2.1. Transcription Initiation of HBV cccDNA
Peng et al. studied the HBV transcriptome by using 5′ RNA sequencing on about 3000 hepatocytes from liver-humanized FRG mice infected with HBV. Their findings indicated that transcription from cccDNA likely starts within the X/Enhancer I domain in a random way. The buildup of X transcripts, especially those that can produce functional X protein, may stimulate the productive transcription of cccDNA. The transcription start site for HBx is close to the start codon of the X open reading frame.
The researchers also found that HBx and related transcripts can be produced even with the presence of the Smc5/6 complex. By infecting primary human hepatocytes (PHHs) with a virus that had a stop codon mutation in HBx, they noted that most viral transcripts came from the HBx/enhancer I domain. This suggests that HBx transcription begins regardless of SMC5/6 complex presence and occurs before the transcription of viral structural genes. The mechanism of HBx transcription is still unclear, but G-quadruplexes in HBx regions and nucleosome occupancy may play a role. Notably, HBx transcripts were detected as early as 4 hours after infection. These levels remained comparable for the first 24 hours, even when cccDNA was barely detectable.
Further research is needed to see if any HBx transcripts exist within the viral nucleocapsid. In vivo, liver cells show varied transcriptome and proteomic patterns, but it remains largely unknown if liver zonation affects HBV transcription.
2.2. Coordinated HBV cccDNA Transcription in Single Cells
HBV transcripts derive from both cccDNA and integrated viral DNA. cccDNA is the main template for viral transcripts, while integrated HBV can also generate smaller transcripts. In mice infected with HBV, over 99% of viral transcripts were found to come from cccDNA. Analysis of the 3.5 kb and 2.1 kb transcripts indicated coordinated expression at the single-cell level.
Studies reveal that HBV-infected hepatocytes usually contain only a few cccDNA molecules. It is crucial to understand whether these cccDNA support transcription from all promoters or subsets while generating all transcripts. By aligning viral transcripts from different HBV genotypes in individual hepatocytes, Peng et al. demonstrated that cccDNA could generate all viral transcripts or primarily those from the X domain without notable structural gene expression.
Although coordination of cccDNA transcription appears evident at the cellular level, human liver samples show different expression patterns of HBsAg and HBV core protein in infected cells. In one study, HBeAg-negative patients showed a strong presence of HBsAg-positive cells without HBV core protein, likely due to HBsAg originating mostly from integrated HBV.
2.3. Transcription of Inactive cccDNA and Implications for Therapy
In chronic HBV infection cases, some patients experience low-level viremia or occult HBV infection, characterized by low HBV cccDNA levels in hepatocytes and minimal viral RNA transcription. Transcriptionally inactive cccDNA has been seen in patients co-infected with HIV who had long-term NUC treatment. A study found that prolonged NUC treatment often led to a higher number of hepatocytes with inactive viral transcription.
When patients ceased NUC treatment, a viral rebound usually occurred, even if they had no detectable HBV before stopping the treatment. The patterns of this rebound vary among patients, indicating different HBV replication dynamics within the liver.
The existence of inactive cccDNA has significant clinical implications. Many investigational drugs lead to viral relapse after treatment ends. Assessing inactive or latent cccDNA remains challenging due to limited liver biopsy samples and minimal available technology. However, some animal studies have shown promise in suppressing cccDNA transcription through blocking viral entry.
In liver-humanized mice, a recent study found a small proportion of hepatocytes having cccDNA with low levels of HBx-related RNAs without any structural gene transcription. Enhanced treatment approaches may alter chronic hepatitis B by blocking persistent infection cycles. Given that HBV-infected hepatocytes change in metabolism and gene expression, entry inhibitors may effectively support combination therapies aimed at reducing treatment duration.
