Synchronized infection identifies early rate-limiting steps in the hepatitis B virus life cycle

Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral life cycle. We employed a synchronized infection protocol to quantify HBV entry kinetics. HBV attachment to cells at 4°C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation at 12h post-infection. HBV uptake is clathrin- and dynamin dependent with actin and tubulin playing a role in the first 6h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6h of infection and cccDNA was first detected at 24h post-infection. Our studies show the majority (83%) of cell bound particles enter HepG2-NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting this as a rate-limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell culture systems and will inform the design and evaluation of physiologically relevant models that support efficient HBV replication.


40
Hepatitis B Virus (HBV) infects 257 million individuals worldwide and is a major driver of end-stage liver disease, 41 cirrhosis, and hepatocellular carcinoma (HCC). HBV is an enveloped DNA and prototypic member of the levels and inoculation time that persisted after 3 and 7 days of culture (Fig.3d), demonstrating that the amount 119 of internalized virus defines virus replication.

121
To extend and validate these observations we assessed HBV internalization kinetics in HepG2-NTCP cells by 122 monitoring HBV particle associated core or surface envelope glycoproteins (Fig.3e). Densitometric scanning of 123 western blots showed a peak of intracellular core and surface glycoprotein-associated particles at 8h and a 124 subsequent decline over the duration of the assay. Intracellular HBV DNA showed a delayed peak at 12h post 125 internalization. Given the semi-quantitative nature of western blots, these data are in good agreement with 126 earlier PCR data and show a time-dependent internalization of HBV particles that reaches saturation at 8-12h post 127 inoculation.
dHepaRG cells with HBV in the presence of PEG showed comparable viral uptake kinetics over a 6h period to 137 HepG2 and Huh-7 cells over-expressing NTCP (Supp Fig.2b). These data suggest that NTCP expression levels per 138 se have a negligible impact on the kinetics of HBV internalization but may regulate the absolute levels of 139 internalized virus. Our attempts to study HBV internalization into PHHs yielded poor quality data with limited 140 evidence of viral uptake even in the in the presence of PEG and showed high donor variability.

143
To study the cellular pathways that regulate HBV internalization we evaluated a panel of pharmacological agents 144 that target various cellular trafficking pathways: Dynasore inhibits dynamin and arrests vesicle formation from 145 the plasma membrane 33 ; Pitstop 2 inhibits clathrin-mediated endocytosis 34 and ethyl-isopropyl amiloride (EIPA) 146 targets the Na + /H + exchanger, inhibiting macropinocytosis 35,36 . We confirmed the specificity of Pitstop and agents were evaluated for their ability to inhibit HBV internalization into HepG2-NTCP or Huh-7 NTCP cells after 150 6h inoculation and HBeAg expression measured after 5 days. Heparin and MyrB were included as positive controls 151 known to inhibit HBV uptake. Pre-treating cells with Dynasore and Pitstop and maintaining compounds during the 152 viral inoculation stage inhibited HBV uptake into both cell lines ( Fig.4a-b), suggesting a dynamin and clathrin dependent endocytic uptake process. In contrast, EIPA, had no effect on HBV internalization, suggesting a 154 negligible role for micropinocytosis in viral uptake into hepatoma cells. These observations were validated by 155 measuring HBeAg expression 5 days post inoculation ( Fig.4a-b). To study the role of the host cytoskeleton in 156 regulating HBV uptake, HepG2-NTCP cells were treated with either nocodazole or cytochalasin D that interfere 157 with microtubule and actin dynamics, respectively 37,38 (Fig.4c). Both of these agents significantly inhibited HBV 158 uptake in the first 6h following infection and HBeAg levels, suggesting a role for microtubule and actin filaments 159 in regulating intracellular capsid trafficking.

161
Kinetics of HBV trafficking from the cytoplasm to the nucleus.

162
To study the early steps in the HBV life cycle that precede cccDNA genesis we analyzed subcellular fractions for 163 intracellular HBV DNA. A synchronized HBV infection was performed and cytoplasmic and nuclear fractions 164 harvested at early (1, 3, 6, 8 and 12h) and late (24, 48 and 72h) time points post-trypsinization for quantification 165 of HBV DNA and cccDNA. Cellular fractionation was confirmed by probing cytoplasmic and nuclear samples for 166 α-tubulin and lamin A/C, respectively and DNA samples for presence of the housekeeping gene PRNP (Suppl Fig.4).

167
Intracellular HBV DNA was first detected in the cytoplasm within 1h of incubating the cells at 37°C and particle 168 trafficking to the nucleus was detected after 3h (Fig.5a). HBV DNA levels in the nucleus and cytoplasm were 169 saturated by 12h and we noted 3.5-fold higher levels of viral DNA in the cytoplasm compared to the nucleus as 170 well as a loss of viral DNA in both nuclear and cytoplasmic fractions after 24h (Fig.5a). We used published PCR particles entered HepG2-NTCP cells. We observed a surprisingly high level (46%) of intracellular HBV DNA in 180 HepG2 cells and since we failed to detect any cccDNA in these cells, this most likely reflects a non-productive 181 uptake pathway in these cells (Table 1). Finally, we noted that less than 1% of the intracellular HBV DNA detected 182 at 6h was converted into cccDNA by 72h. It is worth noting that the detection limit of our assays for quantifying 183 rc-and cccDNA is 100 copies per reaction, suggesting that our earlier conclusion is not biased by differences in 184 the sensitivity of the PCR methods. In summary, these data show that particle internalization is efficient with the 185 majority of cell-bound particles entering NTCP expressing cells, with at least 22% of particle associated DNA 186 reaching the nucleus within 12h. In contrast, the subsequent conversion of incoming rcDNA to cccDNA is 187 inefficient, identifying a rate-limiting step in establishing productive infection.  recently reported HBV in clathrin-coated pits and vesicles using electron microscopy (EM) and cryo-EM with 217 immunogold labelling 18 consistent with a clathrin-dependent endocytic entry route.

219
We first detect cccDNA in the synchronized infection assay after 24h, consistent with reports for DHBV infection 44 .

220
Since our PCR method to quantify cccDNA uses a T5 exonuclease to remove non-cccDNA species, this treatment 221 may result in a loss of >20% of cccDNA 21 . Given these caveats our data suggests that a minority of intracellular

232
Our results support a role for a clathrin and dynamin in defining HBV particle uptake into HepG2-NTCP and Huh-