Venezuelan equine Encephalitis virus capsid protein forms a tetrameric complex with CRM1 and importin alpha/beta that obstructs nuclear pore complex function

Abstract

Development of the cellular antiviral response requires nuclear translocation of multiple transcription factors and activation of a wide variety of cellular genes. To counteract the antiviral response, several viruses have developed an efficient means of inhibiting nucleocytoplasmic traffic. In this study, we demonstrate that the pathogenic strain of Venezuelan equine encephalitis virus (VEEV) has developed a unique mechanism of nuclear import inhibition. Its capsid protein forms a tetrameric complex with the nuclear export receptor CRM1 and the nuclear import receptor importin alpha/beta. This unusual complex accumulates in the center channel of the nuclear pores and blocks nuclear import mediated by different karyopherins. The inhibitory function of VEEV capsid protein is determined by a short 39-amino-acid-long peptide that contains both nuclear import and supraphysiological nuclear export signals. Mutations in these signals or in the linker peptide attenuate or completely abolish capsid-specific inhibition of nuclear traffic. The less pathogenic VEEV strains contain a wide variety of mutations in this peptide that affect its inhibitory function in nuclear import. Thus, these mutations appear to be the determinants of this attenuated phenotype. This novel mechanism of inhibiting nuclear transport also shows that the nuclear pore complex is vulnerable to unusual cargo receptor complexes and sheds light on the importance of finely adjusted karyopherin-nucleoporin interactions for efficient cargo translocation.

FIG. 1.

Identification of the VEEV capsid-specific peptide that inhibits nuclear import. (A) (Top) Amino acid sequences of H68 and H60 peptides derived from VEEV capsid protein. Capsid-specific sequences are indicated by uppercase letters, and the glycine linker between the peptides and GFP is indicated by lowercase letters. Methionine was introduced to promote translation initiation. (Bottom) Representative confocal images demonstrating the distribution of the nuclear import reporter 4×Tomato-3×NLS in the presence of H68-GFP or H60-GFP. BHK-21 cells were infected with the packaged replicons VEErep/H68-GFP or VEErep/H60-GFP and VEErep/4×Tomato-3×NLS and fixed at 4 h postinfection. Scale bars, 20 μm. (B) Representative confocal images of cells expressing 4×Tomato-3×NLS or 4×Tomato from corresponding VEEV replicons at 4 h postinfection. Scale bars, 20 μm. (C) Box plot showing the nuclear/cytoplasmic distribution of 4×Tomato and 4×Tomato-3×NLS (4×Tom and 4×Tom-3×NLS, respectively) alone or when 4×Tomato-3×NLS was expressed in the presence of H60-GFP or H68-GFP (H60 and H68, respectively). The nuclear/cytoplasmic ratio for distribution of 4×Tomato was used as a control demonstrating the distribution of the large protein that is incapable of translocation to the nucleus. The P values were calculated using the Mann-Whitney test (n = 30 for all experiments). Red line, median. (D) Schematic representation of the VEEV replicons used in this study. H indicates the position of a wild-type or mutant peptide derived from VEEV capsid.

FIG. 2.

Identification of the NLS in VEEV capsid protein. (A) Sequence alignment of a highly positively charged VEEV capsid fragment (WT) with corresponding sequences of the previously identified frameshift noncytopathic mutant protein (Frsh) (19). The mutated fragment is underlined. The sequence used for analysis of NLS localization is indicated in boldface. N52-85, N52-71, N72-85, and N52-71AA represent peptides fused with a 4×Tomato reporter for dissecting the position of the NLS. The mutated amino acids in N52-71AA are indicated in blue. (B) Representative images of cells expressing different 4×Tomato fusions. The images were acquired on a Nikon Ti-U inverted fluorescence microscope using a 60× CFI Super Plan Fluor objective. The nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole). (C) Representative confocal images demonstrating the distribution of 4×Tomato-3×NLS in the presence of a H68AA2-GFP fusion containing mutations in the NLS. (D) Box plot demonstrating nuclear/cytoplasmic distribution of 4×Tomato-3×NLS alone or in the presence of H68-GFP or H68AA2-GFP. Note that in the presence of mutant H68AA2-GFP protein, accumulation of the reporter protein in the nucleus remained at almost the same level. (E) Box plot of the nuclear/cytoplasmic distribution of H68-GFP and H68AA2-GFP. Mutation of the NLS inhibits accumulation of the H68AA2-GFP in nuclei. The P values were calculated using the Mann-Whitney test (n = 30 for all experiments). (F) Mutations in the VEEV capsid NLS inhibit its translocation into the nuclei. BHK-21 cells were infected with a replicon expressing VEEV capsid-GFP containing mutations in the NLS. The mutations are indicated in blue. The images were acquired on a Nikon Ti-U inverted fluorescence microscope with a 60× CFI Super Plan Fluor objective. The nuclei were stained with DAPI. Mut, mutant. Scale bars, 20 μm.

FIG. 3.

Identification of the NES in VEEV capsid protein. (A) Sequence alignment of H68 peptides derived from different New World alphaviruses with a consensus NES sequence (NES), functional NES of cyclic-AMP (c-AMP)-dependent kinase inhibitor (PKI) (51) and supraphysiological NES (S1) (13). The sequence alignment was performed using ClustalW. Conservative hydrophobic amino acids in the NES are shown in red. Amino acids that are identical between different members of the New World alphaviruses are shaded in green. Asterisks indicate identical residues; colons indicate conserved substitutions; periods indicate semiconserved substitutions. WEEV, western equine encephalitis virus. (B) BHK-21 cells were coinfected with packaged replicons expressing VEEV capsid-GFP and 4×Tomato-3×NLS and treated with leptomycin B at 2 h postinfection as described in Materials and Methods. The images were acquired after 4 h of leptomycin B treatment on a Zeiss LSM510 confocal microscope. Scale bars, 20 μm. (C) (Top) Amino acid alignments of mutated H68-GFP fusions. Conserved hydrophobic amino acids are indicated in red, and introduced point mutations are shown in blue. (Bottom) Representative confocal images of cells expressing 4×Tomato-3×NLS and mutant proteins. Scale bars, 20 μm. (D) Box plot demonstrating the nuclear/cytoplasmic distribution of 4×Tomato-3×NLS when expressed alone and the distribution of the same protein when coexpressed with mutant peptide-GFP. A small but statistically significant increase in nuclear-reporter accumulation was detected for single-amino-acid mutants. The double mutant no longer affected nuclear accumulation of 4×Tomato-3×NLS. (E) Box plot demonstrating the nuclear/cytoplasmic distributions of H68-GFP and its mutants. The increase in nuclear accumulation of the mutant-peptide-GFP fusions was correlated with their reduced efficiencies in nuclear import inhibition. The P values were calculated using the Mann-Whitney test (n = 30 for all experiments).

FIG. 4.

H68 forms a tetrameric complex with CRM1, importin α1, and importin β. (A) Surface plasmon resonance sensograms of 6×His-H68-GFP (left) and 6×His-H68AA1-GFP, an NES mutant (right), upon exposure to the indicated concentrations of CRM1. RU, relative units. (B) Surface plasmon resonance response of 6×His-H68-GFP upon exposure to importin α1 (imp a) or β (imp b) or CRM1 alone or their combinations as indicated. The red dashed line indicates the increase in response of the tetrameric complex compared to the trimeric complex. The error bars indicate standard deviations. (C) Surface plasmon resonance response of 6×His-H68-GFP or its NLS (6xHis-H68AA2-GFP) or NES (6x-His-H68AA1-GFP) mutant upon exposure to importin α1 or β or CRM1 alone or their combinations.

FIG. 5.

H68-GFP, CRM1, and importin β are readily detectable in the NPC. (A) HeLa cells were transfected with the plasmid expressing 6×His-H68-GFP. At 24 h posttransfection, they were fixed and processed for immuno-EM using anti-GFP antibodies. The inset demonstrates 6×His-H68-GFP localization near the center of the NPC (boxed area). The cytoplasm is at the top of the image. (B) The distribution of gold particles in the midplane of the NPC and at nuclear and cytoplasmic sites was calculated using multiple images. (C) BHK-21 cells expressing H68-GFP from VEEV replicon- or mock-infected cells were stained with anti-CRM1 antibodies, followed by Alexa Fluor 555-labeled secondary antibodies. The CRM1 and H68-GFP are clearly colocalized at the nuclear rim and in small aggregates in the cytoplasm and nuclei. The box plot presents the medians of the fluorescence intensities of the CRM1-specific signals in the nuclear envelope and nucleus. (D) BHK-21 cells expressing H68-GFP from VEEV replicon- or mock-infected cells were stained with anti-importin β antibodies and Alexa Fluor 555-labeled secondary antibodies. The importin β and H68-GFP are clearly colocalized at the nuclear rim and in small aggregates in the cytoplasm, but not in the nuclei. The box plot presents the medians of the fluorescence intensities of importin β-specific signals in the nuclear envelope. The P values were calculated using the Mann-Whitney test (n = 8 for importin β; n = 6 for CRM1). Scale bars, 20 μm. Rim, nuclear rim; Nuc, nucleoplasm.

FIG. 6.

Mutations in the NES- and NLS-connecting linker affect the ability of H68 peptide to inhibit nuclear import. In all of the experiments presented, BHK-21 cells were coinfected with packaged VEEV replicons encoding mutant-peptide-GFP fusions and 4×Tomato-3×NLS. The cells were fixed at 4 h postinfection, and the distribution of fluorescent proteins was analyzed using a Leica SP1 confocal microscope. (A) (Top) Amino acid sequences of the wild-type H68 peptide and the deletion or substitution mutants. The positions of the deletions are indicated by dashed lines. (Bottom) The deletions of 3 or 5 aa in the linker peptide completely abolished nuclear import inhibition by mutant-peptide-GFP fusions. H68del54-56 accumulated more efficiently in the nuclei than H68-GFP (compare Fig. 1A and panel E). (B) (Top) Amino acid sequences of the wild-type H68 peptide and the designed mutants. Four or 7 amino acids of the linker were replaced with glycines, which are highlighted in blue. (Bottom) Substitution of 4 aa (H68G4) partially restored the nuclear import of 4×Tomato-3×NLS. Substitution of 7 amino acids (H68G7) completely abolished the ability of the mutant peptide (H68G7) to inhibit nuclear import. (C) (Top) Amino acid sequences of the wild-type H68 peptide and the insertion mutants. Five- and 10-aa-long linkers were inserted between the supra-NES and NLS. (Bottom) In the presence of both mutated GFP fusions, nuclear import of the 4×Tomato-3×NLS reporter was partially restored. The mutant peptide with the longer linker had less inhibitory effect on nuclear import. The enlarged insets demonstrate the accumulation of 4×Tomato-3×NLS and H68A5-GFP at the nuclear rim. (D) The box plot demonstrates the nuclear/cytoplasmic distribution of 4×Tomato-3×NLS expressed alone or in the presence of a mutant peptide-GFP. (E) The box plot demonstrates the nuclear/cytoplasmic distribution of H68-GFP and mutant proteins. The P values were calculated using the Mann-Whitney test (n = 30 for all experiments). Scale bars, 20 μm.

FIG. 7.

H68 peptide derived from nonpathogenic VEEV Pixuna is incapable of efficient inhibition of nuclear import. (A) Sequence alignment of H68 peptides derived from representative members of different VEEV subtypes. The green-shaded box indicates the most divergent sequence between different subtypes. NLS and NES are indicated by red letters. The asterisks indicate identical residues; the colons indicate conserved substitutions; the periods indicate semiconserved substitutions. (B) (Bottom) Representative confocal image of BHK-21 cells coexpressing 4×Tomato-3×NLS and H68Pix-GFP or H68E51-GFP. (Top) The mutated amino acid in the H68E51 peptide is highlighted in blue. The amino acids that differ between H68 and H68Pix are highlighted in turquoise. Scale bars, 20 μm. (C) Box plot demonstrating the nuclear/cytoplasmic distribution of 4×Tomato-3×NLS when expressed alone or in the presence of a mutant H68 peptide-GFP. The distributions of 4×Tomato-3×NLS in the presence of H68Pix and H68E51 were similar. (D) Box plot presenting the nuclear/cytoplasmic distributions of H68-GFP and H68Pix-GFP. H68Pix-GFP mostly accumulated in the nucleus. The distribution of H68E51 was not analyzed due to its high tendency to aggregate in the cytoplasm. The P values were calculated using the Mann-Whitney test (n = 30 for H68 and H68Pix; n = 29 for H68E51).

FIG. 8.

Model of H68-dependent inhibition of nuclear import. (Left) Binding of CRM1 and H68-GFP does not require RanGTP, and thus, the tetrameric complex H68-GFP/CRM1/importin α/β can form in the cytoplasm. Alternatively, the dimeric complex H68-GFP/CRM1 may preassemble on the cytoplasmic fibers by binding with Nup358, followed by binding with importin α/β. (Right) Next, the H68-GFP complex moves through NPC and blocks the central channel by binding to yet-unidentified nucleoporins. As a result, NPCs become inaccessible to receptor-mediated traffic but still support the diffusion of small molecules.