Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

Li, Hung Cheng; Huang, Er; Su, Pei Yi; Wu, Szu Yao; Yang, Ching Chun; Lin, Young Sun; Chang, Wen Chang; Shih, Chiaho

doi:10.1371/journal.ppat.1001162

Cited by 131 publications

(178 citation statements)

References 58 publications

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“…Heterokaryon assays were performed as described previously (19). Briefly, at 24 h posttransfection, human Huh7 cells were treated with trypsin, and 10 6 Huh7 cells were mixed with 2 ϫ 10 6 untransfected mouse NIH 3T3 cells, before being seeded on glass coverslips in a 6-cm dish.…”

Section: Cells Viruses and Antibodies Madin-darby Canine Kidney (Mmentioning

confidence: 99%

Identification and Characterization of Three Novel Nuclear Export Signals in the Influenza A Virus Nucleoprotein

Liu

Cao

et al. 2012

J Virol

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The nuclear export of the influenza A virus ribonucleoprotein (vRNP) is crucial for virus replication. As a major component of the vRNP, nucleoprotein (NP) alone can also be shuttled out of the nucleus by interacting with chromosome region maintenance 1 (CRM1) and is therefore hypothesized to promote the nuclear export of the vRNP. In the present study, three novel nuclear export signals (NESs) of the NP-NES1, NES2, and NES3-were identified as being responsible for mediating its nuclear export. The nuclear export of NES3 was CRM1 dependent, whereas that of NES1 or NES2 was CRM1 independent. Inactivation of these NESs led to an overall nuclear accumulation of NP. Mutation of all three NP-NESs significantly impaired viral replication. Based on structures of influenza virus NP oligomers, these three hydrophobic NESs are found present on the surface of oligomeric NPs. Functional studies indicated that oligomerization is also required for nuclear export of NP. Together, these results suggest that the nuclear export of NP is important for virus replication and relies on its NESs and oligomerization.T he influenza A virus genome consists of eight negative-sense single-stranded RNA segments (vRNA) (17). Each vRNA segment is associated with multiple copies of the viral nucleoprotein (NP) and three polymerase subunits (PA, PB1, and PB2), forming the viral ribonucleoprotein (vRNP) complex. During an early stage of infection, the vRNPs are released into the cytoplasm from virions following fusion of the viral membrane and endosomal membrane (41). Subsequently, the incoming vRNPs are transported into the nucleus, where viral genome replication and transcription occur (41).One of the determinants for vRNP nuclear import is NP (8, 28, 44, 49, 52, 53), the major protein in the vRNP structure. Thus far, two nuclear localization signals (NLSs) and a nuclear accumulation signal (NAS) have been identified in NP. The stronger NLS is an unconventional signal located in the N-terminal basic region (between residues 3 and 13) of NP (28, 44), and the weaker signal, a classical bipartite NLS, is located between residues 198 and 216 (49). The NAS-spanning residues (327 to 345) were identified through analyses of mutants that lacked both of the NLSs but still exhibited partial nuclear distribution (9).At a late stage of virus infection, the vRNPs exit the nucleus to assemble and bud from the apical plasma membrane of polarized cells (3). The trafficking of the vRNPs into and out of the nucleus is a tightly regulated process (7). The nuclear export of progeny vRNPs is mediated by the CRM1 cellular export receptor (12,23,29,48). Nuclear export protein (NEP; formerly referred to as the NS2 protein), which possesses a nuclear export signal (NES), and the matrix protein (M1) of influenza A virus are deemed responsible for directing export of the vRNPs (29, 31). This process is regulated by the Raf/MEK/ERK pathway (32), which is stimulated by the membrane association of influenza virus hemagglutinin (HA) (25). Interestingly, exogenously expresse...

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Section: Cells Viruses and Antibodies Madin-darby Canine Kidney (Mmentioning

confidence: 99%

Identification and Characterization of Three Novel Nuclear Export Signals in the Influenza A Virus Nucleoprotein

Liu

Cao

et al. 2012

J Virol

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“…A heterokaryon assay was performed as described previously (22). Briefly, the plasmid encoding the SV40 LT-NES fusion protein was transfected into 293T cells.…”

Section: Cells and Reagentsmentioning

confidence: 99%

A Nuclear Export Signal in the Matrix Protein of Influenza A Virus Is Required for Efficient Virus Replication

Cao

Liu

et al. 2012

J Virol

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The influenza A virus matrix 1 protein (M1) shuttles between the cytoplasm and the nucleus during the viral life cycle and plays an important role in the replication, assembly, and budding of viruses. Here, a leucine-rich nuclear export signal (NES) was identified specifically for the nuclear export of the M1 protein. The predicted NES, designated the Flu-A-M1 NES, is highly conserved among all sequences from the influenza A virus subtype, but no similar NES motifs are found in the M1 sequences of influenza B or C viruses. The biological function of the Flu-A-M1 NES was demonstrated by its ability to translocate an enhanced green fluorescent protein (EGFP)-NES fusion protein from the nucleus to the cytoplasm in transfected cells, compared to the even nuclear and cytoplasmic distribution of EGFP. The translocation of EGFP-NES from the nucleus to the cytoplasm was not inhibited by leptomycin B. NES mutations in M1 caused a nuclear retention of the protein and an increased nuclear accumulation of NEP during transfection. Indeed, as shown by rescued recombinant viruses, the mutation of the NES impaired the nuclear export of M1 and significantly reduced the virus titer compared to titers of wild-type viruses. The NES-defective M1 protein was retained in the nucleus during infection, accompanied by a lowered efficiency of the nuclear export of viral RNPs (vRNPs). In conclusion, M1 nuclear export was specifically dependent on the Flu-A-M1 NES and critical for influenza A virus replication. Influenza A virus is a negative-strand RNA virus composed of eight segmented strands of an RNA genome (21). The viral structural components include the viral envelope, the transmembrane proteins (hemagglutinin [HA], neuraminidase [NA], and M2), M1, NS2, and the viral ribonucleoprotein (vRNP), which contains viral RNA (vRNA), nucleoprotein (NP), and viral polymerase proteins (PB1, PB2, and PA) (44).M1 is the most abundant protein in the virus particle, sustaining the virion structure by forming a shell connecting the viral envelope and the nucleocapsid (40). M1 is synthesized in the late stages of infection, shuttles between the nucleus and the cytoplasm (45), and plays multiple roles in various steps of the influenza virus life cycle.Newly synthesized M1 is transported from the cytoplasm into the nucleus via a nuclear localization signal (NLS) located in its N-terminal domain (residues 101 to 105) (38, 48). In the nucleus, M1 is involved in the blocking of the transcription of viral mRNA by binding to vRNPs (4, 49). Aside from terminating viral transcription, M1 also plays an important role in the nuclear export of vRNPs. Indeed, the nuclear presence of M1 is required for vRNPs to be transported to the cytoplasm (8, 27), where the vRNPs are subsequently transported to the budding site. First, M1 binds to histones in the nucleus (51) and may participate in releasing vRNPs from the nuclear matrix. Second, M1 likely bridges the NEP and vRNPs, forming a complex that is in turn exported from the nucleus by the nuclear export signal (NES) ...

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“…For assembled capsids at least, nuclear import requires phosphorylation (Kann et al, 1999). While the nuclear transport capacity of core is well established, a recent publication also reported nuclear export of core protein, which was thought to be implicated in export of HBV specific transcripts by the TAP/NxF1 pathway (Li et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

Deroubaix

Osseman

Cassany

et al. 2015

Journal of General Virology

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Biopsies from patients show that hepadnaviral core proteins and capsids -collectively called core -are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that e, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.

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Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

Cited by 131 publications

References 58 publications

Identification and Characterization of Three Novel Nuclear Export Signals in the Influenza A Virus Nucleoprotein

Identification and Characterization of Three Novel Nuclear Export Signals in the Influenza A Virus Nucleoprotein

A Nuclear Export Signal in the Matrix Protein of Influenza A Virus Is Required for Efficient Virus Replication

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

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