Search

Hepatitis B virus (HBV) exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for virion budding. In addition to enveloped virions, HBV-replicating cells nonlytically release non-enveloped (naked) capsids independent of the integral ESCRT machinery, but the exact secretory mechanism remains elusive. Here, we provide more detailed information about the existence and characteristics of naked capsid, as well as the viral and host regulations of naked capsid egress. HBV capsid/core protein has two highly conserved Lysine residues (K7/K96) that potentially undergo various types of posttranslational modifications for subsequent biological events. Mutagenesis study revealed that the K96 residue is critical for naked capsid egress, and the intracellular egress-competent capsids are associated with ubiquitinated host proteins. Consistent with a previous report, the ESCRT-III-binding protein Alix and its Bro1 domain are required for naked capsid secretion through binding to intracellular capsid, and we further found that the ubiquitinated Alix binds to wild type capsid but not K96R mutant. Moreover, screening of NEDD4 E3 ubiquitin ligase family members revealed that AIP4 stimulates the release of naked capsid, which relies on AIP4 protein integrity and E3 ligase activity. We further demonstrated that AIP4 interacts with Alix and promotes its ubiquitination, and AIP4 is essential for Alix-mediated naked capsid secretion. However, the Bro1 domain of Alix is non-ubiquitinated, indicating that Alix ubiquitination is not absolutely required for AIP4-induced naked capsid secretion. Taken together, our study sheds new light on the mechanism of HBV naked capsid egress in viral life cycle., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shen 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.)

Tripartite motif (TRIM) proteins, comprising a family of over 100 members with conserved motifs, exhibit diverse biological functions. Several TRIM proteins influence viral infections through direct antiviral mechanisms or by regulating host antiviral innate immune responses. To identify TRIM proteins modulating hepatitis B virus (HBV) replication, we assessed 45 human TRIMs in HBV-transfected HepG2 cells. Our study revealed that ectopic expression of 12 TRIM proteins significantly reduced HBV RNA and subsequent capsid-associated DNA levels. Notably, TRIM65 uniquely downregulated viral pregenomic (pg) RNA in an HBV-promoter-specific manner, suggesting a targeted antiviral effect. Mechanistically, TRIM65 inhibited HBV replication primarily at the transcriptional level via its E3 ubiquitin ligase activity and intact B-box domain. Though HNF4α emerged as a potential TRIM65 substrate, disrupting its binding site on the HBV genome did not completely abolish TRIM65's antiviral effect. In addition, neither HBx expression nor cellular MAVS signaling was essential to TRIM65-mediated regulation of HBV transcription. Furthermore, CRISPR-mediated knock-out of TRIM65 in the HepG2-NTCP cells boosted HBV infection, validating its endogenous role. These findings underscore TRIM proteins' capacity to inhibit HBV transcription and highlight TRIM65's pivotal role in this process.

Importance: The biogenesis and clinical application of serum HBV pgRNA have been a research hotspot in recent years. This study further characterized the heterogeneity of the 3' terminus of capsid RNA by utilizing a variety of experimental systems conditionally supporting HBV genome replication and secretion, and reveal that the 3' truncation of capsid pgRNA is catalyzed by cellular ribonuclease(s) and viral RNaseH at positions after and before 3' DR1, respectively, indicating the 3' DR1 as a boundary between the encapsidated portion of pgRNA for reverse transcription and the 3' unprotected terminus, which is independent of pgRNA length and the 3' terminal sequence. Thus, our study provides new insights into the mechanism of pgRNA encapsidation and reverse transcription, as well as the optimization of serum HBV RNA diagnostics., Competing Interests: The authors declare no conflict of interest.

In pursuit of novel therapeutics targeting the hepatitis B virus (HBV) infection, we evaluated a dihydroquinolizinone compound (DHQ-1) that in the nanomolar range reduced the production of virion and surface protein (HBsAg) in tissue culture. This compound also showed broad HBV genotype coverage, but was inactive against a panel of DNA and RNA viruses of other species. Oral administration of DHQ-1 in the AAV-HBV mouse model resulted in a significant reduction of serum HBsAg as soon as 4 days following the commencement of treatment. Reduction of HBV markers in both in vitro and in vivo experiments was related to the reduced amount of viral RNA including pre-genomic RNA (pgRNA) and 2.4/2.1 kb HBsAg mRNA. Nuclear run-on and subcellular fractionation experiments indicated that DHQ-1 mediated HBV RNA reduction was the result of accelerated viral RNA degradation in the nucleus, rather than the consequence of inhibition of transcription initiation. Through mutagenesis of HBsAg gene sequences, we found induction of HBsAg mRNA decay by DHQ-1 required the presence of the HBV posttranscriptional regulatory element (HPRE), with a 109 nucleotides sequence within the central region of the HPRE alpha sub-element being the most critical. Taken together, the current study shows that a small molecule can reduce the overall levels of HBV RNA, especially the HBsAg mRNA, and viral surface proteins. This may shed light on the development of a new class of HBV therapeutics., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The sensitivity and the limit of detection of Raman sensors are limited by the extremely small scattering cross section of Raman labels. Silver nanorice antennae are coupled with a patterned gold triangle nanoarray chip to create spatially broadened plasmonic "hot spots", which enables a large density of Raman labels to experience strong local electromagnetic field. Finite difference time domain simulations have confirmed that the quasi-periodic structure increases the intensity and the area of the surface plasmon resonance (SPR), which enhances the surface-enhanced Raman scattering (SERS) signal significantly. The SERS signal of the nanorice/DNA/nanoarray chip is compared with that of the nanorice/DNA/film chip. The SERS signal is greatly enhanced when the Ag nanorices are coupled to the periodic Au nanoarray instead of the planar film chip. The resulting spatially broadened SPR field enables the SERS biosensor with a limit of detection of 50 aM toward hepatitis B virus DNA with the capability of discriminating a single-base mutant of DNA. This sensing platform can be extended to detect other chemical species and biomolecules such as proteins and small molecules.