Hepatitis B virus (HBV), which can lead to acute and chronic hepatitis, infects more than 2 billion people worldwide, according to serological evidence. Although vaccines and treatments are available, there are approximately 1.5 million new HBV infections each year globally.
A new study has revealed a key step in the HBV life cycle: Researchers found that HBV virions can be released within exosomes, which are capable of infecting neighboring cells. The authors, led by Qingyan Wu of the department of virology, Paul-Ehrlich-Institute, Langen, Germany, suggest this strategy may help the virus escape immune surveillance and target a new hepatocyte.
The study was published online in Cellular and Molecular Gastroenterology and Hepatology.
The researchers isolated exosomes from the supernatants of HBV-producing cells using exosomal and HBV markers. Electron microscopy using ultrathin sectioning along with immunogold labeling confirmed the presence of intact HBV virions in exosomes. The ultracentrifugation enabled the separation of the free virion fraction from the virions enclosed in exosomes. These findings fit in with previous discoveries of quasi-enveloped hepatitis A virus and hepatitis E virus.
The exosomes released free HBV virion and naked capsid after exposure to detergent. Cellular exposure to exosome morphogenesis inhibitors interfered with the release of exosome-packaged HBV. The researchers also observed large HBV surface antigens (LHB) on the external surface of the exosomes and found that the antigens allowed the exosome to infect susceptible cells through interaction with the sodium-taurocholate co-transporting polypeptide. LHB may also play an additional role in infectivity by countering the ability of antibodies to neutralize HBV.
However, the researchers also found that an LHB-specific neutralizing antibody inhibited infection of differentiated HepaRG cells with exosome-containing HBV. One explanation is that the antibody blocks the interaction between LHB and the target cell. Another is that the exosome disassembles near the target cell membrane and releases the virus, which is then blocked by the antibody since it can block entry of released virus.
To investigate the release pathway, the researchers used three different exosome release inhibitors and found that all three interfered with HBV exosomal release. They also found that cells deficient in the exosome proteins Alix and syntenin did not release exosomal HBV.
Alix appears to be involved in HBV exosomal release, as evidenced by the fact that release of exosomal HBV is boosted in Alix-deficient cells following rescue through overexpression of mCherry-Alix fusion construct. Overexpression of mCherry-Alix had no effect on release of free HBV virions.
The researchers also found evidence that two other exosomal proteins, CD63 and TSG101, play a role in incorporation of LHBs in the HBV envelope, as well as release of HBV through interactions with the protein alpha-taxilin. CD63 and TSG101 are also critical to the formation of exosomes, and the authors suggest further research into their functioning could be fruitful.
Whether exosome-released HBV results from crosstalk between the virus and host cells still needs to be determined. If host factors play a role in connecting HBV to exosomes, it will be interesting to work out which conditions trigger this process, as well as determine which events trigger the release of free virus through multivesicular bodies.
The researchers posit that LHBs could perform a similar function as classical hepatitis B surface antigens and filaments in foiling the immune response. Such a function would require that the virus escape from antibodies before opsonin proteins tag the antigens. It’s also possible that LHBs enable infection of nonhepatic tissues, though this would likely be inefficient.
Many other host proteins have been observed in exosomes released by HBV-infected hepatocytes, suggesting that host proteins may play other roles. A proteomics analysis found proteasome subunit proteins in HepAD38-derived exosomes. The authors suggest that those proteins may allow the exosomes to mediate transcellular immune regulation.
Subviral particles may enhance viral infection, and exosomes from HBV-positive cells may contribute, possibly through exosome surface LHBs, according to the authors. They found that an LHB-specific neutralizing antibody inhibited infection of differentiated HepaRG cells. One explanation is that the antibody blocks the interaction between LHB and the target cell. Another is that the exosome disassembles near the target cell membrane and releases the virus, which is then blocked by the antibody since it can block entry of released virus.
“This previously undiscovered strategy of sequestering HBV particles in exosomes could be a strategy to escape from the immune response and to target them, protected by the exosomal membrane, to the hepatocyte. Exosomes that carry HBV particles seem also to have the potential to deliver HBV to nonpermissive cells with low efficiency. This suggests that exosomes could be an additional factor that contributes to the spread of HBV,” the authors wrote.
The authors had no financial conflicts. This research was funded by the LOEWE Center ACLF, DRUID, the Germany Research Foundation, and the China Scholarship Council.