The C-Terminal Region of Lymphocytic Choriomeningitis Virus Nucleoprotein Contains Distinct and Segregable Functional Domains Involved in NP-Z Interaction and Counteraction of the Type I Interferon Response^▿

Cells and viruses.

Baby hamster kidney (BHK-21) cells (ATCC CCL-10), human embryonic kidney (293T) cells (ATCC CRL-11268), Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34), and African green monkey kidney epithelial (Vero) cells (ATCC CCL-81) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, l-glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were grown at 37°C in a 5% CO₂ atmosphere. For viral infections, cells were maintained in a mixture (1:1) of Opti-MEM reduced serum medium and the normal medium for each cell type. LCMV (Armstrong strain ARM53b) was produced by infecting BHK-21 cells (multiplicity of infection [MOI] = 0.1) and harvesting the supernatants on day 3 or 4 postinfection (55). LCMV titers were determined by immunofocus centers on Vero cells as described previously (2).

Plasmids.

Hemagglutinin (HA)- and FLAG-tagged versions of NP and Z of LCMV and LASV were obtained by cloning the corresponding open reading frames (ORFs) into modified versions of the pCAGGs multicloning site (MCS) plasmid (46). To that end, the pCAGGs MCS plasmid was modified by cloning the HA (YPYDVPDYA) and FLAG (DYKDDDDK) epitopes, allowing the generation of N- or C-terminus-tagged versions of proteins as described previously (45).

For the mammalian two-hybrid (M2H) system, the herpes simplex virus VP16 transactivating domain and the GAL4 DNA-binding domain ORFs were amplified by PCR and cloned into the pCAGGs MCS plasmid to generate pCAGGs VP16 and pCAGGs GAL4 plasmids, respectively, containing two flanking MCSs to facilitate the generation of N-terminal and C-terminal fusion proteins. HA-tagged versions of LCMV and LASV NP and Z ORFs were amplified by PCR and cloned into pCAGGs VP16 and pCAGGs GAL4 plasmids. Previously described N-terminal and C-terminal deletion mutants, as well as single amino acid substitutions of LCMV NP to alanine (38), were subcloned into the pCAGGs VP16 plasmid to generate NP-VP16 fusion proteins. Amino acids participating in the active site of the 3′-5′ exonuclease domain located within the C terminus of LCMV NP (25, 54) were replaced by alanine (D382A, E384A, D459A, H519A, and D522A) by site-directed mutagenesis (Stratagene), using a pGEM-T LCMV NP template, and subcloned into the pCAGGs VP16 plasmid. The pG5Luc reporter plasmid (Promega) was modified by fusing the green fluorescent protein (GFP) ORF to the N-terminal region of the firefly luciferase (FFL) coding sequence to generate pG5 GFP/Luc.

For the bimolecular fluorescence complementation (BiFC) system, N-terminal (EYN) and C-terminal (EYC) sequences of the yellow fluorescent protein (YFP) ORF were amplified by PCR from pCAGGs EYN-NS1 and pCAGGs EYC-NS1 and cloned into the pCAGGs MCS vector to generate pCAGGs EYN and pCAGGs EYC, respectively, containing two flanking MCSs to facilitate the generation of N- and C-terminal fusion proteins. LCMV NP and Z were subcloned into EYN and EYC pCAGGs to generate N- and C-terminal fusion proteins.

Primers for the generation of the described plasmids are available upon request. The coding regions of the generated constructs were verified by DNA sequencing, and protein expression was verified by Western blotting.

M2H assay.

293T cells (6.5 × 10⁵ per transfection) were cotransfected in suspension with 2 μg of the indicated pCAGGs VP16 and GAL4 expression plasmids, 1 μg of the reporter pG5 GFP/Luc plasmid, and 0.1 μg of the simian virus 40 (SV40)-Renilla luciferase expression vector pRL SV40 (Promega), to normalize transfection efficiencies, using 1 μg of Lipofectamine 2000 per μg of plasmid DNA. Transfected cells were seeded onto 12-well tissue culture plates. At 48 h posttransfection, protein-protein interaction was evaluated by GFP expression using a Zeiss fluorescence microscope. After imaging, cell lysates were prepared to determine luciferase activities and protein expression. Luciferase activities were determined using the dual-luciferase reporter assay (Promega) and a Lumicount luminometer (Packard). Reporter gene activation is expressed as fold induction over the negative controls (pCAGGs NP-VP16- and pCAGGs GAL4-transfected cells). The percentage of interaction of LCMV NP mutants was calculated on the basis of the wild-type NP-Z interaction. All M2H experiments were performed in triplicate. The mean and standard deviation were calculated using Microsoft Excel software. Protein expression was determined by Western blotting using the indicated antibodies.

BiFC assay.

MDCK cells (10⁵) were transfected in suspension with 1 μg of the indicated pCAGGs EYN and EYC plasmids using 1 μg of Lipofectamine 2000 per μg of plasmid DNA and seeded onto coverslips placed on 24-well tissue culture plates. At 24 h posttransfection, cells were cultured for 3 h at 30°C in a 5% CO₂ atmosphere to allow maturation of the YFP (27). Cells were then fixed with 100% methanol for 5 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked in 2.5% bovine serum albumin (BSA) in 1× phosphate-buffered saline (1× PBS) for 1 h at room temperature. Samples were incubated for 1 h at 37°C with a 1:500 dilution in 2.5% BSA of a monoclonal antibody against GFP (AB1218; AbCAM) that recognizes only the reconstituted form of the fluorescent protein. Subsequently, cells were washed with 1× PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI; Research Organics) and a 1:1,000 dilution of secondary goat anti-mouse immunoglobulin G (IgG)-Alexa Fluor 647 (Invitrogen) for 30 min at 37°C. Cells were washed with 1× PBS, and coverslips were mounted with Mowiol solution onto glass slides and analyzed using a 63× oil immersion objective and a Zeiss fluorescence microscope. Images were colored using Adobe Photoshop CS4 (version 11.0) software. Representative images of at least three independent transfections are shown.

Immunofluorescences.

Vero cells (10⁵ per transfection) on coverslips were cotransfected with 1.5 μg of the indicated LCMV and LASV NP and Z expression plasmids using 1 μg of Lipofectamine 2000 per μg of DNA. Empty pCAGGs MCS plasmid was included to maintain a constant amount of transfected plasmid DNA. At 48 h posttransfection, cells were fixed with 4% formaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 for 10 min at room temperature, followed by an overnight blocking step with 2.5% BSA in 1× PBS. After blocking, cells were incubated for 1 h at 37°C with the indicated primary antibodies: an anti-HA mouse monoclonal antibody (1:500; H9658; Sigma) and an anti-FLAG rabbit polyclonal serum (1:500; F7425; Sigma). After incubation, cells were washed with 1× PBS and incubated with DAPI (Research Organics), a 1:500 dilution of goat anti-mouse IgG-Alexa Fluor 488 (for the monoclonal antibody), and a 1:300 dilution of goat anti-rabbit IgG-rhodamine red secondary antibodies for 30 min at 37°C. Cells were then washed with 1× PBS, and coverslips were mounted with Mowiol solution onto glass slides and analyzed by florescence microscopy using a 63× oil immersion objective. For NP-Z colocalization during LCMV infection, subconfluent monolayers of Vero cells on glass coverslips were infected with LCMV (MOI = 0.1), and at 48 hpi, cells were fixed, permeabilized, and blocked as described above. After blocking, cells were incubated for 1 h at 37°C with a monoclonal antibody against LCMV NP (1.1.3) and a polyclonal rabbit serum against LCMV Z. After incubation, cells were washed 3 times with 1× PBS and incubated with DAPI, goat anti-mouse IgG-Alexa Fluor 488 (for NP monoclonal antibody), and goat anti-rabbit IgG-rhodamine red (for Z polyclonal antibody). Coverslips were mounted with Mowiol solution and analyzed as described above. Mock-infected cells were included as controls. Representative images of three independent transfections or infections are shown.

Protein gel electrophoresis and Western blotting analysis.

Proteins were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad) overnight at 4°C. After blocking for 1 h at room temperature with 10% dry milk in 1× PBS, membranes were incubated with anti-HA mouse monoclonal antibody (1:1,000; H9658; Sigma), anti-FLAG polyclonal antibodies (1:1,000; F7425; Sigma), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) monoclonal antibody (1:1,000; AB9484; AbCAM), anti-VP16 polyclonal antibody (1:5,000; V4388; Sigma), and anti-GFP polyclonal antibody (1:500; Santa Cruz, SC8334). Incubation with antibodies was done overnight at 4°C. After overnight incubation, membranes were washed three times with 1× PBS containing 0.1% Tween 20 and incubated with a 1:2,000 dilution of the respective secondary horseradish peroxidase-conjugated anti-mouse and/or anti-rabbit Ig antibodies (GE Healthcare United Kingdom) for 1 h at room temperature. After 3 washes with 1× PBS containing 0.1% Tween 20, proteins were detected using a chemiluminescence kit and autoradiography films from Denville Scientific Inc. Protein band intensities were quantified using ImageJ software (NIH) and are represented as a percentage of wild-type NP expression levels.

Generation and isolation of VLPs.

To generate virus-like particles (VLPs), 293T cells were cotransfected in suspension (6-well plate format, 10⁶ cells/well) with 2.5 μg of each indicated plasmid using 1 μg of Lipofectamine 2000 per μg of DNA. Empty pCAGGs MCS plasmid was used to keep a constant amount of transfected plasmid DNA. At 72 h posttransfection, cells and tissue culture supernatants were collected. Supernatants were clarified at 10,000 rpm for 30 min and then layered on top of a 20% sucrose cushion and centrifuged at 35,000 rpm on an SW-41 rotor for 2.5 h. VLP-containing pellets were resuspended in 100 μl of 1× PBS, and 20 μl was analyzed by Western blotting. Cell pellets were lysed with 400 μl of lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 100 mM NaCl, 1% NP-40, complete cocktail of protease inhibitors; Roche) for 30 min on ice. Cell lysates were clarified by centrifugation at 14,000 rpm for 30 min at 4°C. Twenty microliters (5% of total cell lysates) of each sample was analyzed (input) by Western blotting.

Assessment of LCMV NP-Z interaction.

Z is the driving force of arenavirus budding and in the absence of other viral proteins mediates formation of VLPs (50, 51, 61, 63). In addition, the Z proteins of LASV (15), Mopeia virus (MOPV) (59), and TCRV (34) have been shown to mediate incorporation of NP into VLPs. To characterize the NP-Z interaction for the prototypic arenavirus LCMV, we used two complementary approaches: the M2H system and the BiFC assay. For the M2H system (Fig. 1), we cotransfected 293T cells with pCAGGs plasmids expressing NP-VP16 and GAL4-Z (Fig. 1A), together with the reporter plasmid pG5 GFP/Luc and with pRL SV40 to normalize transfection efficiencies. In this system, the LCMV NP-Z interaction would allow GAL4 binding to the reporter plasmid, while the VP16 transactivation domain would recruit the machinery necessary for reporter gene expression (GFP fused to FFL). We used pCAGGs VP16 and GAL4 plasmids as negative controls, alone or in combination with LCMV NP- and Z-tagged expression plasmids, to demonstrate the specificity of the LCMV NP-Z interaction. We detected the LCMV NP-Z interaction using both GFP (Fig. 1B) and FFL (Fig. 1C) reporter gene expression. Reporter gene expression was not detected when either GAL4 or VP16 was used alone or in combination with LCMV NP or Z protein, demonstrating the specificity of the interaction.

To confirm and further characterize this LCMV NP-Z interaction, we used the BiFC assay (Fig. 2). For this, we fused the N terminus (residues 1 to 155; EYN) and C terminus (residues 156 to 239; EYC) of YFP to either the NP or Z protein from LCMV (Fig. 2A). The NP-Z interaction should restore the YFP ternary structure and associated fluorescence properties. We confirmed that all fusion constructs were expressed, as determined by Western blotting using an anti-GFP polyclonal antibody that detects the N- and C-terminal domains of both GFP and YFP (Fig. 2B). The differences in the apparent molecular mass of LCMV Z observed depending on whether EYN or EYC was fused to the N or C terminus were likely due to differences in the links between the N- or C-terminal EYN or EYC expression plasmids. In the case of construct Z-EYC, we observed an extra band with faster mobility that likely reflects a degradation product. We then cotransfected MDCK cells with the indicated plasmid combinations (Fig. 2C). In cells cotransfected with NP-EYN and EYC-Z and with NP-EYC and EYN-Z, we observed YFP expression indicative of LCMV NP-Z interaction (Fig. 2C, YFP). To confirm the reconstitution of YFP, we used a monoclonal antibody that recognizes only the reconstituted ternary fluorescent structure of YFP (Fig. 2C, α-GFP). Consistent with our previous data using the M2H assay (Fig. 1), LCMV NP-Z interaction was detected when LCMV NP was fused to the N terminus of EYN or EYC and LCMV Z was fused to the C terminus of EYN or EYC. In contrast, we did not detect LCMV NP-Z interaction in the other combinations, which further supported the specificity of this NP-Z interaction.

Subcellular colocalization of LCMV NP and LCMV Z.

Results from BiFC showed that reconstituted YFP fluorescence mediated by NP-Z interactions was restricted to the cell cytoplasm (Fig. 2C). To confirm that NP-Z interaction occurred, as predicted, in the cell cytoplasm, we cotransfected Vero cells with LCMV NP-HA and LCMV Z-FLAG pCAGGs-based expression plasmids alone or in combination (Fig. 3). Empty pCAGGs plasmid was added in the single transfections to normalize the amount of total transfected DNA. C-terminal epitope tagging of both LCMV NP and Z proteins has previously been shown to not affect the function of these proteins (40, 50). When expressed individually, both LCMV NP and Z were distributed diffusely throughout the cytoplasm, but Z also exhibited clear plasma membrane localization (Fig. 3A). Interestingly, in cells expressing both NP and Z, we observed the formation of cytoplasm aggregates where both NP and Z colocalized. We next examined whether the formation of these NP- and Z-containing inclusion bodies also occurred in LCMV-infected cells. To that end, we infected Vero cells with LCMV (MOI = 0.1 PFU/cell), and at 48 hpi we examined the cells by double immunofluorescence staining using an anti-NP mouse monoclonal antibody and an anti-Z rabbit polyclonal serum (Fig. 3B). We observed colocalization of NP and Z within cytoplasmic inclusion bodies.

Mapping regions within LCMV NP required for its interaction with LCMV Z.

To identify the LCMV NP domains involved in NP-Z interaction, we used a series of previously described N-terminal (Fig. 4) and C-terminal (Fig. 5) LCMV NP deletion mutants (38). We fused these mutants to VP16 and used them in the M2H assay to assess their ability to interact with Z.

Deletion of the N-terminal first 300 amino acids of LCMV NP (Fig. 4A) did not significantly affect the ability of LCMV NP to interact with LCMV Z (Fig. 4B). However, further N-terminal deletions (e.g., ΔN350) disrupted the ability of LCMV NP to interact with LCMV Z (Fig. 4B). We observed differences in expression levels among the NP N-terminal deletion mutants, but all were detected by Western blotting using an anti-VP16 polyclonal antibody (Fig. 4C). It should be noted that mutant ΔN350 was impaired in its ability to interact with Z but ΔN350 was expressed at levels similar to NP wild type. Likewise, we also examined the ability of LCMV NP C-terminal deletion mutants to interact with LCMV Z (Fig. 5A). Deletion of the last 5 amino acids did not affect the ability of LCMV NP to interact with LCMV Z (Fig. 5B). However, deletion of more than 5 amino acids in the C terminus of LCMV NP (e.g., ΔC10) significantly affected its interaction with LCMV Z (Fig. 5B). This lack of interaction was not due to significant differences in protein expression since all LCMV NP C-terminal deletion mutants were expressed similarly to wild-type LCMV NP (Fig. 5C), a finding consistent with previously published data (47). These results indicated that the C-terminal region (amino acids 300 to 553) of LCMV NP is required and sufficient for its interaction with LCMV Z.

Examining the relationship between anti-IFN activity of LCMV NP and its ability to interact with LCMV Z.

We have previously shown that the C-terminal region of LCMV NP (amino acids 370 to 553) is required for its anti-IFN activity (38). Since the C-terminal domain of NP is also involved in its interaction with LMCV Z, we examined whether both NP activities could be separated in the primary structure of LCMV NP. Support for the feasibility of segregating these two NP activities was provided by the observation that TCRV NP lacks the ability, compared to other arenavirus NPs, to counteract the IFN response (39) and the rescue of a viable recombinant LCMV carrying a D382A mutation in NP (rLCMVD382A) that lacks the ability to counteract the IFN response (38), but both TCRV NP-Z and LCMV NP-Z interactions are required for production of infectious TCRV and LCMV, respectively. To experimentally test this hypothesis, we assessed in the M2H assay the ability of LCMV NPs with mutations in the DIEGR motif (D382A, G385A, and R386A) (38) and the recently described 3′-5′ exonuclease motif (D382A, E384A, D459A, H517A, and D552A) (25, 54), all of which were previously shown to play a critical role in the IFN-counteracting activity of NP, to interact with Z (Fig. 6). As a control, we used the LCMV NP I383A mutant that we previously showed retains its anti-IFN function (38). All of the NP mutants interacted with LCMV Z to levels comparable to those for the LCMV NP wild type (Fig. 6A) and were expressed at levels comparable to those for the LCMV NP wild type (Fig. 6B). These results revealed that residues playing key roles in the anti-IFN activity of NP are not critical for NP-Z interaction.

Assessing homotypic and heterotypic NP-Z interactions between LCMV and LASV.

Coinfection of the same cells with two phenotypically different arenavirus strains (or variants) results in the generation of virus progeny containing reassortant viruses where S and L genome segments are exchanged. This genetic approach has been widely used to define the function of viral gene products in arenavirus pathogenesis. However, to produce a viable reassortant arenavirus, proteins encoded by the L and S genome segments of the two viral strains coinfecting the same cell should permit the virus protein-protein interactions required for production and multiplication of infectious viral progeny. Arenavirus NP and Z proteins are encoded by the S and L genome segments, respectively, and need to interact to allow incorporation of vRNPs into Z-mediated budding viruses. Generation of reassortant viruses between OWAs has been previously described (36). To assess heterotypic NP-Z interactions between LASV and LCMV, we first confirmed the NP-Z interaction in LASV using the M2H assay system (Fig. 7). LCMV NP interacted with LASV Z, but LASV NP did not interact with LCMV Z, suggesting a unidirectional interaction between NP and Z proteins from LASV and LCMV.

Z has been documented to be the driving force of arenavirus budding (50, 51, 61, 62). Accordingly, NP-Z interaction is predicted to play a critical role in packaging vRNPs into Z-mediated budding viral particles. Therefore, we predicted that LASV NP would not be incorporated into LCMV Z-induced VLPs. To examine NP incorporation into Z-mediated production of VLPs, we used a previously described VLP assay (33) (Fig. 8). For this, we generated HA-tagged versions of LCMV and LASV NP and FLAG-tagged versions of LCMV and LASV Z that were cotransfected alone or in combination into 293T cells. Seventy-two hours after transfection, tissue culture supernatants and cell lysates were collected. Supernatants were pelleted through a 20% sucrose cushion, and VLP-containing pellets were analyzed by Western blotting. We observed incorporation of both LCMV and LASV NPs into LASV Z-induced VLPs. In contrast, LCMV NP but not LASV NP was incorporated into LCMV Z-mediated VLPs. As expected, expression of LCMV or LASV NPs alone was not detected in the tissue culture supernatants, demonstrating specific NP incorporation in Z-induced VLPs. All tagged proteins were expressed to similar levels, as determined by Western blotting of cell lysates (Fig. 8, Input, α-HA and α-FLAG).

Colocalization of LCMV and LASV NP-Z.

Since the observed unidirectional NP-Z heterotypic interaction was unexpected, we attempted to confirm these results by assessing colocalization of LCMV and LASV NP and Z proteins in plasmid-transfected cells (Fig. 9). We cotransfected Vero cells with HA-tagged LCMV and LASV NP with FLAG-tagged LCMV and LASV Z alone or in different combinations. LCMV NP (Fig. 9A) and LASV NP (Fig. 9B) showed a diffuse distribution in the cytoplasm of transfected cells, whereas LCMV Z (Fig. 9C) and LASV Z (Fig. 9D) were distributed throughout the cytoplasm but were also present in the cell membrane. Cotransfection of NP and Z from LCMV (Fig. 9E) or from LASV (Fig. 9H) resulted in the formation in the cell cytoplasm of inclusion bodies that contained both NP and Z proteins. We observed similar inclusion bodies when we replaced LCMV Z by LASV Z in the presence of LCMV NP (Fig. 9F). On the other hand, coexpression of LASV NP and LCMV Z resulted in minimal colocalization of both viral proteins and the absence of the large inclusion bodies (Fig. 9G), confirming our results with the M2H and VLP assays.