Abstract
Extracellular HBV RNA has been detected in both HBV-replicating cell culture media and sera from chronic hepatitis B (CHB) patients, but its exact origin and composition remain controversial. Here, we demonstrated that extracellular HBV RNA species were of heterogeneous lengths, ranging from the length of pregenomic RNA to a few hundred nucleotides. In cell models, these RNAs were predominantly associated with naked capsids, although virions also harbored a minority of them. Moreover, HBV RNAs in hepatitis B patients' blood circulation were localized in unenveloped capsids in the form of capsid-antibody complexes (CACs) and in virions. Furthermore, we showed that extracellular HBV RNAs could serve as the template for viral DNA synthesis. In conclusion, extracellular HBV RNAs mainly consist of pgRNA or the pgRNA species degraded by the RNase H domain of the polymerase in the process of viral DNA synthesis and circulate as CACs and virions. Their presence in blood circulation of CHB patients may be exploited to develop novel biomarkers for HBV persistence.IMPORTANCE Although increasing evidence suggests the presence of extracellular HBV RNA species, their origin and molecular forms are still under debate. In addition to the infectious virions, HBV is known to secrete several species of incomplete viral particles, including hepatitis B surface antigen (HBsAg) particles, naked capsids, and empty virions, during its replication cycle. Here, we demonstrated that extracellular HBV RNAs were associated with naked capsids and virions in HepAD38 cells. Interestingly, we found that unenveloped capsids circulate in the blood of hepatitis B patients in the form of CACs and, together with virions, serve as vehicles carrying these RNA molecules. Moreover, extracellular HBV RNAs are heterogeneous in length and represent either pregenomic RNA (pgRNA) or products of incomplete reverse transcription during viral replication. These findings provide a conceptual basis for further application of extracellular RNA species as novel biomarkers for HBV persistence.
Highlights
IMPORTANCE increasing evidence suggests the presence of extracellular Hepatitis B virus (HBV) RNA species, their origin and molecular forms are still under debate
Extracellular HBV RNAs are heterogeneous in length and predominantly integral to naked capsids instead of virions in HepAD38 cell culture supernatant
Employing the HepAD38 cell culture system, we demonstrated the presence of extracellular HBV RNAs and immature and mature viral DNA packaged in both the naked capsids and virions
Summary
IMPORTANCE increasing evidence suggests the presence of extracellular HBV RNA species, their origin and molecular forms are still under debate. In. addition to DNA-containing virions, a large amount of incomplete viral particles, such as hepatitis B surface antigen (HBsAg) particles, empty virions, and naked capsids, can be released from cells in the process of virus replication [9]. We demonstrate that extracellular HBV RNAs are heterogeneous in length, ranging from full-length pgRNA (3.5 kilonucleotides [knt]) to RNA fragments with merely several hundred nucleotides These RNA molecules represent 3= receding pgRNA fragments that have not been completely reverse transcribed to DNA and pgRNA fragments hydrolyzed by the RNase H domain of polymerase in the process of viral replication. Extracellular HBV RNAs are localized in naked capsids and in virions in culture supernatants of HBV replicating cells and circulate as CACs and virions in blood of hepatitis B patients
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