Importin-α7 Is Involved in the Formation of Ebola Virus Inclusion Bodies but Is Not Essential for Pathogenicity in Mice

Abstract

Ebola virus (EBOV) protein 24 antagonizes the host interferon (IFN) response by hijacking select nuclear importin-α isoforms. Thereby, it blocks STAT1-mediated IFN-α/β and IFN-γ synthesis. However, owing to the lack of importin-α knockout animal models in the past, their role in EBOV pathogenesis remained largely unknown. Here, we demonstrate that importin-α7 is involved in the formation of EBOV inclusion bodies and replication. However, deletion of the gene encoding importin-α7 was not sufficient to increase survival rates among mice infected with EBOV.

Ebola virus (EBOV; species Zaire ebolavirus, genus Ebolavirus, family Filoviridae) can cause severe Ebola hemorrhagic fever (EHF) in humans and nonhuman primates. Currently, we are experiencing the largest outbreak of EBOV infection, which started in late 2013 and has already affected multiple countries in West Africa. To date, approximately 26 000 cases with >11 000 deaths have been reported [1]. Thus, it is mandatory to understand the molecular determinants involved in EBOV pathogenicity. Several viral determinants are known to contribute to EBOV pathogenicity, such as the glycoprotein (GP), viral protein 24 (VP24), and VP35 [2–8]. However, the role of cellular factors involved in EBOV pathogenicity remains largely unknown [9].

Importin-α isoforms belong to the best-described cellular interaction partners of EBOV VP24, which is a key viral pathogenicity determinant. VP24 interacts with and sequesters human importin-α5, importin-α6, and importin-α7 isoforms (also known as KPNA1, KPNA5, and KPNA6, respectively) in the cytoplasm [10, 11], thus inhibiting importin-α–mediated interaction and transport of tyrosine-phosphorylated STAT1 (PY-STAT1) into the nucleus. This blockage of interferon α/β (IFN-α/β)– and IFN-γ–induced nuclear accumulation of PY-STAT1 leads to the inhibition of IFN-induced gene expression. The direct competition between VP24 and PY-STAT1 for importin-α binding allows VP24 to promote viral escape from the host innate immune response. This inhibition of the cell-intrinsic innate immune response renders EBOV-infected cells nonresponsive to IFN-α/β or IFN-γ treatment [12]. Interestingly, this VP24/importin-α interaction was observed with several members of the genus Ebolavirus [10]. Marburg virus (MARV; species Lake Victoria marburgvirus, genus Marburgvirus, family Filoviridae), on the other hand, has evolved mechanisms to counteract IFN signaling that differ from those for EBOV [13]. MARV infection does not detectably block STAT1 nuclear import but instead inhibits phosphorylation of STAT1 and STAT2. Surprisingly, this function was not associated with MARV VP24 but with the MARV matrix protein VP40. It was shown that expression of MARV VP40 was sufficient to block IFN signaling, probably by targeting the Janus kinase family kinase 1 that mediates STAT1 and STAT2 phosphorylation [13]. Thus, it is believed that suppression of the IFN-α/β– and IFN-γ–mediated antiviral response allows sustained filovirus replication, which further promotes viral disease progression and severity [14]. However, owing to the absence of animal models lacking certain importin-α isoforms, the contribution of these cellular host factors to EBOV pathogenicity remains unknown.

Recently, importin-α7 knockout (α7^−/−) mice became available [15], which allowed us to examine the involvement of importin-α7 in EBOV replication and pathogenesis. We used the mouse-adapted EBOV (MA-EBOV) Mayinga strain [16], which is closely related to the EBOV Makona strain currently circulating in West Africa.

MATERIALS AND METHODS

Biosafety and Ethics Statement

All work with infectious EBOV was performed in the Maximum Containment Laboratory of the Rocky Mountain Laboratories (RML) according to standard operation protocols (SOP) approved by the institutional biosafety committee. All animal work was conducted in compliance with the guidelines of and under a protocol approved by the RML Institutional Animal Care and Use Committee. The RML facility is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International and has an approved Office of Laboratory Animal Welfare assurance number.

Animal Experiments

Generation of importin-α7 knockout (α7^−/−) mice in the C57BL/6 genetic background was described before [15]. Mice were bred at the animal facilities of the Max Delbrück Center for Molecular Medicine, in Berlin, and the Heinrich Pette Institute, Leibniz Institute for Experimental Virology, in Hamburg, Germany. To assess MA-EBOV pathogenicity in wild type (WT) and α7^−/− mice, groups of 16–25 animals were infected intraperitoneally with 1×, 10×, and 100× mouse lethal dose that can produce death in 50% of the animals (MLD₅₀). Weight loss and survival were monitored for 14 and 28 days after infection, respectively, as described before [4]. To assess virus replication in mice, groups of 4–6 animals were euthanized on day 4 after infection, and spleen, liver, and blood samples were collected for virologic and pathologic analyses. Virus titers were determined as 50% tissue culture infective doses on Vero E6 cells as described before [4].

Immunohistochemical Analyses

To assess viral pathology in the infected organs, the spleens from 4–6 infected WT and α7^−/− mice were fixed and inactivated in 10% formalin according to SOP. Subsequently, samples were removed from high containment for immunohistochemical staining. Briefly, formalin-fixed tissues were embedded in paraffin and thin sections were stained using a monoclonal anti-VP40 antibody as described before [9]. Tissues were counterstained with hematoxylin, as previously described [17]. Tissue sections were then analyzed in a blinded fashion by the pathologists at the Heinrich Pette Institute, Leibniz Institute for Experimental Virology.

Transmission Electron Microscopy

Tissue samples were fixed and inactivated with 2.5% glutaraldehyde according to approved SOP. Subsequently, samples were removed from high containment, washed in phosphate-buffered saline, and postfixed with 1% osmium tetroxide. For better structure preservation, specimens were treated with 1% gallic acid after osmification, as described elsewhere [18]. Thereafter, samples were stained with 1% uranyl acetate in double-distilled water, dehydrated in graded ethanol, and embedded in Epon. Ultrathin sections were prepared on a Leica Ultracut UCT microtome. The images were acquired with a FEI Eagle 4 k camera on a Tecnai G20 microscope operated at 80 kV.

Statistical Analysis

The statistical significance of obtained data was calculated with GraphPad Prism 5 (v5.03), using the unpaired, 2-tailed Student t test or, when all values in one group were identical (eg, 0), the 1-sample, 2-tailed Student t test. Statistical significance was defined as a P value of <.05.

RESULTS

Importin-α7 Promotes MA-EBOV Replication in Mice

To assess whether importin-α7 plays a role in MA-EBOV replication in mice, we infected WT and α7^−/− mice [15] with 1×, 10×, and 100× MLD₅₀ (Figure 1A–C). Control mice were mock infected with Dulbecco's modified Eagle's medium. On day 4 after infection, we collected spleen, liver, and blood specimens from infected or uninfected control groups and determined virus titers. None of the mock-infected control groups were infected (data not shown). At a low infection dose of 1× MLD₅₀, none of the MA-EBOV–infected α7^−/− mice showed virus titers in spleen and liver, in contrast to WT mice (Figure 1A). Virus titers were significantly reduced in the blood of α7^−/− mice infected with 1× MLD₅₀, compared with WT mice (Figure 1A). Upon infection with 10× MLD₅₀, virus titers were generally reduced in α7^−/− mice, compared with WT mice, albeit not significantly (Figure 1B). At the highest infection dose (100× MLD₅₀), no differences in organ and blood titers were observed among the infected groups (Figure 1C). Thus, importin-α7 seems to be involved in efficient MA-EBOV replication in mice.

Figure 1.

Ebola virus replication and visualization of inclusion bodies in wild-type (WT) and importin-α7–deficient (α7^−/−) mice. A–C, WT and α7^−/− mice were intraperitoneally infected with 1× mouse lethal dose that can produce death in 50% of the animals (MLD₅₀; A; n = 4–6), 10× MLD₅₀ (B; n = 5–6), or 100× MLD₅₀ (C; n = 5–6) of the mouse-adapted Ebola virus (MA-EBOV) strain as described before [4]. Virus titers were measured by determining the 50% tissue culture infective dose (TCID₅₀) on day 4 after infection in the spleen, liver, and blood specimens from infected mice [4]. No virus was detected in control groups, which were mock treated with Dulbecco's modified Eagle's medium (data not shown). Each data point represents an individual organ sample per animal. Additionally, means of the logarithmic virus titers are shown (n = 4–6; *P < .05). D, Transmission electron microscopy of spleen sections for WT mice (upper panel) and α7^−/− mice (lower panel) infected with 10× MLD₅₀ (n = 4) of the MA-EBOV strain. The left panel shows an overview of the infected cell, the middle panel shows magnification of inclusion bodies, and the right panel shows magnifications of virus particles. Inclusion bodies are indicated with arrows. Abbreviation: N, nucleus.

Importin-α7 Plays a Role in the Formation of Viral Inclusion Bodies

We then analyzed the effect of importin-α7 on organ pathology in MA-EBOV–infected mice. Therefore, thin sections of spleens, as one of the major site of EBOV replication [19, 20], collected from experimental groups on day 4 after infection were stained immunohistochemically for the presence of VP40, an abundant EBOV protein [19]. The presence of viral antigen was predominantly detected in macrophages (Supplementary Figure 1). The number of viral antigen–positive cells was generally reduced in α7^−/− mice, compared with WT mice that were infected with 10 or even 100× MLD₅₀. Interestingly, dense granular structures were observed at higher magnification in macrophages of WT mice but not in macrophages in infected α7^−/− mice. These granular structures resembled inclusion bodies found in tissues infected with EBOV or MARV [21, 22] and have been identified as a site of EBOV replication [23]. The reduced formation of these granular structures, most likely representing inclusion bodies, further correlates with the generally reduced presence of viral antigen in splenic macrophages of MA-EBOV–infected α7^−/− mice.

Next, we assessed whether the granular structures present in WT splenic macrophages represent EBOV inclusion bodies [23]. Therefore, we performed transmission electron microscopy of murine spleen sections infected with 10× MLD₅₀ of MA-EBOV on day 4 after infection (Figure 1D). After analyzing large areas of spleen sections, we could visualize the symmetrical formation of these structures, indicating inclusion bodies in WT tissues (Figure 1D). In contrast, such symmetrical structures were rarely detected in tissues of α7^−/− mice, and instead, mostly large and homogenous dense structures were present (Figure 1D). In both WT and α7^−/− spleen tissues, viral particles were detected (Figure 1D). Thus, our findings show that importin-α7 is involved in efficient formation of inclusion bodies.

Deletion of the Gene Encoding Importin-α7 Does Not Increase Survival in Mice

Finally, we addressed whether importin-α7 plays a role in MA-EBOV pathogenicity. Therefore, we infected WT and α7^−/− mice with 1×, 10×, or 100× MLD₅₀ of MA-EBOV and monitored weight loss and survival for 14 and 28 days after infection, respectively. With all infection doses tested, weight loss in α7^−/− mice was slightly less than that in WT mice, although the difference was not statistically significant (Figure 2). Survival rates did not differ between WT and α7^−/− mice in any of the infected groups independently of the infection dose. These data show that importin-α7 is not essential for MA-EBOV pathogenicity in mice.

Figure 2.

Pathogenicity of Ebola virus in wild-type (WT) and importin-α7–deficient (α7^−/−) mice. A–C, WT mice (filled square) and α7^−/− mice (open square) were intraperitoneally infected with 1× mouse lethal dose that can produce death in 50% of the animals (MLD₅₀; A; n = 13–19), 10× MLD₅₀ (B; n = 13–21), or 100× MLD₅₀ (C; n = 10) of the MA-EBOV strain as described before [4]. Animals were monitored daily for weight loss (14 days) and survival (28 days). Control groups that were mock infected with Dulbecco's modified Eagle's medium did not show any weight loss (data not shown) or pathological alterations between WT and α7^−/− mice (Supplementary Figure 1), and all animals survived (gray line). Data are mean values ± SD for weight loss.

DISCUSSION

Here, we show that importin-α7, previously proposed to function as a potential EBOV pathogenicity factor [10, 11], is not essential for MA-EBOV pathogenicity in mice. However, importin-α7 seems to play a role in the formation of viral inclusion bodies, which are a known site of EBOV replication. Macrophages play a pivotal role in viral dissemination. EBOV spreads from the initial infection site to regional lymph nodes via monocytes, macrophages, and dendritic cells and probably through the lymphatic system. The virus can then further spread via the blood to the liver and spleen [20]. Thus, the impaired inclusion body formation, particularly in macrophages, might be reflected in significantly reduced blood titers early in infection. Moreover, extensive virus replication was still observed later in infection, albeit with reduced formation of inclusion bodies. Thus, one might speculate whether additional sites of replication exist within the host cell that do not involve inclusion bodies. Moreover, it would be interesting to address in future studies whether importin-α7 might also function as a cytoplasmic chaperone, as has been reported for nonclassical importins [24], and whether this function might be used by viruses for the formation of replication complexes, similar to inclusion bodies.

In contrast to EBOV infection, importin-α7, but not importin-α4 or -α5, was described to be a major determinant of influenza virus pathogenicity in mice [15]. Therefore, one might speculate whether importin-α isoforms other than importin-α7 might contribute to EBOV pathogenicity. Interestingly, VP24 has previously been shown to interact with importin-α5 and importin-α6 [10, 11]. However, since there is no homologue of human importin-α6 in mice and human importin-α6 is only expressed in testes [25], the focus should be on importin-α5, which is one of the major transport proteins of STAT-1 [26]. Future studies should address the impact of a VP24/importin-α5 interaction on EBOV pathogenicity by using the corresponding knockout mice.

Recently, it was shown that WT mouse strains generally used in experimental studies, such as BALB/c or C57BL/6 (used here), do not represent the best animal disease model for Ebola infection. In these mice, hallmark symptoms of the human disease, such as coagulation abnormalities, are not present. However, certain recombinant, inbred (CC-RIX) mice from the Collaborative Cross panel have been shown to exhibit a disease similar to human EHF [9] and might serve as a more suitable small-animal disease model to study the role of individual importin-α isoforms in EBOV pathogenesis.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.