The translocation motif of hepatitis B virus envelope proteins is dispensable for infectivity

Abstract

The early events of hepatitis B virus (HBV) infection remain unclear. In 2006, Stoeckl et al. proposed a new entry mechanism involving a translocation motif (TLM) present in the pre-S2 domain of envelope proteins (L. Stoeckl, A. Funk, A. Kopitzki, B. Brandenburg, S. Oess, H. Will, H. Sirma, and E. Hildt, Proc. Natl. Acad. Sci. USA 103:6730-6734, 2006). After receptor binding and internalization into the endosomal compartment, this motif would allow the translocation of HBV particles through the endosomal membrane into the cytosol. In this study we have used two different mutated viruses containing a truncated TLM and showed their ability to infect human hepatocytes in primary culture, thus demonstrating the dispensability of the TLM for HBV infectivity.

FIG. 1.

Amino acid deletions in the pre-S2 region of the S gene. Deletions (shown in the form Δx/y, where x is the position of the first deleted amino acid and y is the position of the last deleted amino acid) are indicated as thin lines. Positions are given relative to the first N-terminal amino acid of the L protein (subtype ayw, EMBL accession no. X02496).

FIG. 2.

Viral titers of inocula determined by quantitative PCR analysis of viral DNA. Results are expressed in GEq per ml of inoculum. The two controls, L− and Myr−, consist of HBV with the L protein deleted, impairing virion production, and of HBV with an unmyristoylated L protein, resulting in the production of noninfectious viruses, respectively. Mutant viruses, with deletions in the pre-S2 domain, are identified as Δx/y. Statistical analysis was performed by the Mann-Whitney test; significant differences compared to the WT condition are indicated by asterisks (P < 0.05). The dotted horizontal line indicates the level of the WT condition.

FIG. 3.

Infectivity of mutant and WT viruses. The infectivity is expressed as a ratio between the level of infection markers (HBe or HBV RNA) and the number of GEq used for infection. The control Myr− is described in the legend to Fig. 2. Mutant viruses with deletions in the pre-S2 domain are identified as Δx/y. Statistical analysis was performed using the Mann-Whitney test; significant differences compared to the WT condition are indicated by asterisks (P < 0.05). The dotted horizontal line indicates the level of the WT condition. (A) Infection was assessed by measuring HBeAg in the culture supernatant of infected cells 10 days postinfection. One arbitrary unit (AU) corresponds to the HBe concentration in the positive control of the assay kit. (B) Infection was assessed by measuring intracellular viral RNA by Q-PCR after reverse transcription 10 days postinfection. Expression data are normalized to 18S rRNA, and the relative viral RNA expression levels are calculated according to the ΔΔCt (comparative Ct) method (Applied Biosystems user bulletin 2 on relative quantification of gene expression) in which the reference corresponds to the WT condition.