E2 Site Mutations in S Protein Strongly Affect Hepatitis B Surface Antigen Detection in the Occult Hepatitis B Virus

Abstract

The mechanism of occult hepatitis B infection (OBI) has not yet been fully clarified. Our previous research found that novel OBI-related mutation within S protein, E2G, could cause the hepatitis B surface antigen (HBsAg) secretion impairment, which resulted in intracellular accumulation in OBI of genotype B. Here, to further explore the role of E2 site mutations in the occurrence of OBI, we analyzed these site mutations among 119 OBI strains identified from blood donors. Meanwhile, 109 wild-type HBV strains (HBsAg positive/HBV DNA positive) were used as control group. Furthermore, to verify the E2 site mutations, two conservative 1.3-fold full-gene expression vectors of HBV genotype B and C (pHBV1.3B and pHBV1.3C) were constructed. Then, the E2 mutant plasmids on the basis of pHBV1.3B or pHBV1.3C were constructed and transfected into HepG2 cells, respectively. The extracellular and intracellular HBsAg were analyzed by electrochemical luminescence and cellular immunohistochemistry. The structural characteristics of S proteins with or without E2 mutations were analyzed using relevant bioinformatics software. E2 mutations (E2G/A/V/D) existed in 21.8% (26/119) of OBIs, while no E2 mutations were found in the control group. E2G/A/V/D mutations could strongly affect extracellular and intracellular level of HBsAg (p < 0.05). Notably, unlike E2G in genotype B that could cause HBsAg intracellular accumulation and secretion decrease (p < 0.05), E2G in genotype C could lead to a very significant HBsAg decrease both extracellularly (0.46% vs. pHBV1.3C) and intracellularly (11.2% vs. pHBV1.3C) (p < 0.05). Meanwhile, for E2G/A mutations, the relative intracellular HBsAg (110.7–338.3% vs. extracellular) and its fluorescence intensity (1.5–2.4-fold vs. with genotype-matched pHBV1.3B/C) were significantly higher (p < 0.05). Furthermore, N-terminal signal peptides, with a typical cleavage site for peptidase at positions 27 and 28, were exclusively detected in S proteins with secretion-defective mutants (E2G/A). Our findings suggest that: (1) E2G/A/V/D mutations were confirmed to significantly influence the detection of HBsAg, (2) the underlying mechanism of OBI caused by E2G mutation is quite different between genotype B and genotype C, and (3) E2G/A could produce a N-terminal truncated S protein, which might attribute to the HBsAg secretion impairment in the OBIs.

Reactivation of Occult Hepatitis B Virus Infection During Long-Term Entecavir Antiviral Therapy

“…Furthermore, because the S gene’s open reading frame partially overlaps with that of the reverse transcriptase (RT) gene, mutations in the S gene cause antiviral resistance mutations to appear in the RT domain. The variation of S gene not only Strongly Affect HBsAg Detection ( Kuhns et al, 2021 ; Wang et al, 2021 ), but also leads to HBV drug resistance and immune escape ( Delfino et al, 2021 ). Drug resistance must be considered when a patient experiences a poor curative effect during a follow-up visit.…”

M133S mutation possibly involve in the ER stress and mitophagy pathway in maintenance hemodialysis patients with occult hepatitis B infection

“…Infection with OBI can be transmitted by blood transfusion and organ transplantation, including liver transplantation, and the clinical outcome of OBI is not known, and most of the results suggest that OBI is a significant risk factor for accelerated liver disease progression and the development of cirrhosis and hepatocellular carcinoma 31 .The www.nature.com/scientificreports/ molecular mechanism of OBI is not completely understood, and several possible mechanisms have been proposed, including mutations in the HBV gene resulting in undetectable HBsAg commercial assays; robust host suppression of HBV replication and transcription as well as immune surveillance; and coinfection with other viruses such as hepatitis C virus (HCV) 32,33 . Indeed, it has been reported that deletion of HBsAg in serum can be associated with mutations in the HBV-borne gene S [34][35][36] . Our preliminary study identified mutations in the S genes Q129R, T131N, M133S, F134L, and D144E in 7 serologically positive patients with MHD in combination with OBI, and no mutations in M133S have been reported to date.…”

Host immunity and HBV S gene mutation in HBsAg-negative HBV-infected patients

“…Thirty-one HBsAg-negative samples in the TMD region exhibited multiple amino acid substitution sites, many of which were newly discovered, such as E2K/R/D, G10D, A17R, F20L/S, L21V, L22V, F200Y, and S204N. E2 site mutations in the S protein are confirmed to impair secretion of HBsAg, which significantly affected detection of HBsAg ( 31 ). For genotype B, the highest mutation frequency of the S protein in HBsAg-negative patients was sF20L/S; regarding genotype C, sF19V/S had the highest frequency.…”