Single Hepatocyte Hepatitis B Virus Transcriptional Landscape in HIV Coinfection

Abstract

Background: Hepatitis B virus (HBV) is a leading cause of liver failure and hepatocellular carcinoma. Approximately 10% of people with HIV also have HBV and are at higher risk of liver disease progression than in HBV monoinfection. Antivirals, common to HIV and HBV, suppress HBV DNA levels but do not eradicate virus because the transcriptional template, covalently closed circular DNA (cccDNA), is long lived in infected hepatocytes.

Methods: Using single-cell laser capture microdissection, we isolated >1100 hepatocytes from 5 HIV/HBV coinfected persons with increasing exposure to HBV antivirals (HB1-HB5; no exposure to >7 years exposure), quantifying cccDNA and pregenomic RNA (pgRNA) in each cell using droplet digital polymerase chain reaction.

Results: The proportion of infected hepatocytes decreased with antiviral exposure from 96.4% (HB1) to 29.8% (HB5). Upper cccDNA range and median pgRNA decreased from HB1 to HB5 (P < .05 for both). The amount of pgRNA transcribed per cccDNA also decreased from HB1 to HB5 (P < .05). Cells with inactive pgRNA transcription were enriched from 0% (HB1) to 14.3% (HB5) of infected hepatocytes.

Conclusions: cccDNA transcription is reduced in HIV/HBV coinfected people with longer antiviral duration. Understanding HBV transcriptional regulation may be critical to develop a functional cure.

Keywords: HBV transcription; HIV/HBV coinfection; intrahepatic hepatitis B; single-cell laser capture microdissection; viral landscape; viroscape.

Figure 1.

Total hepatitis B virus (HBV) DNA and dually active antiretroviral therapy exposure. Total HBV DNA was quantified in hundreds of single hepatocytes from 5 HIV/HBV coinfected persons (HB1–HB5) using droplet digital polymerase chain reaction (ddPCR). The amount of total HBV DNA is shown for each hepatocyte as an individual point and is denoted within each participant separately. Indicated by the black horizontal line is the lower limit of detection (LLOD) for the ddPCR assay. Hepatocytes with undetectable total HBV DNA were assigned a value just below the LLOD. Kruskal-Wallis nonparametric testing was used to test for significant differences in total HBV DNA amounts across the participants. Denoted by lines in boxplots, from top to bottom, are Q1+1.5*IQR, Q1, Q2 (Median), Q3, and Q3–1.5*IQR. Individual points representing each analyzed cell are overlaid on the boxplots.

Figure 2.

Covalently closed circular DNA (cccDNA) and dually active antiretroviral therapy exposure. cccDNA was quantified in hundreds of single hepatocytes among 5 human immunodeficiency virus (HIV)/hepatitis B virus (HBV) coinfected persons (HB1–HB5) using droplet digital polymerase chain reaction (ddPCR) after exonuclease I/III treatment of total extracted DNA. The amount of cccDNA is shown for each hepatocyte as an individual point and is denoted within each participant separately. Infected hepatocytes that also had detectable pregenomic RNA (pgRNA) are indicated by solid red circles, while those that had cccDNA but did not have detectable pgRNA are indicated by open red circles (inactive pgRNA transcription). Uninfected hepatocytes are indicated by solid black circles. Indicated by the black horizontal line is the lower limit of detection (LLOD) for the ddPCR assay. Hepatocytes with undetectable cccDNA were assigned a value just below the LLOD. Kruskal-Wallis nonparametric testing was used to test for significant differences in cccDNA amounts across the participants. Denoted by lines in boxplots, from top to bottom, are Q1+1.5*IQR, Q1, Q2 (Median), Q3, and Q3–1.5*IQR. Individual points representing each analyzed cell are overlaid on the boxplots.

Figure 3.

Pregenomic RNA (pgRNA) and dually active antiretroviral therapy exposure. pgRNA was quantified in hundreds of single hepatocytes among 5 human immunodeficiency virus (HIV)/hepatitis B virus (HBV) coinfected persons (HB1–HB5) using droplet digital polymerase chain reaction (ddPCR). The amount of log₁₀(pgRNA) is shown for each hepatocyte as an individual point and is denoted within each participant separately, infected hepatocytes by magenta circles and uninfected hepatocytes by light blue circles. Indicated by the black horizontal line is the lower limit of detection (LLOD) for the ddPCR assay. Hepatocytes with undetectable pgRNA were assigned a value just below the LLOD. Kruskal-Wallis nonparametric testing was used to test for significant differences in pgRNA amounts across the participants. Denoted by lines in boxplots, from top to bottom, are Q1+1.5*IQR, Q1, Q2 (Median), Q3, and Q3–1.5*IQR. Individual points representing each analyzed cell are overlaid on the boxplots.

Figure 4.

Pregenomic RNA (pgRNA) transcription does not depend quantitatively on covalently closed circular DNA (cccDNA). A, The amount of log₁₀(pgRNA) and corresponding cccDNA was plotted for each hepatocyte among the 5 participants. The horizontal and vertical solid black lines indicate the respective LLOD for each assay. There is no clear relationship between pgRNA quantities and cccDNA quantities in the same cell. B, The amount of pgRNA was adjusted per molecule of cccDNA within each cell to calculate a transcriptional index. The transcriptional index for each cell is plotted as an individual point and is denoted within each participant separately. Kruskal-Wallis nonparametric testing was used to test for significant differences in the transcriptional indices across the participants. Denoted by lines in boxplots, from top to bottom, are Q1+1.5*IQR, Q1, Q2 (Median), Q3, and Q3–1.5*IQR. Individual points representing each analyzed cell are overlaid on the boxplots.