome takes place in the cytoplasm, inside protein cores that represent intermediates in the morphogenesis of the virion. Replication of the dsDNA genome occurs via a unique mechanism involving the reverse transcription of DNA from an RNA intermediate. Thus hepadnaviruses are sometimes categorized as "retroid" viruses because of the similarity in replication strategy to the retroviruses, although in a sense the two strategies are mirror images of one another. The key difference is that, in the case of the retroviruses, the plus sense ssRNA is packaged as the genome of the virion, whereas in the case of the hepadnaviruses, the ssRNA is the intracellular intermediate in the replication of the dsDNA genome (Fig. 22-3).

The virion attaches to the hepatocyte via a sequence in the pre-Sl protein and enters by receptor-mediated endocytosis. Following removal of the envelope, the nucleocapsid is translocated to the nucleus and the viral genome released. The short (plus) strand of viral DNA is then completed to produce a full-length relaxed circular (RC) dsDNA molecule. This in turn is converted to a cpvalaitly closed circular (CCC) form by removal of the protein primer from the minus strand and ol the oligoribonucleotide primer from the plus strand, elimination of the terminal redundancy from the minus strand, and ligation of the two ends of the DNA. This closed circular form becomes twisted to yield what is known as supercoiled (SO DNA, which is the template for transcription by cellular RNA polymerase II. The "minus" strand only is transcribed to give mRNAs of 2.1 and 2.4 kb, plus a 3.4 kb RNA transcript known as the preg-enome that is actually longer than the genome itself because it contains terminally redundant sequences. Following transport to the cytoplasm, the 3.4 kb species is translated to yield the C (core) antigens and the polymerase, while

/mmtmmmtmmmmmam^^

Nucleus \ Supercoiled viral DNA

(Transcription

Translation

DNA polymerase Viral proteins Assembly of core

Pregenome packaged into nucleocapsid

DNA polymerase Viral proteins Assembly of core

Pregenome packaged into nucleocapsid

Synthesis ol (-) DNA

Synthesis of (+) DNA

Synthesis ol (-) DNA

Synthesis of (+) DNA

Fig. 22-3 Replication cycle of hepatitis B virus. See text for details [Modified from G, Civttico, Y Y Wang, C Luscombe, N. Bishop, G Tachedjian, I Gust, and S. Locarnim, / Med Virol 31,90 (1990) Courtesy Dr S Locarnini I

the 2.1 and 2.4 kb transcripts are translated from three different initiation codons to yield the three forms of S (surface) antigens.

Replication of the viral genome occurs via a mechanism absolutely distinct from that of any other DNA virus. The RNA pregenome associates with the polymerase and core protein to form an immature core particle in the cytoplasm. Within this structure the reverse transcriptase, primed by the virus-coded terminal protein, transcribes a complementary (minus) strand of DNA. Meanwhile, the RNase H progressively degrades the RNA template from its 3' end, leaving only a short 5' oligoribonucleotide which, following transposition to base-pair with a complementary site on the newly synthesized minus strand, serves as the primer for the DNA polymerase to transcribe a DNA plus strand. Some of the core particles, containing newly synthesized viral DNA, are recycled back into the nucleus to amplify the pool ol HBV genomes available for transcription. The remainder are assembled into virions before the plus strand of the genome has been completed. The cores bud through those areas of endoplasmic reticulum into which the L, M, and S antigens have been inserted, thereby acquiring an HBsAg-containing lipid envelope. Vesicles transport the virions to the exterior without cell lysis.

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