Recombinant Duck hepatitis B virus Large envelope protein (S)

What is the structure and function of the DHBV large envelope protein?

The large envelope protein (L protein) of Duck Hepatitis B Virus is encoded by the Pre-S/S open reading frame. It is initiated from the AUG at position 801 in the pre-S region of the pre-S/S coding sequence, which yields an N-terminal consensus sequence for myristylation . The L protein typically appears as a doublet at 36 and 35 kDa on Western immunoblots .

The L protein plays critical roles in:

• Mediating virus adsorption to host cells

• Participating in virion morphogenesis

• Contributing to the viral infectious cycle

Structurally, the L protein contains a pre-S domain that is essential for viral infectivity and can undergo significant conformational changes during the viral life cycle. These conformational changes are regulated by post-translational modifications, particularly phosphorylation .

How does phosphorylation affect the function of DHBV large envelope protein?

Phosphorylation of the large envelope protein is a critical post-translational modification that regulates its function in the viral life cycle. Key aspects of this phosphorylation include:

• The L protein heterogeneity (appearance as a doublet) is due to phosphorylation of threonine and/or serine residues within the pre-S domain .

• At least one possible phosphorylation site is located at a novel (S/T)PPL motif which is conserved near the carboxyl end of the pre-S1 domain in all hepadnavirus sequences .

• Two to three additional (S/T)P motifs are present in the carboxyl half of the pre-S1 domain of all hepadnaviruses .

Functionally, phosphorylation affects:

• Topological shifts within the L protein during morphogenesis

• The L protein in serum-derived particles is resistant to phosphatase digestion in the absence of detergents, reflecting an internal disposition of the phosphorylated pre-S domain .

• The relative amount of the phosphorylated form increases with time in the viral growth cycle .

These findings demonstrate that phosphorylation-dephosphorylation of the L protein is an important, regulated mechanism necessary for correct virion morphogenesis .

What are the most effective methods for expressing recombinant DHBV envelope proteins?

Researchers have employed several strategies for expressing recombinant DHBV envelope proteins, with varying levels of success:

Vaccinia Virus Expression System:

• The envelope (Pre-S/S) gene of DHBV can be amplified by polymerase chain reaction (PCR) and cloned into plasmids under the control of vaccinia virus promoters (e.g., P7.5) .

• Recombination in cell culture, followed by screening in human TK- 143 cells in the presence of 5-bromouracil deoxyriboside (5-BUdR), can generate recombinant vaccinia viruses bearing the envelope gene .

• This approach has successfully produced DHBsAg that can be detected by dot enzyme immunoassay (EIA) in infected cell lysates .

Direct Plasmid Transfection:

• Eukaryotic recombinant plasmids (e.g., pCAGGS-based constructs) can be used to express viral proteins in duck embryo fibroblasts (DEFs) .

• This approach is particularly useful for studying the individual effects of viral proteins on host cells .

When designing expression systems, researchers should consider:

• The need for proper post-translational modifications

• The potential cytotoxicity of viral proteins

• The specific objectives of the study (e.g., protein characterization vs. vaccine development)

How can recombinant DHBV be engineered as a gene delivery vector?

Engineering DHBV as a gene delivery vector involves several strategic approaches to insert cargo genes while maintaining viral functions. The main approaches identified in the literature include:

Polymerase Spacer Region Insertion:

• The polymerase spacer region can tolerate insertions without abolishing polymerase activity and self-sufficient replication .

• Early studies demonstrated that insertion of coding sequences (e.g., bacterial protein A - 369 nt) in-frame into the spacer region, between preS and S in the overlapping ORF, allowed for viral replication .

• More recent approaches have used a clinically-isolated, highly-replicative HBV mutant with a large in-frame deletion in the polymerase spacer as a base vector .

• This vector can tolerate insertions of up to 675 nt while retaining wild-type-level replicative competence .

ORF Duplication Strategy:

• Duplicating the overlapping part of C/P ORFs creates non-overlapping C and P ORFs .

• ORFs encoding reporter proteins (e.g., blastidicin resistance protein - 399 nt, or GFP - 720 nt) can be inserted between C and P ORFs .

• Short IRES units can be used to separate the three ORFs .

• Smaller inserts (e.g., BsdR) allow for replication and progeny virion production comparable to wild-type HBV, but longer inserts (e.g., GFP) significantly reduce replication efficiency .

S ORF Replacement:

• Replacing most of the S ORF with foreign genes (e.g., GFP or interferon coding sequences) in-frame .

• This approach prematurely terminates the overlapping P ORF and requires trans-complementation with both polymerase and envelope proteins .

• This strategy has shown therapeutic potential, as demonstrated by recombinant DHBV expressing duck interferon inhibiting co-infecting wild-type DHBV in primary duck hepatocyte infection assays .

The choice of strategy depends on the research objectives, cargo size, and required replication efficiency. Trans-complementation of viral proteins is often necessary to produce infectious recombinant virions.

What are the challenges in generating infectious recombinant DHBV particles?

Producing infectious recombinant DHBV particles faces several challenges:

• Maintaining Replication Efficiency:

  • Insertion of foreign sequences often reduces viral replication efficiency .

  • The extent of reduction depends on the size and nature of the insertion, with larger inserts generally causing more severe impairment .

  • Recombinant genomes typically replicate at efficiencies ranging from 1.5%-4% of wild-type when inserting sequences in the polymerase spacer region .

• Structural Protein Requirements:

  • Many recombinant designs disrupt viral structural protein coding regions, necessitating trans-complementation .

  • When the preS/S ORF is interrupted by cargo insertion, the recombinant genome requires trans-complemented envelope proteins to form mature progeny virions .

  • Similarly, disruption of the core protein coding region requires trans-complementation with functional core proteins .

• Insertion Site Limitations:

  • The polymerase spacer region offers flexibility, but cargo sequences must not introduce stop codons in the P ORF, which is often difficult and sometimes impossible .

  • Genome size constraints limit the capacity for foreign sequence insertion, particularly in designs that duplicate portions of viral ORFs .

• Cargo Expression Levels:

  • Achieving sufficient cargo gene expression is challenging, as viral promoters may not provide optimal expression levels .

  • Attempts to enhance expression through exogenous promoters have not consistently demonstrated improved expression in infection assays .

Despite these challenges, researchers have developed strategies to optimize recombinant virus production, including using viral mutants with replication-enhancing deletions and employing IRES elements to isolate polymerase translation.

How can DHBV envelope proteins be utilized in vaccine development?

DHBV envelope proteins show promising potential for vaccine development through several strategies:

Recombinant Vaccinia Virus Expressing Pre-S/S Protein:

• Recombinant vaccinia virus bearing the envelope gene of DHBV (pGDHBV-5) can replicate in cell cultures and express DHBsAg .

• After multiple-site intradermal injections of this recombinant virus, DHBsAg can be detected in the serum of immunized adult ducks, indicating successful replication and expression in vivo .

• When used as a therapeutic vaccine in persistently DHBV-infected ducks, a transient significant decrease of serum DHBsAg has been observed, suggesting immunological clearance .

Key Considerations for Vaccine Development:

• The recombinant virus must be capable of expressing sufficient levels of the envelope proteins to elicit strong immune responses.

• The immunogenicity of the expressed proteins may be affected by their conformational state and post-translational modifications.

• The safety profile of the vaccine vector must be carefully evaluated.

• Optimization of administration routes and vaccination schedules is crucial for maximizing efficacy.

Therapeutic Potential:

• Beyond preventative applications, DHBV envelope protein-based vaccines may have therapeutic value for chronic infections.

• The observed decrease in serum DHBsAg after vaccination of persistently infected ducks suggests potential for immune-mediated viral clearance .

While promising, challenges remain in optimizing expression systems, ensuring proper protein folding, and enhancing immune responses to achieve durable protection or therapeutic effects.

What molecular techniques are used to detect phosphorylation of the DHBV large envelope protein?

Researchers employ several sophisticated techniques to detect and characterize phosphorylation of the DHBV large envelope protein:

Metabolic Labeling with Radioisotopes:

• 32P labeling can be used to directly identify phosphorylated proteins .

• Cells expressing the L protein are cultured in the presence of 32P-orthophosphate, allowing incorporation of the radioisotope into phosphorylated proteins.

• The labeled proteins are then isolated by immunoprecipitation and visualized by autoradiography after gel electrophoresis .

Phosphatase Digestion Assays:

• Residue-specific phosphatases can be used to confirm phosphorylation and identify the type of phosphorylated amino acids .

• Treatment of the purified L protein with these enzymes results in mobility shifts on SDS-PAGE if phosphate groups are removed.

• This approach has been used to demonstrate that L protein heterogeneity is due to phosphorylation of threonine and/or serine residues within the pre-S domain .

Western Immunoblotting:

• Western blots often reveal the L protein as a doublet at 36 and 35 kDa, with the higher molecular weight band representing the phosphorylated form .

• This technique can be used to track changes in the relative abundance of phosphorylated and non-phosphorylated forms during viral replication.

Detergent Treatments:

• L protein in serum-derived particles is resistant to phosphatase digestion in the absence of detergents, reflecting an internal disposition of the phosphorylated pre-S domain .

• Detergent treatments can expose the phosphorylated domains, making them accessible to phosphatase digestion.

These techniques have revealed important insights, such as the increased relative amount of the phosphorylated form of L protein over time in the viral growth cycle, suggesting temporal regulation of phosphorylation during viral replication .

How does the virulence of duck hepatitis viruses correlate with host mRNA modifications?

Recent research has revealed intriguing correlations between viral virulence and host mRNA modifications, particularly N6-methyladenosine (m6A) modification:

Virulence-Dependent m6A Modification Levels:

• m6A-modified mRNA exists in both virulent and attenuated duck hepatitis A virus (DHAV)-infected duckling livers .

• Importantly, m6A levels in mRNA are much higher in attenuated DHAV-infected livers compared with virulent DHAV-infected livers, suggesting virulence-dependent regulation of m6A modification .

Modification Motifs and Patterns:

• Analysis of modification motifs indicates that GAAGAAG is the most enriched motif in m6A modifications during DHAV infection .

• Combined m6A-seq and RNA-seq data analysis shows a generally positive correlation between m6A and mRNA expression levels in DHAV-infected duckling livers .

Biological Processes Affected:

• Genes with altered m6A levels during infection are enriched in various biological processes, including:

  • Oxidation-reduction processes

  • Antiviral immune responses

While this research specifically addresses DHAV rather than DHBV, it provides valuable insights into how hepadnaviruses may interact with host RNA modification machinery. The observed virulence-dependent coordination between m6A modification and mRNA expression suggests that virus-host interactions at the RNA modification level may be important determinants of disease severity and outcomes.

These findings have implications for understanding the molecular mechanisms of viral pathogenesis and may inform the development of novel antiviral strategies targeting RNA modifications.

What role does the DHBV large envelope protein play in host cell adsorption?

The DHBV large envelope protein plays a crucial role in viral adsorption to host cells, though it is not the only viral protein involved in this process:

Evidence for L Protein Involvement in Adsorption:

• Studies with the related Duck Hepatitis A Virus (DHAV) have shown that structural proteins mediate host cell adsorption .

• For DHBV, the pre-S domain of the L protein is particularly important for receptor binding and viral entry.

Experimental Approaches to Study Adsorption:

• Antibody-blocking experiments provide valuable insights into the role of specific viral proteins in adsorption.

• In studies with DHAV, virus copy numbers after treatment with antibodies against specific viral proteins (such as VP3) were significantly lower than in negative control groups .

• Similar approaches can be applied to study DHBV L protein's role in adsorption.

Quantification Methods:

• qRT-PCR can be used to measure virus copy numbers and assess the effect of antibodies against specific viral proteins on virion adsorption .

• This technique allows for precise quantification of the impact of blocking specific viral proteins on the adsorption process.

While the large envelope protein is crucial for viral attachment, research suggests that it functions as part of a complex process involving multiple viral components. Understanding these interactions is essential for developing strategies to block viral entry and prevent infection.

What are the latest advances in recombinant DHBV vector design for gene delivery to hepatocytes?

Recent advances in recombinant DHBV vector design have focused on improving efficiency, safety, and cargo capacity for targeted gene delivery to hepatocytes:

Enhanced Vector Designs:

• Vectors based on clinically-isolated, highly-replicative HBV mutants with large in-frame deletions in the polymerase spacer have demonstrated superior replication efficiency compared to wild-type virus .

• These vectors can tolerate insertions of up to 675 nt while maintaining wild-type-level replicative competence .

• The combination of replication-enhancing deletions and strategic use of IRES elements has improved vector performance .

Non-Protein Cargo Delivery:

• Recent developments have demonstrated that recombinant HBV vectors can deliver and express functional RNA in infected primary tupaia hepatocytes (PTH) .

• This represents an important advance as it expands the potential applications beyond protein expression to include RNA-based therapeutics .

Safety Enhancements:

• Premature termination of all viral ORFs in vectors designed for gene therapy applications improves safety by preventing the expression of viral proteins .

• Such vectors require trans-complementation with wild-type HBV proteins for virus production but express only the cargo gene product upon infection .

Therapeutic Applications:

• Recombinant hepadnaviruses expressing interferon have shown the ability to inhibit co-infecting wild-type virus in hepatocyte infection assays, demonstrating therapeutic potential .

• This represents the first demonstration of the therapeutic value of recombinant hepadnavirus vectors .

Reporter Systems:

• Vectors carrying fluorescent (DsRed, GFP, RFP) or bioluminescent reporters have been developed, facilitating tracking of infection and expression in target cells .

• These systems are valuable for optimizing vector design and studying infection dynamics .

Despite these advances, challenges remain in balancing cargo capacity with replication efficiency and ensuring targeted delivery to hepatocytes in vivo. Ongoing research continues to refine these vectors for potential clinical applications.

Table 1: Comparison of Major Recombinant DHBV Vector Design Strategies

Note: Data compiled from search result

Table 2: Post-translational Modifications of DHBV Large Envelope Protein