RESUMO
Hepatitis B virus (HBV) is a global pathogen. We report here that the cellular CRM1 machinery can mediate nuclear export of entire HBV core (HBc) particles containing encapsidated viral RNAs. Two CRM1-mediated nuclear export signals (NESCRM1) cluster at the conformationally flexible spike tips of HBc particles. Mutant NESCRM1 capsids exhibit strongly reduced associations with CRM1 and nucleoporin358 in vivo. CRM1 and NXF1 machineries mediate nuclear export of HBc particles independently. Inhibition of nuclear export has pleiotropic consequences, including nuclear accumulation of HBc particles, a significant reduction of encapsidated viral RNAs in the cytoplasm but not in the nucleus, and barely detectable viral DNA. We hypothesize an HBV life cycle where encapsidation of the RNA pregenome can initiate early in the nucleus, whereas DNA genome maturation occurs mainly in the cytoplasm. We identified a druggable target for HBV by blocking its intracellular trafficking.
Assuntos
Vírus da Hepatite B , RNA Viral , Transporte Ativo do Núcleo Celular/genética , Capsídeo/metabolismo , Citoplasma/metabolismo , Vírus da Hepatite B/genética , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.
Assuntos
Vírus da Hepatite B/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo , Transporte Ativo do Núcleo Celular/genética , Linhagem Celular , Núcleo Celular , Citoplasma/genética , Replicação do DNA , Vírus da Hepatite B/genética , Interações Hospedeiro-Patógeno , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Sinais de Exportação Nuclear , Proteínas de Transporte Nucleocitoplasmático/genética , Estrutura Terciária de Proteína , RNA , Transporte de RNA/genética , RNA Mensageiro/genética , RNA Interferente Pequeno , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas do Core Viral/genéticaRESUMO
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.
