Drastic Reduction in the Production of Subviral Particles Does Not Impair Hepatitis B Virus Virion Secretion

Abstract

Hepatitis B virus (HBV) contains three coterminal envelope proteins on the virion surface: large (L), middle (M), and small (S). The M and S proteins are also secreted as empty "subviral particles," which exceed virions by at least 1,000-fold. The S protein serves as the morphogenic factor for both types of particles, while the L protein is required only for virion formation. We found that cotransfecting replication constructs with a small dose of the expression construct for the missing L, M, and S proteins reconstituted efficient virion secretion but only 5 to 10% of subviral particles. The L protein inhibited secretion of subviral particles in a dose-dependent manner, whereas a too-high or too-low L/S protein ratio inhibited virion secretion. Consistent with the results of cotransfection experiments, a point mutation at the ؊3 position of the S gene AUG codon reduced HBsAg secretion by 60 to 70% but maintained efficient virion secretion. Surprisingly, ablating M protein expression reduced virion secretion but markedly increased the maturity of virion-associated genomes, which could be reversed by providing in trans both L and M proteins but not just M protein. M protein stability was dependent on the coexpression of S protein. Our findings suggest that efficient HBV virion secretion could be maintained despite drastic reduction in subviral particle production, which supports the recent demonstration of separate secretion pathways adopted by the two types of particles. The M protein appears to facilitate core particle envelopment, thus shortening the window of plus strand DNA elongation.Hepatitis B virus (HBV) is a small enveloped DNA virus that infects only humans and higher primates (17). It is estimated that 2 billion people worldwide have been exposed to HBV and more than 350 million are chronically infected. Inside the 42-nm virion is a nucleocapsid or core particle that shields the relaxed circular double stranded DNA genome. The minus-strand DNA is full length (3.2-kb) and terminally redundant, whereas the plus-strand DNA is incomplete. Upon infection of hepatocytes, the genome is repaired to form the unit-length, fully double-stranded covalently closed circular DNA in the nucleus (54), which serves as the template for transcription. The four major transcripts of 3.5, 2.4, 2.1, and 0.7 kb are termed pre-C/C, pre-S1, preS2/S, and X, respectively, all sharing the same 3Ј end but different 5Ј initiation sites (4). The mRNAs are exported to cytoplasm for protein translation and genome replication. The 3.5-kb transcript is over the genome length and therefore contains all of the genetic information of the viral genome. The longer and less abundant version (preC RNA) directs the expression of hepatitis B e antigen (HBeAg), a secreted viral protein with immunomodulatory function (37). The shorter one or pregenomic RNA (pgRNA) serves as the messenger for core protein and viral polymerase, as well as the template for DNA synthesis. To this end, the nascent core protein assembles into core particle, packaging bot...

HBV secretion is regulated through the activation of endocytic and autophagic compartments via Rab7 stimulation

“…In control experiments, the expression of FLAG-tagged mock protein or an addition of control antibody did not increase the HBV DNA levels after immunoprecipitation. There was no significant difference in the amount of HBsAg in the supernatant from cells with or without Rab7 depletion (data not shown), suggesting that the secretion pathways of HBV particles and HBsAg subviral particles (SVPs) are different, as reported previously (Garcia et al, 2009), and Rab7 does not affect the secretion pathway of SVPs. The expression of a GTPase-defective Rab7 mutant (Rab7T22N) also increased the amount of intracellular LHBs and released viral genome, as with the Rab7 knockdown (Fig.…”

Unusual Features of Sodium Taurocholate Cotransporting Polypeptide as a Hepatitis B Virus Receptor

“…Covalently closed circular DNA (cccDNA) was extracted according to an established method (33). Virions were immunoprecipitated from culture supernatant by a combination of rabbit antipreS1 antibody (R271) and horse anti-Ad/Ay antibody according to established protocols (29,31). Virion DNA, intracellular replicative DNA, and cccDNA were detected by Southern blotting, while HBV RNA was detected by Northern blotting.…”

“…The recombinant NTCP protein thus produced contains bovine rhodopsin tag at the carboxyl terminus to enable its detection by a mouse monoclonal antibody (1D4; Santa Cruz), which was used at dilutions of 1:1,000 for Western blotting and 1:100 for immunofluorescence (IF) staining. The other antibodies used included anti-␤ actin (Sigma), rabbit anti-core (Dako), rabbit anti-preS1 (R271) (29), another rabbit antibody against preS1 residues 12 to 46 of genotypes B/C (MGTNLSVPNPLGFFPDHQLDPAFGA NSNNPDWDFN; GenScript), mouse monoclonal anti-preS2 (S26; Virogen), rabbit anti-S (NB100-62652; Novus), goat anti-S (custom-made), and horse anti-S (anti-Ad/Ay; Abcam). ccHBV and sHBV.…”

“…The HBsAg/ HBeAg ratio refers to HBsAg titer in culture supernatant divided by HBeAg titer from the same volume of culture supernatant. Southern and Northern blot analyses were performed as previously described (29,31), and for such purposes the cells were seeded in 6-or 12-well plates for infection experiments. To avoid bias in detecting ccHBV or sHBV, the 3.1-kb HBV DNA of genotypes B, C, and D was mixed at 1:1:2 ratio for probe labeling.…”

“…Virion DNA, intracellular replicative DNA, and cccDNA were detected by Southern blotting, while HBV RNA was detected by Northern blotting. Core, S, and ␤-actin proteins were detected by Western blotting as described previously (29,31).…”