Limits...
Role of hepatitis B virus genotype D & its mutants in occult hepatitis B infection.

Sengupta S, Panda SK, Acharya SK, Durgapal H - Indian J. Med. Res. (2013)

Bottom Line: One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg.This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays.Pre-S2/S promoter deletions do not affect HBV replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, All India Institute of Medical Sciences, New Delhi, India.

ABSTRACT

Background & objectives: Non-detection of hepatitis B virus (HBV) envelope protein (hepatitis B surface antigen, HBsAg) in a chronically HBV infected individual has been described as occult infection. One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg. This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays. Genotype D of HBV has a characteristic 33 nucleotide (nt) deletion upstream of the pre-S2/S promoter. This deletion may reduce HBsAg secretion in occult infection patients infected with genotype D HBV. Additional deletions in the pre-S2/S promoter may further aggravate reduced HBsAg secretion in patients infected with genotype D HBV. Thus, the aim of the present study was to determine the role of genotype D specific 33nt deletion and additional pre-S2/S promoter deletions in causing reduced or no secretion of HBsAg, in occult infection. Since these deletions overlap virus polymerase, their effect on virus replication was also investigated.

Methods: We examined the in vitro expression of HBsAg, ratio of cure and 'e' antigen (HBcAg/HBeAg), their secretion and virus replication, using overlength 1.3 mer/1.86 mer genotype A replicons, and genotype D replicons with and without additional pre-S2/S promoter deletions from cases of occult infection.

Results: Genotype D replicon showed a decrease in HBsAg secretion compared to the wild-type genotype A. Genotype D replicons carrying additional pre-S2/S promoter deletions, showed further reduction in HBsAg secretion, demonstrated presence of intracellular HBcAg/HBeAg, virus replication intermediates and 'e' antigen secretion.

Interpretation & conclusions: The characteristic 33 nt deletion of genotype D HBV reduces HBsAg secretion. Additional pre-S2/S promoter deletions may further diminish HBsAg secretion, leading to occult infection. Pre-S2/S promoter deletions do not affect HBV replication.

Show MeSH

Related in: MedlinePlus

(A) Schematic representation (not drawn to scale) of overlength 1.86mer/1.3mer constructs of HBV. The over-length 1.86mer construct begins at nucleotide 2429 while the 1.3mer construct begins at nucleotide 1045. Block arrows indicate HBV promoters. Grey rectangularboxes depict open reading frames and enhancers of HBV. (B) Schematic representation (not drawn to scale) of deletions of the various occult HBV infection isolates. Genotype D HBV isolates 821.1, 313.1, 761.1 show deletions of 33 nucleotides (Δnt 2859-2878, 2882-2894) upstream of the pre-S2/S promoter in the pre-S1ORF. HBV isolate 313.1 carries pre-S2/S promoter deletions (Δnt 3026-3208). HBV isolate 761.1 carries pre-S2/S promoter deletions (Δnt 3039-3095).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3818595&req=5

Figure 1: (A) Schematic representation (not drawn to scale) of overlength 1.86mer/1.3mer constructs of HBV. The over-length 1.86mer construct begins at nucleotide 2429 while the 1.3mer construct begins at nucleotide 1045. Block arrows indicate HBV promoters. Grey rectangularboxes depict open reading frames and enhancers of HBV. (B) Schematic representation (not drawn to scale) of deletions of the various occult HBV infection isolates. Genotype D HBV isolates 821.1, 313.1, 761.1 show deletions of 33 nucleotides (Δnt 2859-2878, 2882-2894) upstream of the pre-S2/S promoter in the pre-S1ORF. HBV isolate 313.1 carries pre-S2/S promoter deletions (Δnt 3026-3208). HBV isolate 761.1 carries pre-S2/S promoter deletions (Δnt 3039-3095).

Mentions: HBV has a circular genome with overlapping open reading frames. However, studies for subtype adw2, which is the wild-type used in the present study have been carried out using linear overlength (1.3/1.5mer) replication competent constructs consisting of two sets of enhancer, (enhancers I and II) one at the 5’ end and another at the 3’ end of the linear construct1415. The enhancers at the 3’end of the linear construct are required for pre-S1 and pre-S2 promoter function1617. Enhancer I at the 5’end of the construct is required for normal expression of all the viral transcripts18. However, since the 1.3mer constructs carry two sets of enhancers for a single set of pre-S2/S promoters, these have been observed to produce larger amounts of HBsAg compared to constructs with a single set of enhancers16. Over-length 1.86mer HBV constructs were used because we hypothesized that these would be maintaining the large versus small envelope protein ratio (L/S) since for both sets of enhancers there are two sets of pre-S2/S promoters (Fig. 1A). Thus we wanted to determine whether the L/S ratio plays a strict role in regulating HBsAg secretion, or whether the total amounts of surface proteins affect HBsAg synthesis, and secretion. Further, in the overlength 1.86mer construct a single viral polymerase open reading frame is present (Fig. 1A). Hence in the current study the viral replication intermediates are produced by a single viral polymerase.


Role of hepatitis B virus genotype D & its mutants in occult hepatitis B infection.

Sengupta S, Panda SK, Acharya SK, Durgapal H - Indian J. Med. Res. (2013)

(A) Schematic representation (not drawn to scale) of overlength 1.86mer/1.3mer constructs of HBV. The over-length 1.86mer construct begins at nucleotide 2429 while the 1.3mer construct begins at nucleotide 1045. Block arrows indicate HBV promoters. Grey rectangularboxes depict open reading frames and enhancers of HBV. (B) Schematic representation (not drawn to scale) of deletions of the various occult HBV infection isolates. Genotype D HBV isolates 821.1, 313.1, 761.1 show deletions of 33 nucleotides (Δnt 2859-2878, 2882-2894) upstream of the pre-S2/S promoter in the pre-S1ORF. HBV isolate 313.1 carries pre-S2/S promoter deletions (Δnt 3026-3208). HBV isolate 761.1 carries pre-S2/S promoter deletions (Δnt 3039-3095).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818595&req=5

Figure 1: (A) Schematic representation (not drawn to scale) of overlength 1.86mer/1.3mer constructs of HBV. The over-length 1.86mer construct begins at nucleotide 2429 while the 1.3mer construct begins at nucleotide 1045. Block arrows indicate HBV promoters. Grey rectangularboxes depict open reading frames and enhancers of HBV. (B) Schematic representation (not drawn to scale) of deletions of the various occult HBV infection isolates. Genotype D HBV isolates 821.1, 313.1, 761.1 show deletions of 33 nucleotides (Δnt 2859-2878, 2882-2894) upstream of the pre-S2/S promoter in the pre-S1ORF. HBV isolate 313.1 carries pre-S2/S promoter deletions (Δnt 3026-3208). HBV isolate 761.1 carries pre-S2/S promoter deletions (Δnt 3039-3095).
Mentions: HBV has a circular genome with overlapping open reading frames. However, studies for subtype adw2, which is the wild-type used in the present study have been carried out using linear overlength (1.3/1.5mer) replication competent constructs consisting of two sets of enhancer, (enhancers I and II) one at the 5’ end and another at the 3’ end of the linear construct1415. The enhancers at the 3’end of the linear construct are required for pre-S1 and pre-S2 promoter function1617. Enhancer I at the 5’end of the construct is required for normal expression of all the viral transcripts18. However, since the 1.3mer constructs carry two sets of enhancers for a single set of pre-S2/S promoters, these have been observed to produce larger amounts of HBsAg compared to constructs with a single set of enhancers16. Over-length 1.86mer HBV constructs were used because we hypothesized that these would be maintaining the large versus small envelope protein ratio (L/S) since for both sets of enhancers there are two sets of pre-S2/S promoters (Fig. 1A). Thus we wanted to determine whether the L/S ratio plays a strict role in regulating HBsAg secretion, or whether the total amounts of surface proteins affect HBsAg synthesis, and secretion. Further, in the overlength 1.86mer construct a single viral polymerase open reading frame is present (Fig. 1A). Hence in the current study the viral replication intermediates are produced by a single viral polymerase.

Bottom Line: One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg.This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays.Pre-S2/S promoter deletions do not affect HBV replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, All India Institute of Medical Sciences, New Delhi, India.

ABSTRACT

Background & objectives: Non-detection of hepatitis B virus (HBV) envelope protein (hepatitis B surface antigen, HBsAg) in a chronically HBV infected individual has been described as occult infection. One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg. This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays. Genotype D of HBV has a characteristic 33 nucleotide (nt) deletion upstream of the pre-S2/S promoter. This deletion may reduce HBsAg secretion in occult infection patients infected with genotype D HBV. Additional deletions in the pre-S2/S promoter may further aggravate reduced HBsAg secretion in patients infected with genotype D HBV. Thus, the aim of the present study was to determine the role of genotype D specific 33nt deletion and additional pre-S2/S promoter deletions in causing reduced or no secretion of HBsAg, in occult infection. Since these deletions overlap virus polymerase, their effect on virus replication was also investigated.

Methods: We examined the in vitro expression of HBsAg, ratio of cure and 'e' antigen (HBcAg/HBeAg), their secretion and virus replication, using overlength 1.3 mer/1.86 mer genotype A replicons, and genotype D replicons with and without additional pre-S2/S promoter deletions from cases of occult infection.

Results: Genotype D replicon showed a decrease in HBsAg secretion compared to the wild-type genotype A. Genotype D replicons carrying additional pre-S2/S promoter deletions, showed further reduction in HBsAg secretion, demonstrated presence of intracellular HBcAg/HBeAg, virus replication intermediates and 'e' antigen secretion.

Interpretation & conclusions: The characteristic 33 nt deletion of genotype D HBV reduces HBsAg secretion. Additional pre-S2/S promoter deletions may further diminish HBsAg secretion, leading to occult infection. Pre-S2/S promoter deletions do not affect HBV replication.

Show MeSH
Related in: MedlinePlus