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. 2014 Jul;88(13):7455-63.
doi: 10.1128/JVI.00257-14. Epub 2014 Apr 16.

Characteristics of nucleocytoplasmic transport of H1N1 influenza A virus nuclear export protein

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Characteristics of nucleocytoplasmic transport of H1N1 influenza A virus nuclear export protein

Shengyan Gao et al. J Virol. 2014 Jul.

Abstract

The influenza A virus nuclear export protein (NEP) plays crucial roles in the nuclear export of the viral ribonucleoprotein complex through the chromosome region maintenance 1 (CRM1)-mediated cellular protein transport system. However, the detailed mechanism of NEP nucleocytoplasmic trafficking remains incompletely understood. Here, we investigated the subcellular localization of NEP from two strains of H1N1 influenza A virus and found that 2009 swine-origin H1N1 influenza A virus A/California/04/2009 (CA04) NEP displayed a distinct cellular distribution pattern, forming unique nuclear aggregates, compared to A/WSN/33 (H1N1) (WSN) NEP. Characterization of the nucleocytoplasmic transport pathways of these two NEPs showed that they both enter the nucleus by passive diffusion but are exported through the nuclear export receptor CRM1-mediated pathway with different efficiencies. The two identified nuclear export signals (NESs) on the two NEPs functioned similarly despite differences in their amino acid sequences. Using a two-hybrid assay, we confirmed that the CA04 NEP interacts less efficiently with CRM1 and that a threonine residue at position 48 is responsible for the nuclear aggregation. The present study revealed the dissimilarity in subcellular NEP transport processes between the 2009 pandemic (H1N1) influenza A virus CA04 and the laboratory-adapted H1N1 virus WSN and uncovered the mechanism responsible for this difference.

Importance: Because the efficiency of the nucleocytoplasmic transport of viral components is often correlated with the viral RNA polymerase activity, propagation, and host range of influenza viruses, the present study investigated the subcellular localization of NEP from two strains of H1N1 influenza virus. We found that the NEPs of both A/California/04/2009 (H1N1) (CA04) and A/WSN/33 (H1N1) (WSN) enter the nucleus by passive diffusion but are exported with different efficiencies, which were caused by weaker binding activity between the CA04 NEP and CRM1. The results of the present study revealed characteristics of the nuclear import and export pathways of NEP and the mechanism responsible for the differences in the cellular distribution of NEP between two H1N1 strains.

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Figures

FIG 1
FIG 1
The cellular localization pattern of CA04-NEP is different from that of WSN-NEP when overexpressed in cells. (A) N-terminally c-Myc-tagged NEP from WSN or CA04 was transiently expressed in 293T cells. At 24 h posttransfection, the cells were treated with 100 ng/μl of cycloheximide for 3 h to block protein synthesis. LMB (11 nM) was added to the medium along with the cycloheximide for LMB treatment. (B and C) Western blotting confirmed the different cellular localizations of the two NEPs. 293T cells transfected with WSN-NEP or CA04-NEP were lysed with whole-cell lysis buffer (B) or nucleus (“N”)-cytoplasm (“C”) fractionation buffer (C) at 24 h posttransfection and prepared for Western blotting. An anti-c-Myc monoclonal antibody was used for protein detection. DAPI (4′-6-diamidino-2-phenylindole) was used for nuclear staining. Results shown are representative images.
FIG 2
FIG 2
Different subcellular distributions of viral proteins in CA04- and WSN-infected cells. A549 cells were infected with WSN or CA04 at an MOI of 1 and fixed at 8 h, 10 h, and 24 h postinfection (h.p.i). The subcellular localization of NEP, M1 (A), and NP (B) was examined with corresponding antibodies. DAPI was used for nuclear staining.
FIG 3
FIG 3
NEP enters the nucleus by passive diffusion. (A) In vitro NEP transport assay. EGFP-NEP (upper panel) and EGFP-M1 (lower panel) fusion proteins were separately expressed in 293T cells, and cell lysates in transport buffer were added to digitonin-treated 293T cells. For WGA treatment (WGA+), the permeabilized cells were incubated in transport buffer containing 50 μg/ml WGA before addition of cell lysates. Images revealed fluorescence signals of EGFP from fusion proteins. (B) Energy depletion assay of the nucleocytoplasmic transport of NEP. N-terminally myc-tagged NEP was expressed in 293T cells. N-terminally FLAG-tagged SVLT was expressed as a positive control. At 24 h posttransfection, the cells were incubated with energy depletion medium (ATP−) or normal culture medium (ATP+) for 3 h before fixation and visualization. Anti-myc and anti-FLAG monoclonal antibodies were used for the detection of the indicated proteins.
FIG 4
FIG 4
Evaluation of the nuclear export activity of SVLT-NEP fusion proteins. (A) Schematic representation of the SVLT-NEP chimeric protein. (B) 293T cells were transfected with plasmids encoding SVLT, SVLT-NEP (WSN), or SVLT-NEP (CA04). At 24 h posttransfection, the cells were treated with 100 ng/μl cycloheximide for 3 h to block protein synthesis. LMB (11 nM) was added to the medium along with the cycloheximide for LMB treatment. (C) A Western blot assay of 293T cells transfected with FLAG-SVLT or SVLT-NEP fusion protein constructs was carried out at 24 h posttransfection. An anti-FLAG monoclonal antibody was used to detect the target proteins.
FIG 5
FIG 5
The NESs on WSN-NEP and CA04-NEP have comparable functions. (A) Alignment of amino acid sequences of WSN-NEP and CA04-NEP. (B) 293T cells were transfected with plasmids expressing fusion proteins of EGFP and NESs. At 24 h posttransfection, the cells were treated with 100 ng/μl cycloheximide for 3 h to block protein synthesis prior to fixation. The cells were then permeabilized with PBS-T, mounted with DAPI-containing medium, and imaged by fluorescence microscopy.
FIG 6
FIG 6
The nuclear aggregation of CA04-NEP is caused by inefficient interaction with Crm1. (A) Mammalian cell two-hybrid assays were used to detect the binding of NEPs (and their mutants) with Crm1. 293T cells were cotransfected with the same amounts of pBIND-NEP encoding point mutants, pACT-Crm1, and pG5luc. At 24 h posttransfection, the cells were lysed, and luciferase activity assays were performed. The length of each bar represents the relative level of luciferase activity, calculated as the ratio of firefly activity to Renilla activity. Results shown are the means ± standard deviations (SD) from three independent experiments. (B) Western blotting with an anti-NEP monoclonal antibody confirmed that all constructs expressed similar levels of NEP. (C and D) 293T cells were transfected with a plasmid expressing myc-tagged CA04-NEP or point mutants. At 24 h posttransfection, the cells were treated with 100 ng/μl cycloheximide for 3 h to block protein synthesis and processed for immunofluorescence microscopy. (C) The numbers of cells transfected with myc-CA04-NEP or point mutants exhibiting nuclear aggregation were counted, and ratios to total examined cells were calculated. Approximately 90 to 100 cells were counted for each replicate. Results shown are the averages from three independent experiments. (D) Cellular localizations of wild-type and point mutant proteins were detected with anti-myc monoclonal antibodies, and images representing the whole population are shown.

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