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Probing the spatiotemporal patterns of HBV multiplication reveals novel features of its subcellular processes

['Lei Yue', 'Key Laboratory Of Medical Molecular Virology', 'Moe Nhc Cams', 'School Of Basic Medical Sciences', 'Shanghai Medical College', 'Fudan University', 'Shanghai', 'Research Unit', 'Shanghai Public Health Clinical Center', 'Chang Li']

Date: 2021-08

Through evolution, Hepatitis B Virus (HBV) developed highly intricate mechanisms exploiting host resources for its multiplication within a constrained genetic coding capacity. Yet a clear picture of viral hitchhiking of cellular processes with spatial resolution is still largely unsolved. Here, by leveraging bDNA-based fluorescence in situ hybridization (FISH) combined with immunofluorescence, we developed a microscopic approach for multiplex detection of viral nucleic acids and proteins, which enabled us to probe some of the key aspects of HBV life cycle. We confirmed the slow kinetics and revealed the high variability of viral replication at single-cell level. We directly visualized HBV minichromosome in contact with acetylated histone 3 and RNA polymerase II and observed HBV-induced degradation of Smc5/6 complex only in primary hepatocytes. We quantified the frequency of HBV pregenomic RNAs occupied by translating ribosome or capsids. Statistics at molecular level suggested a rapid translation phase followed by a slow encapsidation and maturation phase. Finally, the roles of microtubules (MTs) on nucleocapsid assembly and virion morphogenesis were analyzed. Disruption of MTs resulted in the perinuclear retention of nucleocapsid. Meanwhile, large multivesicular body (MVB) formation was significantly disturbed as evidenced by the increase in number and decrease in volume of CD63 + vesicles, thus inhibiting mature virion secretion. In conclusion, these data provided spatially resolved molecular snapshots in the context of specific subcellular activities. The heterogeneity observed at single-cell level afforded valuable molecular insights which are otherwise unavailable from bulk measurements.

HBV is a hepatotropic, enveloped virus with a partially double-stranded relaxed circular DNA genome. Studies on the molecular biology of HBV mainly rely on biochemical extraction and bulk quantification methods. Detailed spatiotemporal information on virus components in subcellular context is still lacking. Here, we re-evaluated the reproduction schemes of HBV by fluorescence in situ hybridization (FISH). We visualized cccDNA minichromosome formation in an epigenetic context, identified pgRNA associated with actively translating ribosomes and capsids. Moreover, the active participation of microtubules in nucleocapsid transport and MVB-mediated virion secretion was identified. These observations have broad implications for understanding the HBV replication cycle and may facilitate the identification of novel antiviral targets.

Funding: This work was supported by the National Natural Science Foundation of China for XZ (81873962, 32070152) and for ZY (91842309), National Science and Technology Major project of China for ZY (2017ZX10302-201-001-005), Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program for ZY (No. 2017BT01S131), CAMS Innovation Fund for Medical Sciences for ZY (No. 2019-I2M-5-040), and Major Special Projects of Basic Research of Shanghai Science and Technology Commission for ZY (No. 18JC1411100). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Yue et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

With the aid of a series of classical analytical methods such as ultrafiltration, Southern and Northern blot etc. [ 1 ], the aforementioned framework of HBV life cycle was established. However, this picture still lacks many key spatial and molecular contexts which involves essential co-opted host factor, and there is also a general lack of observations at single-cell level in the context of cellular architectures. Previously, by optimizing the viewRNA in situ hybridization assay, we developed an assay for visualizing HBV RNA, DNA and cccDNA in liver specimens and in cell model [ 8 – 10 ]. Here, by combining this assay with immuno-detection of proteins, we were able to analyze the formation of intranuclear minichromosome, viral transcription, translation, packaging and viral egress in a multiplexed and spatially resolved manner. We observed the distinct localization of nuclear HBV DNA within the milieu of K27 acetylated Histone H3 and RNA Polymerase II (Pol II). In addition, we surveyed the occupancy of ribosome and capsids on pgRNA and confirmed the mutually exclusive nature of pgRNA translation and encapsidation. In addition, we inferred from the quantitative data the relatively quick pgRNA translation and slow capsid maturation process. Finally, we found that although nucleocapsids can still be assembled after MTs disruption, their transport was arrested in the peri-nuclear region. Moreover, virion morphogenesis and release via the MVB route were significantly impaired as vesicle fusion was disrupted in the absence of functional MTs. The ability to visualize HBV nucleic acids within sophisticated subcellular architectures enabled us to observe cell-to-cell variability of HBV infection and to probe the subcellular as well as molecular details of viral activity.

HBV is a hepatotropic, enveloped virus of the hepadnaviridae family with a partially double-stranded relaxed circular DNA (rcDNA) genome [ 1 ]. The rcDNA in incoming virion is repaired by cellular enzymes and transformed into covalently closed circular DNA (cccDNA), the template for all HBV transcripts, including pregenomic RNA (pgRNA) and subgenomic RNAs [ 2 ]. The pgRNA serves not only as the template for viral reverse transcription but also as the messenger encoding HBcAg (Core) and the polymerase (Pol) essential for its genome replication [ 3 ]. Nucleocapsid assembly is initiated by the binding of viral polymerase to pgRNA together with cellular factors such as Heat shock protein 90 (Hsp90) [ 4 ]. Subsequently, viral DNA synthesis is initiated, and mature nucleocapsid is then enveloped by viral surface antigens. Cellular microtubules (MTs) network mediates the delivery of nucleocapsid into the nucleus following viral entry [ 5 , 6 ], they were also proposed to be required for nucleocapsid formation [ 7 ].

Results

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009838

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