Infection with hepatitis B virus (HBV) leads to a wide spectrum of clinical presentations ranging from an asymptomatic carrier state, acute self-limited or fulminant hepatitis, to chronic hepatitis with progression to cirrhosis and hepatocellular carcinoma (HCC). Both viral factors as well as the host immune response have been implicated in the pathogenesis and clinical outcome of HBV infection. Evidence has been accumulating that certain HBV mutants are associated with unique clinical manifestations, may affect the natural course of the infection and confer resistance to antiviral agents.1–3 Naturally occurring mutations in the context of various genotypes have been identified in the structural and nonstructural genes as well as regulatory elements of the virus. The best characterized mutants are the pre-core (pre-C) stop codon mutation resulting in a loss of hepatitis B e antigen,4 defined clusters of mutations in the core promoter resulting in enhanced viral replication,5–7 and mutations in the reverse transcriptase/polymerase conferring resistance to antivirals.3 Furthermore, several mutations in the HBV surface gene have been identified which alter the antigenicity of the viral surface proteins (HBsAg) and structure of the viral envelope.2, 8
Molecular Biology of HBV Surface Expression.
The viral envelope plays a key role in the viral life cycle and pathogenesis of HBV infection. First, HBV surface-host cell interaction represents the very first step of viral infection and thus is central to the host cell tropism of the virus. Second, the HBV surface proteins represent a key target of host antiviral immune responses. Finally, the HBV surface antigen (HBsAg) is used as a diagnostic tool because of its high circulating level in active HBV infection.8, 9
The three viral envelope proteins are expressed from a single open reading frame.8–10 Transcription of this frame starts at an upstream promoter resulting in the formation of preS1 RNA, and at a second internal promoter resulting in synthesis of preS2/S RNAs (Fig. 1A). Splicing of transcripts is not required for expression of surface proteins. The transcripts contain many potential splice donor and acceptor sites, but they also contain a splice-suppressing sequence, homologous to the rev-responsive element (RRE) of HIV. The corresponding sequence in the HBV genome was termed posttranscriptional regulatory element (PRE).11 Translation of preS1 RNA yields the large envelope protein (LHBs). The 5′ ends of the second transcript -preS2/S RNA- are heterogeneous.10 Translation of the longer preS2/S transcript from the first start codon yields the middle-sized envelope protein MHBs, whereas translation of the shorter transcript begins from the second start codon results in synthesis of the small surface protein SHBs.8–10 (Fig. 1A). The domain present in all three proteins is referred to as the S domain (Fig. 1A). The additional N-terminal domain of MHBs is called preS2, and the N-terminal domain unique to LHBs is named preS1 (Fig. 1A).10
The envelope proteins have a complex transmembrane topology and are subjected to extensive posttranslational modifications including glycosylation.8, 10 Both the virion and the surface antigen particles are assembled at the endoplasmic reticulum (ER) and bud to the lumen of a post-ER intermediate compartment.8, 10
Mutant-induced Inhibition of Surface RNA Splicing: A Novel Target for Modulation of HBsAg Expression.
In this issue of HEPATOLOGY, Hass et al. describe a novel mutation affecting HBsAg expression by a posttranscriptional mechanism.12 Aiming to study the functional role of HBV mutations in HBsAg-negative HBV infection, Hass et al. functionally characterized viral strains from two HBsAg-negative patients with reactivation. Starting from this clinical presentation, Hass et al. cloned several full-length HBV genomes from the time of HBV reactivation and functionally characterized the sequenced genomes by transfection into human hepatoma cells.12
Interestingly, a large fraction of genomes from one of the two patients did not express pre-S2/S mRNA and HBsAg, suggesting the presence of mutations resulting in an altered S gene expression. Sequence analysis and site-directed mutagenesis revealed a single G→A mutation within the S gene at position 458 (G458A) to be responsible for this effect.12 To further characterize the mechanism of this mutant-induced downregulation of preS2/S transcription and HBsAg expression, the authors performed a series of experiments demonstrating that the G458A mutation exerts a posttranscriptional effect on preS2/S mRNA processing. By studying the viral sequences, the authors noticed that the G458A mutation is adjacent to a 5′ splice site of the S mRNA.12
Although functional subgenomic S transcripts have not been previously shown to undergo splicing, splicing of HBV pregenomic RNA has been described.13–15 Splicing of HBV transcripts does not appear to be essential for HBV replication,13–15 but whether this process may affect other aspects of viral life cycle has not been rigorously studied. By detecting spliced preS2/S RNA in HBV transfected hepatoma cells, Hass et al. demonstrate that preS2/S splicing occurs during replication of authentic full-length HBV genomes (Fig. 1B). In further experiments the authors demonstrate that G458A inhibits pre-S2/S splicing (Fig. 1B). Surprisingly, inhibition of splicing resulted in a marked decrease of the unspliced pre-S2/S transcript and its translation products. While the mechanism of this finding has not been elucidated, the authors hypothesize that the mutations may alter the interaction of RNA with RNA binding proteins thus resulting in reduced RNA stability or nuclear export.12
To function properly, mRNAs must contain, in addition to a string of codons, information that specifies their nuclear export, subcellular localization, translation and stability.16 This information is provided by specific RNA-binding proteins. These proteins — collectively referred to as heterogeneous nuclear ribonucleoproteins (hnRNP proteins) or mRNA-protein complex proteins (mRNP proteins) — are PRE-mRNA/mRNA-binding proteins that associate with these transcripts and strongly affect their function and fate.16, 17 These proteins influence pre-mRNA processing as well as the transport, localization, translation and stability of mRNAs. It was recently been shown that one group of these proteins — the exon junction complex (EJC) — marks exon–exon junctions and has a role in mRNA export.16, 17
The authors hypothesize that the G458A mutation may alter the interaction of preS2/S RNA with EJC proteins, thus modulating posttranscriptional RNA processing. Furthermore, they suggest that upon formation of the spliceosome, the mutation may also affect the RNA protein-interaction at the 3′ splice site.12 Noting that the 3′ splice site is in close proximity to the HBV PRE, which plays a crucial role in splicing inhibition and export,11 the authors then suggest that mutant-induced alteration of pre-S2/S mRNA-protein interaction at the 3′ splice site may facilitate unspliced pre-S2/S RNA export via PRE.12
Further studies elucidating the mechanism of this unique observation may provide important insights into the regulation of posttranscriptional HBV RNA processing. These studies may also address the question of whether the observed preS2/S splicing is present in all genotypes and why the identified mutation appears to specifically inhibit preS2/S RNA splicing. Furthermore, it would be of interest to study whether the mutation impairs envelopment of viral capsids and subsequently the formation of infectious virions.
Clinical Implication of HBV Mutations Preventing S RNA Splicing.
Another key question is whether the G458A mutation may play a functional role in the clinical course of disease. In this context, it is of interest to note, that a variety of mutations have been identified in the HBV surface proteins resulting in differential antigen recognition and immune response. The best-characterized mutations are vaccine or immune escape mutations resulting in escape from immune responses following active or passive immunization or during HBV reactivation following immunosuppression.18–21
Pathogenesis and viral immune response during acute hepatitis B are mediated mainly by a strong T cell response directed against a variety of epitopes of viral proteins including surface proteins.22, 23 Since G458A results in a marked down-regulation of HBsAg expression, it is conceivable that mutant-induced alteration of surface protein expression may facilitate escape from host antiviral humoral and cellular immune responses (immune escape variant) and thus contributing to a different clinical course. Interestingly, the patient harboring the G458A mutation experienced HBV reactivation following immunosuppression by coinfection with HIV.12 Further studies are needed to answer this question.
Finally, the presence of defined mutations in the surface gene may lead to negative tests in diagnostic assays for HBsAg.24 These mutations include the alteration of HBsAg antigenicity escaping anti-HBs based detection assays as well as mutations in surface gene promoters resulting in downregulation of HBsAg expression by a cotranscriptional mechanism.25, 26 In this study, Hass et al. demonstrate that diagnostic escape may also be the result of a posttranscriptional effect of viral mutations on HBsAg expression.
In summary, the detailed molecular and functional analysis of a novel HBV surface mutation isolated from a patient with impaired cellular immunity and HBV re-activation by Hass et al. has paved a new avenue to gain novel insight into the regulation of HBsAg expression. Further epidemiological and functional studies based on the findings of Hass et al. may also define the impact of the biological phenotype of G458A for diagnostic escape and HBV re-activation following immunosuppression.
The authors acknowledge the support of the European Union (EU NoE VIRGIL), the Deutsche Forschungsgemeinschaft (DFG) and the Bundesministerium für Forschung und Technologie (BMBF).