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Journal of Virology, March 2008, p. 2699-2704, Vol. 82, No. 6
0022-538X/08/$08.00+0     doi:10.1128/JVI.02344-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Inhibition of IRF-3 Activation by VP35 Is Critical for the High Level of Virulence of Ebola Virus{triangledown}

Amy L. Hartman,1 Brian H. Bird,3 Jonathan S. Towner,1 Zoi-Anna Antoniadou,2 Sherif R. Zaki,2 and Stuart T. Nichol1*

Special Pathogens Branch,1 Infectious Disease Pathology Branch, Division of Viral and Rickettsial Diseases, National Center for Zoonotic, Vector-Borne, and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, 30329,2 University of California, Davis, School of Veterinary Medicine, Davis, California 956163

Received 30 October 2007/ Accepted 21 December 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Zaire ebolavirus causes a rapidly progressing hemorrhagic disease with high mortality. Identification of the viral virulence factors that contribute to the severity of disease induced by Ebola virus is critical for the design of therapeutics and vaccines against the disease. Given the rapidity of disease progression, virus interaction with the innate immune system early in the course of infection likely plays an important role in determining the outcome of the disease. The Ebola virus VP35 protein inhibits the activation of IRF-3, a critical transcription factor for the induction of early antiviral immunity. Previous studies revealed that a single amino acid change (R312A) in VP35 renders the protein unable to inhibit IRF-3 activation. A reverse-genetics-generated, mouse-adapted, recombinant Ebola virus that encodes the R312A mutation in VP35 was produced. We found that relative to the case for wild-type virus containing the authentic VP35 sequence, this single amino acid change in VP35 renders the virus completely attenuated in mice. Given that these viruses differ by only a single amino acid in the IRF-3 inhibitory domain of VP35, the level of alteration of virulence is remarkable and highlights the importance of VP35 for the pathogenesis of Ebola virus.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Ebola and Marburg viruses are members of the Filoviridae family and are responsible for periodic outbreaks of severe hemorrhagic fever characterized by rapid disease progression and mortality approaching 80 to 90% (26). Identification of the viral virulence factors contributing to such severe disease and high mortality is critical for the design of therapeutics and vaccines against the disease.

Fatal human cases of Ebola hemorrhagic fever are exemplified by very high viral titers in the blood, liver, and spleen as well as profound immunosuppression (2, 20, 21, 29). Virus-specific antibody responses are found rarely or at low levels in fatally infected humans. In contrast, a small number of survivors have lower viral titers as well as detectable virus-specific antibody responses (29). The factors that contribute to death or survival are not well understood, but given the rapid viral replication and disease progression, the clinical outcome is likely determined very early after infection.

Considering that Ebola hemorrhagic fever progresses rapidly and that death or survival is determined within a week to 10 days after the onset of symptoms (26), the innate immune system likely plays an important role in the control, or lack thereof, of Ebola virus replication at these early time points (reviewed in reference 23). Host cells have the innate antiviral immune system as the first line of defense against invading viruses. The innate immune system also serves as the bridge to development of effective adaptive immune responses. Cells detect viral infection through one of two mechanisms: the cytosolic sensor proteins RIG-I and MDA-5 or the Toll-like receptor pathway (reviewed in references 10, 15, and 18). Both mechanisms result in the phosphorylation and nuclear translocation of IRF-3 and IRF-7, two transcription factors that induce the expression of alpha/beta interferon (IFN-{alpha}/β) and other early antiviral proteins and chemokines, such as ISG15, ISG54, ISG56, inducible nitric oxide synthase, MDA-5, interleukin-15, and RANTES. Through a positive-feedback loop, IFN-{alpha}/β induces the expression of a large number of antiviral and immunomodulatory proteins. The end result is that the infected cell limits viral replication, induces an antiviral state in neighboring cells, and activates macrophages and dendritic cells to help stimulate the adaptive immune system. Because of the important role IRF-3 plays in the early induction of antiviral gene expression, many viruses have developed various mechanisms to inhibit, either directly or indirectly, the activation of IRF-3 (1, 27, 31, 32).

Ebola virus has several mechanisms to inhibit different aspects of the innate immune system. The VP35 protein of Ebola virus has an essential role in the ribonucleoprotein complex during virus transcription/replication (24) and also has at least three mechanisms to inhibit the innate antiviral immune system. VP35 inhibits the activation of both IRF-3 and protein kinase R and serves as an RNA-silencing suppressor (RSS) to inhibit RNA interference (3, 4, 9, 14). Besides VP35, the VP24 protein, a minor matrix protein, inhibits signaling from the IFN-{alpha}/β and -{gamma} receptors by preventing nuclear translocation of phosphorylated STAT1 (25). The relative importance of the different mechanisms by which Ebola virus evades the innate immune system is unclear, but the possession of several immunosuppressive components illustrates the critical nature of this function for Ebola virus propagation and survival.

As IRF-3 plays an early role in the induction of antiviral gene expression, the inhibition of IRF-3 activation by VP35 may make a major contribution to the virulence of Ebola virus via inhibition of innate immunity. We recently mapped an IRF-3 inhibitory domain in the C terminus of VP35 (17). Within this domain, the mutation of arginine at position 312 to alanine (R312A) significantly reduced the ability of VP35 to inhibit IRF-3 activation. This mutation also appears to disrupt the RNA binding capacity of VP35 as well as RSS function, but it does not affect the inhibition of protein kinase R activation (7, 9, 14). A recombinant Ebola virus with the R312A mutation in VP35 (recEbo-VP35/R312A) is unable to efficiently inhibit activation of IRF-3 (16). We have also recently shown that infection of liver cells with the recEbo-VP35/R312A virus results in a dramatic increase in IRF-3-dependent gene expression compared to cells infected with the wild-type (WT) virus (A. L. Hartman et al., submitted for publication). IFN, chemokine, and antiviral gene expression pathways were all significantly activated in cells infected with recEbo-VP35/R312A virus relative to cells infected with the WT virus. Therefore, inhibition of IRF-3 activation by VP35 appears to be critically important for controlling host antiviral gene expression in vitro. Based on these findings, our hypothesis is that a virus containing the R312A mutation in VP35 would be greatly reduced in virulence relative to a virus identical to the mutated one in all regards except for the possession of WT VP35.

It was previously shown that WT Zaire ebolavirus is not pathogenic for adult mice, and a lethal disease model required adaptation of the virus by serial passage in progressively older mice (5). The disease induced in mice recapitulates many aspects of Ebola virus-induced disease seen in humans and nonhuman primates (NHPs), including similar levels of virus replication, target cells and organs, and clinical laboratory values (6). The main difference between the mouse model and the NHP model is the lack of coagulation abnormalities as seen in NHPs. Despite this difference, the mouse model of Ebola virus infection has been a useful small-animal model for understanding the basic features of the disease and immune response to this virus (11-13, 22).

In this study, we used the mouse model to evaluate the role of VP35-mediated IRF-3 inhibition in the pathogenesis of Ebola virus infection. Adaptation of the virus to mice (5) resulted in nine nucleotide changes relative to the nucleotides of WT Zaire ebolavirus (8). Of these nucleotide changes, two were found to be the minimal requirements for inducing severe illness and death in mice (8). These two nucleotide substitutions resulted in amino acid changes within the NP gene (S72G) and the VP24 gene (T50I). Using a minimally mouse-adapted (MA) recombinant Ebola virus containing the R312A mutation in VP35, we found that the single R312A amino acid change in VP35 renders the virus completely attenuated compared to the WT virus. Given that these viruses differ by a single amino acid, the level of alteration of virulence is remarkable and highlights the importance of VP35 in the pathogenesis of Ebola virus.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Generation and rescue of MA Ebola virus. All work with infectious virus and infected animals was performed within the biosafety level 4 laboratory at the Centers for Disease Control and Prevention and followed strict biosafety guidelines outlined in reference 31a. Generation of the parental full-length infectious cDNA clone of the Ebola virus genome has been described previously (28). This parental cDNA clone was modified (16) to encode two specific amino acid changes associated with severe illness in mice (8). These changes were Ser 72 to Gly within NP and Thr 50 to Iso within VP24, and the resulting clone was termed the parental MA2 clone. In the new parental MA2 clone, the R312A amino acid change was introduced into VP35 (16, 17). Each clone was sequenced entirely to ensure that no additional unintentional mutations were introduced. Virus was rescued from both clones as described previously (28), and the resulting viruses were named recEboMA2-VP35/WT and recEboMA2-VP35/R312A. Stocks of both rescued viruses as well as the authentic (non-reverse-genetics-generated) MA virus (5) were grown in Vero E6 cells and titrated by the immunoplaque assay (29). Stocks of recEboMA2-VP35/WT and recEboMA2-VP35/R312A viruses were sequenced completely prior to animal infections.

Animal infections. All animal work was performed under the approval of the CDC Institutional Animal Care and Use Committee. Female BALB/c mice (5 to 6 weeks old) were obtained from the Jackson Laboratory or Charles River Laboratories. All mice were housed in groups of five in microisolator cages during the experiments. Mice were allowed to acclimatize for 7 days prior to infection. Mice were anesthetized with isoflurane and infected intraperitoneally (IP) with 10 or 100 PFU of the indicated viruses diluted in 200 µl sterile saline. Mice were weighed daily and checked for signs of illness. For some experiments, mice were bled (10 µl) via their tail veins for measurement of viral RNA in the blood by quantitative reverse transcription-PCR (Q-RT-PCR), as described previously (30). The measurement of viral RNA is expressed as PFU equivalents per milliliter of blood. The PFU equivalent was determined by comparing the experimental sample threshold cycle value to that of a standard curve generated using 10-fold dilutions (in whole blood) of the stock virus with the known PFU titer. Surviving mice were bled retro-orbitally at either day 21 or day 35 postinfection to obtain serum for immunoglobulin G (IgG) analysis. Ebola virus IgG enzyme-linked immunosorbent assays with mouse species-specific modifications were performed essentially as described previously (21). For some experiments, mice were euthanized at 5 days postinfection (d.p.i.). and samples of liver and spleen were harvested for Q-RT-PCR analysis and histology. Immunohistochemistry to detect Ebola virus antigens was performed as described previously (19).


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
In order to use the mouse as a small-animal model to evaluate the role of VP35 in the pathogenesis of Ebola virus, we created a minimally MA full-length cDNA clone of the Ebola virus genome to be used for reverse genetics. We modified our existing cDNA clone (28) to encode the NP and VP24 mutations that are associated with illness and death in mice (8). This MA2 full-length cDNA clone was then additionally modified to encode the R312A mutation within VP35 (16). The rescued viruses were termed recEboMA2-VP35/WT and recEboMA2-VP35/R312A.

Groups of five mice were infected with 100 PFU of either recEboMA2-VP35/WT, recEboMA2-VP35/R312A, the authentic MA virus (5), or saline only. The authentic MA virus is the original adapted virus that was not generated by reverse genetics and contains all nine nucleotide changes as a result of adaptation (5). Mice were monitored closely for signs of illness and weight loss (Fig. 1). Mice infected with the authentic MA virus and the recEboMA2-VP35/WT virus began to appear ruffled at 2 d.p.i., and severe illness was evident by 4 and 5 d.p.i. Mice in both groups lost 15 to 20% of their original body weights by 6 to 7 d.p.i. All mice infected with the authentic MA virus died by 5 to 7 d.p.i. Despite severe illness, no deaths were observed in any of the recEboMA2-VP35/WT virus-infected mice, in contrast to predictions based on published data (8). This is likely due to subtle differences in the cDNA backbone clone used for reverse genetics by the two different research groups (8, 28). There are three amino acid substitutions encoded by GP in the clone by Ebihara et al. (8) that are not encoded by WT Zaire ebolavirus or by the cDNA clone by Towner et al. (28).


Figure 1
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  		FIG. 1. Weight loss in Ebola virus-infected mice. Mice were infected IP with 100 PFU of each of the indicated viruses. Weight loss was monitored daily and is expressed as a percent change of original body weight. Bars represent standard errors from an average of five mice per group.

 
As hypothesized, mice infected with the recEboMA2-VP35/R312A virus gained weight and appeared normal throughout the study. There was no weight loss in this group nor were there any outward signs of illness (Fig. 1). Similar results were obtained when mice were infected with 0.1, 1, and 10 PFU per animal (data not shown). Mice infected with the recEboMA2-VP35/R312A virus remained healthy for all doses of virus. All mice infected with recEboMA2-VP35/R312A virus seroconverted, as determined by an Ebola virus IgG enzyme-linked immunosorbent assay (data not shown). Therefore, the lack of illness or weight loss in mice infected with the recEboMA2-VP35/R312A virus was not due to a lack of exposure to the virus.

In a second experiment, groups of 10 mice were infected with 10 PFU of the indicated viruses. A small sample of blood (10 µl) was taken by tail snip of each mouse on 2, 3, 4, and 6 d.p.i. Viral RNA levels in the blood were measured using a sensitive Q-RT-PCR assay (30). Mice infected with both the authentic MA virus and the recEboMA2-VP35/WT virus had high levels of viral RNA in the blood by 6 d.p.i. (108 and 105 PFU equivalents/ml blood, respectively; see Materials and Methods) (Fig. 2). Remarkably, no viral RNA was detected in the blood of any of the mice infected with the recEboMA2-VP35/R312A virus at any time point tested (Fig. 2), despite confirmation of infection by the presence of Ebola virus-specific IgG antibodies (data not shown).


Figure 2
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  		FIG. 2. Viral RNA levels in the blood of infected mice. BALB/c mice were infected IP with 10 PFU of the indicated viruses. Blood samples were taken via tail snips on 2, 3, 4, and 6 d.p.i. A sensitive TaqMan Q-RT-PCR assay was performed to estimate viral RNA levels. RNA copy number is expressed as PFU equivalents per milliliter of blood (see Materials and Methods). Each data point represents the average of groups of 10 mice, and the standard errors (error bars) are shown.

 
The authentic MA Ebola virus is lethal for mice after IP infection with very small quantities of virus (0.03 PFU, or approximately 1 virion) (5). After injection of a small amount of virus into the peritoneum, it is likely that the virus rapidly enters the peritoneal venous drainage system and is transported directly to the liver and subsequently to the spleen. Liver and spleen cells as well as macrophages within these organs are highly susceptible to Ebola virus infection. The very low infectious dose is likely the result of IP injected virus proceeding rapidly to this highly susceptible pool of cells. Therefore, a very small number of virions can initiate rapid viral replication, leading to high titers in the liver, spleen, and blood within a few days after infection.

In this study, liver and spleen tissues were harvested from mice sacrificed at 5 d.p.i. RNA was extracted from liver tissue to quantify the amount of viral RNA present within the tissue. Extremely high levels of viral RNA were found in the livers of mice infected with both the authentic MA virus and the recEboMA2-VP35/WT virus (1010 and 108 PFU equivalents/g tissue, respectively) (Fig. 3). Immunohistochemistry of liver and spleen tissue from these mice revealed extensive Ebola virus antigen within mononuclear phagocytic cells as well as inflammation in both tissues (Fig. 4A through D). For mice infected with the recEboMA2-VP35/R312A mutant virus, only small amounts of viral RNA were detected in liver samples (102 PFU equivalents/g tissue) (Fig. 3). The amount of viral RNA found in the liver samples from these mice was reduced by 6 to 8 logs compared to the two viruses expressing WT VP35 protein. Liver and spleen samples from mice infected with the recEboMA2-VP35/R312A mutant virus had no detectable Ebola virus antigen and only a small amount of inflammation (Fig. 4E and F). Since the recEboMA2-VP35/R312A virus is unable to control IRF-3-inducible antiviral gene expression (Hartman et al., submitted; 16), it is likely that extensive viral replication and spread was effectively limited by the innate immune response. This study suggests that a potent innate antiviral immune response can sufficiently limit viral replication and spread in the liver, spleen, and blood.


Figure 3
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  		FIG. 3. Viral RNA load in the mouse liver. RNA was extracted from ground liver tissue harvested at 5 d.p.i. Q-RT-PCR analysis estimated the viral RNA copy number per gram of liver tissue. Each bar represents the average of three mice, and the standard errors (error bars) are shown.

 

Figure 4
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  		FIG. 4. Detection of Ebola virus antigen in liver and spleen tissues. Immunohistochemistry was performed to detect Ebola virus antigen (red) in liver (magnification, x40) and spleen (magnification, x20) tissues from mice at 5 d.p.i. (A and B) Authentic MA virus. (C and D) recEboMA2-VP35/WT virus. (E and F) recEboMA2-VP35/R312A virus. (G and H) Saline-infected negative control.

 
Out of the nine nucleotide changes that occurred as a result of the adaptation of Ebola virus to mice (5), two resulted in amino acid substitutions (S72G in NP and T50I in VP24) that appear to be the minimal requirements for causing severe illness in mice (8). The recEboMA2-VP35/WT virus used in this study contains these two amino acid substitutions within NP and VP24. A direct comparison of recEboMA2-VP35/WT with the authentic MA virus in this study revealed that the recombinant virus induces severe illness and weight loss in mice, but it is clearly not as robust an infection because no deaths were observed and viral RNA levels in the blood and other organs were not as high as the levels for authentic MA virus. However, virus levels in the blood and livers of mice infected with recEboMA2-VP35/WT were still significant, and the mice became severely ill and lost nearly 20% of their initial body weights. This study suggests that while the illness induced by a virus containing only the NP and VP24 mutations is severe, more than these two nucleotide changes are required to completely recapitulate the viral replication levels and dose range seen with the authentic MA virus. Regardless, the recEboMA2-VP35/WT virus is a valuable tool to evaluate the role of the R312A mutation in VP35 on disease in mice.

By using precisely defined viruses generated from cDNA clones of Ebola virus, this study presents the first definitive evidence that disruption of the IRF-3 inhibitory domain within VP35 by replacement of arginine at position 312 with alanine has a profound effect on disease induction. As predicted based on our previous finding that the inhibition of IRF-3 activation is critically important for controlling host antiviral gene expression in vitro (Hartman et al., submitted; 16), we found that the recEboMA2-VP35/R312A mutant virus was severely attenuated in mice. Since the difference between the recEboMA2-VP35/WT and recEboMA2-VP35/R312A viruses is one single amino acid within the IRF-3 inhibitory domain of VP35, the attenuated phenotype appears to be specifically due to this single amino acid change and is likely the result of the inability of the mutant virus to effectively suppress the host's innate antiviral gene expression. Based on these findings as well as our previous results (Hartman et al., submitted; 16), the inhibition of IRF-3 activation is most likely the predominant mechanism contributing to these results. However, given that the R312A mutation in VP35 also disrupts RSS function (14) and RNA binding (7), we cannot rule out the possibility of the additive effect of both mechanisms on these results.

The data presented here strongly suggest that innate antiviral immunity can effectively suppress Ebola virus replication and dissemination, but that this process is normally inhibited by VP35 to allow rapid viral replication. Animal studies using two viruses that differ by only one amino acid are ideal tools to definitively assess the individual contribution of viral virulence factors to pathogenesis. A similar approach can be used to assess the contribution of the other immune inhibition mechanisms of Ebola virus to the pathogenesis of this deadly disease.


   ACKNOWLEDGMENTS
 
B.B. was supported in part by fellowships from the Students Training in Advanced Research program and the Veterinary Scientist Training Program of the University of California, Davis, School of Veterinary Medicine (UCD SVM), and the Oak Ridge Institute for Science and Engineering.

We thank Tom Ksiazek, Chief of Special Pathogens Branch, for support and assistance. We also thank Archer Miller for assistance with animal studies. B.B. thanks N. James MacLachlan (UCD SVM) for his steadfast support during the completion of these studies.

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency.


   FOOTNOTES
 
* Corresponding author. Mailing address: Centers for Disease Control and Prevention, 1600 Clifton Rd., MS G-14, Atlanta, GA 30329. Phone: (404) 639-1122. Fax: (404) 639-1118. E-mail: stn1{at}cdc.gov Back

{triangledown} Published ahead of print on 16 January 2008. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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Journal of Virology, March 2008, p. 2699-2704, Vol. 82, No. 6
0022-538X/08/$08.00+0     doi:10.1128/JVI.02344-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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