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The Envelope Gene of Hepatitis B Virus Is Implicated in Both Differential Virion Secretion and Genome Replication Capacities between Genotype B and Genotype C Isolates.

ABSTRACT: Chronic infection by hepatitis B virus (HBV) genotype C is associated with a prolonged replicative phase and an increased risk of liver cancer, compared with genotype B infection. We previously found lower replication capacity but more efficient virion secretion by genotype C than genotype B isolates. Virion secretion requires interaction between core particles and ENVELOPE proteins. In the present study, chimeric constructs between genotype B and genotype C clones were generated to identify the structural basis for differential virion secretion. In addition to dimeric constructs, we also employed 1.1mer constructs, where the cytomegalovirus (CMV) promoter drove pregenomic RNA transcription. Through transient transfection experiments in Huh7 cells, we found that exchanging the entire envelope gene or just its S region could enhance virion secretion by genotype B clones while diminishing virion secretion by genotype C. Site-directed mutagenesis established the contribution of genotype-specific divergence at codons 108 and 115 in the preS1 region, as well as codon 126 in the S region, to differential virion secretion. Surprisingly, exchanging the envelope gene or just its S region, but not the core gene or 3' S region, could markedly increase intracellular replicative DNA for genotype C clones but diminish that for genotype B, although the underlying mechanism remains to be clarified.

SUBMITTER: Jia H

PROVIDER: S-EPMC5408668 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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The Envelope Gene of Hepatitis B Virus Is Implicated in Both Differential Virion Secretion and Genome Replication Capacities between Genotype B and Genotype C Isolates.

Jia Haodi H Qin Yanli Y Chen Chaoyang C Zhang Fei F Li Cheng C Zong Li L Wang Yongxiang Y Zhang Jiming J Li Jisu J Wen Yumei Y Tong Shuping S

Viruses 20170328 4

Chronic infection by hepatitis B virus (HBV) genotype C is associated with a prolonged replicative phase and an increased risk of liver cancer, compared with genotype B infection. We previously found lower replication capacity but more efficient virion secretion by genotype C than genotype B isolates. Virion secretion requires interaction between core particles and ENVELOPE proteins. In the present study, chimeric constructs between genotype B and genotype C clones were generated to identify the ...[more]

PMID: 28350327

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Project description:The core promoter mutants of hepatitis B virus (HBV) emerge as the dominant viral population at the late HBeAg and the anti-HBe stages of HBV infection, with the A1762T/G1764A substitutions as the hotspot mutations. The double core promoter mutations were found by many investigators to moderately enhance viral genome replication and reduce hepatitis B e antigen (HBeAg) expression. A much higher replication capacity was reported for a naturally occurring core promoter mutant implicated in the outbreak of fulminant hepatitis, which was caused by the neighboring C1766T/T1768A mutations instead. To systemically study the biological properties of naturally occurring core promoter mutants, we amplified full-length HBV genomes by PCR from sera of HBeAg(+) individuals infected with genotype A. All 12 HBV genomes derived from highly viremic sera (5 x 10(9) to 5.7 x 10(9) copies of viral genome/ml) harbored wild-type core promoter sequence, whereas 37 of 43 clones from low-viremia samples (0.2 x 10(7) to 4.6 x 10(7) copies/ml) were core promoter mutants. Of the 11 wild-type genomes and 14 core promoter mutants analyzed by transfection experiments in human hepatoma cell lines, 6 core promoter mutants but none of the wild-type genomes replicated at high levels. All had 1762/1764 mutations and an additional substitution at position 1753 (T to C), at position 1766 (C to T), or both. Moreover, these HBV clones varied greatly in their ability to secrete enveloped viral particles irrespective of the presence of core promoter mutations. High-replication clones with 1762/1764/1766 or 1753/1762/1764/1766 mutations expressed very low levels of HBeAg, whereas high-replication clones with 1753/1762/1764 triple mutations expressed high levels of HBeAg. Experiments with site-directed mutants revealed that both 1762/1764/1766 and 1753/1762/1764/1766 mutations conferred significantly higher viral replication and lower HBeAg expression than 1762/1764 mutations alone, whereas the 1753/1762/1764 triple mutant displayed only mild reduction in HBeAg expression similar to the 1762/1764 mutant. Thus, core promoter mutations other than those at positions 1762 and 1764 can have major impact on viral DNA replication and HBeAg expression.

Project description:BackgroundHepatitis B virus (HBV) is a major human pathogen worldwide. To date, there is no curative treatment for chronic hepatitis B. The mechanism of virion secretion remains to be investigated. Previously, we found that nuclear export of HBc particles can be facilitated via two CRM1-specific nuclear export signals (NES) at the spike tip.MethodsIn this study, we used site-directed mutagenesis at the CRM1 NES, as well as treatment with CRM1 inhibitors at a low concentration, or CRM1-specific shRNA knockdown, in HBV-producing cell culture, and measured the secretion of various HBV viral and subviral particles via a native agarose gel electrophoresis assay. Separated HBV particles were characterized by Western blot analysis, and their genomic DNA contents were measured by Southern blot analysis. Secreted extracellular particles were compared with intracellular HBc capsids for DNA synthesis and capsid formation. Virion secretion and the in vivo interactions among HBc capsids, CRM1 and microtubules, were examined by proximity ligation assay, immunofluorescence microscopy, and nocodazole treatment.ResultsWe report here that the tip of spike of HBV core (HBc) particles (capsids) contains a complex sensor for secretion of both HBV virions and naked capsids. HBV virion secretion is closely associated with HBc nuclear export in a CRM1-dependent manner. At the conformationally flexible spike tips of HBc particles, NES motifs overlap extensively with motifs important for secretion of HBV virions and naked capsids.ConclusionsWe provided experimental evidence that virions and naked capsids can egress via two distinct, yet overlapping, pathways. Unlike the secretion of naked capsids, HBV virion secretion is highly CRM1- and microtubule-dependent. CRM1 is well known for its involvement in nuclear transport in literature. To our knowledge, this is the first report that CRM1 is required for virion secretion. CRM1 inhibitors could be a promising therapeutic candidate for chronic HBV patients in clinical medicine.

Project description:UnlabelledIn hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1-TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein.ImportanceHCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1 could be the protein responsible for the process of fusion between viral and cellular membranes. Here we investigated the oligomeric state of HCV envelope glycoproteins. We demonstrate that E1 forms functional trimers after virion assembly and that in addition to the requirement for E2, a determinant for this oligomerization is present in a conserved GxxxG motif located within the E1 transmembrane domain. Taken together, these results indicate that a rearrangement of E1E2 heterodimer complexes likely occurs during the assembly of HCV particles to yield a trimeric form of the E1E2 heterodimer. Gaining structural information on this trimer will be helpful for the design of an anti-HCV vaccine.

Dataset Information

The Envelope Gene of Hepatitis B Virus Is Implicated in Both Differential Virion Secretion and Genome Replication Capacities between Genotype B and Genotype C Isolates.

Publications

The Envelope Gene of Hepatitis B Virus Is Implicated in Both Differential Virion Secretion and Genome Replication Capacities between Genotype B and Genotype C Isolates.

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