Abstract
Mutations in HBsAg, the surface antigen of the hepatitis B virus (HBV), might affect the serum HBV DNA level of HBV-infected patients, since the reverse transcriptase (RT) domain of HBV polymerase overlaps with the HBsAg-coding region. We previously identified a diagnostic escape mutant (W3S) HBV that produces massively glycosylated HBsAg. In this study, we constructed an HBV-producing vector that expresses W3S HBs (pHB-W3S) along with a wild-type HBV-producing plasmid (pHB-WT) in order to analyze the physicochemical properties, replication, and antiviral drug response of the mutant. Transfection of either pHB-WT or W3S into HepG2 cells yielded similar CsCl density profiles and eAg expression, as did transfection of a glycosylation defective mutant, pHB-W3S (N146G), in which a glycosylation site at the 146aa asparagine (N) site of HBs was mutated to glycine (G). Virion secretion, however, seemed to be severely impaired in cases of pHB-W3S and pHB-W3S (N146G), compared with pHB-WT, as determined by qPCR and Southern blot analysis. Furthermore, inhibition of glycosylation using tunicamycinTM on wild-type HBV production also reduced the virion secretion. These results suggested that the HBV core and Dane particle could be formed either by massively glycosylated or glycosylation-defective HBsAg, but reduced and/or almost completely blocked the virion secretion efficiency, indicating that balanced glycosylation of HBsAg is required for efficient release of HBV, and mutations inducing an imbalanced glycosylation of HBs would cause the virion to become stuck in the cells, which might be associated with various pathogeneses due to HBV infection.
Highlights
Hepatitis B virus (HBV) infection is a serious global health problem, with two hundred million people currently infected worldwide
Several amino acid mutations in the reverse transcriptase (RT) region of the HBVpol were found in the W3S, since the HBs sequence was overlapped with RT (Figure 1A,B, and the bottom)
We constructed an HBV replication-competent plasmid pHB-W3S, which was thought to be massively glycosylated on the HBs with several RT mutations (Figure 1B), and the plasmid pHB-W3S (N146G), which was glycosylation-deficient with the same RT mutations as pHB-W3S on the backbone of pHB-wild type (WT) with exception at K152R (Figure 1B) [33,34]
Summary
Hepatitis B virus (HBV) infection is a serious global health problem, with two hundred million people currently infected worldwide. HBV is one of the smallest enveloped animal viruses, and consists of an icosahedral nucleocapsid surrounded by three kinds of envelope or surface proteins [2]. The viral genome within the nucleocapsid is composed of partially double-stranded circular DNA and encodes four known overlapping open reading frames (ORFs): C (core), P (DNA polymerase, HBV pol), S (surface or envelope proteins: large S [LS], middle S [MS], and small S [SS] or HBs) and X (HBx) [3]. The uncoated nucleocapsids are transported to the nucleus, where the covalently closed circular DNA (cccDNA) is formed from relaxed circular DNA (rcDNA), which is the viral genome in the infectious particle. The pgRNA is encapsidated together with HBV pol and is reversely transcribed by pol into minus (−) strand DNA; rcDNA is formed along with (+) strand DNA synthesis. The newly formed nucleocapsid occasionally reenters the nucleus (internal cycle) and otherwise is enveloped to be released (egress) through the ER-Golgi pathway [6,7]
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