It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signals (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES.

Mutations in hepatitis B virus (HBV) reverse transcriptase (RT) are associated with nucleos(t)ide analogue (NA) resistance during long-term antiviral treatment. However, the characterization of mutations in HBV RT in untreated patients has not yet been well illustrated. The objective of this study was to investigate the characterization and clinical significance of natural variability in HBV RT in treatment-naive patients. HBV RT sequences were analyzed in 427 patients by Sanger sequencing and in 66 patients by next-generation sequencing. Primary or secondary NA resistance (NAr) mutations were not found, except A181T in RT (rtA181T) by Sanger sequencing, but they were detected by next-generation sequencing. Mutations were found in 56 RT amino acid (aa) sites by Sanger sequencing, 36 of which had mutations that could lead to changes in B or T cell epitopes in the RT or S protein. The distribution of mutations was diverse in different sections within the RT region. Multiple mutations showed significant association with HBV DNA, HBsAg, HBeAg, age, and severity of liver fibrosis. Mutations at rt251, rt266, rt274, rt280, rt283, rt284, and rt286 were found most in the advanced liver disease (ALD) group by next-generation sequencing. The present study demonstrates that next-generation sequencing (NGS) was more suitable than Sanger sequencing to monitor NAr mutations at a low rate in the treatment-naive patients, and that mutations in the RT region might be involved in the progression to ALD.

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BackgroundAnti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is the most common type of autoimmune encephalitis. Early recognition and treatment, especially distinguishing from viral encephalitis (VE) in the early stages, are crucial for the outcomes of patients with anti-NMDAR encephalitis. Compared with plasma microRNAs (miRNAs), exosomal miRNAs are more abundant and not easy to degrade. Moreover, exosomes can pass through the blood–brain barrier. This study aimed to explore the clinical value of serum exosomal miRNAs in the differential diagnosis of anti-NMDAR encephalitis with VE.MethodSerum samples from a total of 30 patients with anti-NMDAR encephalitis, 30 patients with VE, and 30 cases of control patients hospitalized in the same period were collected. Firstly, the serum exosomes were isolated and identified by transmission electron microscope (TEM), nanoparticle-tracking analyzer (NTA), and Western blot (WB). The expression levels of let-7b and miR-140-5p from serum exosomes were detected by real-time quantitative PCR (qPCR). At the same time, we also detected complement 3 (C3), complement 4 (C4), and high sensitivity CRP (hs-CRP) expression levels in three groups. Finally, we analyzed the difference and diagnostic value of the test results.ResultsIsolated particles showed identical characteristics to the exosomes through TEM, NTA, and WB analyses. Compared with the VE group and control group, the expression of miR-140-5p was significantly upregulated in serum exosomes of the NMDAR group. In contrast, the serum C3 in the NMDAR group was significantly lower than the other two groups. ROC curve analysis showed the area under the curve (AUC) of serum exosomal miR-140-5p and serum C3 was 0.748 (76.67% sensitivity and 73.33% specificity) and 0.724 (76.67% sensitivity and 60% specificity) to distinguish anti-NMDAR encephalitis from VE, respectively. The AUC of serum exosomal miR-140-5p combined with serum C3 was 0.811, the sensitivity was 70.00%, and the specificity was 86.67%.ConclusionSerum exosomal miR-140-5p combined with serum C3 would be a promising marker in the differential diagnosis of anti-NMDAR encephalitis with VE.