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Journal of Virology, March 2007, p. 2995-2998, Vol. 81, No. 6
0022-538X/07/$08.00+0     doi:10.1128/JVI.02486-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Critical for Ebola Virus Replication in Nonhuman Primates

Gabriele Neumann,¹ Thomas W. Geisbert,^2,3 Hideki Ebihara,^4,5 Joan B. Geisbert,² Kathleen M. Daddario-DiCaprio,^2,3 Heinz Feldmann,⁴ and Yoshihiro Kawaoka^1,5*

Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, Madison, Wisconsin,¹ United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland,² Uniformed Services University of the Health Sciences, Bethesda, Maryland,³ Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, and Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada,⁴ Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan⁵

Received 12 November 2006/ Accepted 19 December 2006

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Enveloped viruses often require cleavage of a surface glycoprotein by a cellular endoprotease such as furin for infectivity and virulence. Previously, we showed that Ebola virus glycoprotein does not require the furin cleavage motif for virus replication in cell culture. Here, we show that there are no appreciable differences in disease progression, hematology, serum biochemistry, virus titers, or lethality in nonhuman primates infected with an Ebola virus lacking the furin recognition sequence compared to those infected with wild-type virus. We conclude that glycoprotein cleavage by subtilisin-like endoproteases is not critical for Ebola virus infectivity and virulence in nonhuman primates.

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Ebola virus, a member of the family Filoviridae, causes hemorrhagic fever in human and nonhuman primates, with up to 90% lethality (2, 9). Although the molecular basis for the extreme virulence of Ebola virus remains largely unknown, many enveloped viruses require cleavage of their surface glycoprotein by cellular endoproteases for infectivity and virulence (3). This cleavage event produces two subunits and exposes a fusion peptide that inserts into the host cell membrane, thereby "fusing" the cellular and viral membranes. The glycoprotein (GP) of Zaire ebolavirus (ZEBOV) contains a highly conserved consensus sequence for the subtilisin-like endoprotease furin (10, 12). ZEBOV GP cleavage by this protease has been previously demonstrated (12). By contrast, the glycoprotein of Reston ebolavirus, the species least pathogenic for humans, lacks an optimal furin cleavage site (containing lysine, but not arginine, at position –4). Furin cleavage was therefore considered an important determinant of Ebola virus virulence (2). However, studies of pseudotyped viruses expressing Ebola GP with an altered furin recognition sequence demonstrated that GP cleavage by furin is dispensable for in vitro infectivity of the pseudotyped viruses (7, 14).

Using reverse genetics, which allows the artificial generation of Ebola virus from cloned cDNA (8, 13), we previously generated a ZEBOV mutant based on the Mayinga strain in which the multibasic sequence motif for furin cleavage (RRTRR at amino acids 497 to 501 of GP) was replaced with nonbasic amino acids (AGTAA) (8). This mutation prevented the generation of GP1 and GP2, the two cleavage products, and yet the mutant virus grew in cell culture to titers similar to those of wild-type virus, albeit with mildly attenuated growth early in infection (8). These findings indicated that furin-mediated cleavage of Ebola virus GP is not essential for virus replication in cell culture. However, does this finding also hold true in animals?

Nonhuman primates, including cynomolgus and rhesus macaques, develop disease with a pathology that resembles that observed in Ebola virus-infected humans. Because these animals display similar symptoms and disease progression and succumb to infection with wild-type ZEBOV without prior adaptation, they are considered the "gold standard" animal model for Ebola virus infection.

Here, we assessed our recombinant Ebola virus that lacks the conserved furin recognition sequence (ZEBOVCleav) in rhesus macaques. Two healthy, filovirus-seronegative, adult rhesus macaques were inoculated in the right caudal thigh with 10³ PFU of recombinant ZEBOVCleav virus. Two additional animals were inoculated with the same dose of wild-type ZEBOV generated by reverse genetics. Animals were observed twice daily for signs of illness, including cutaneous rashes, hemorrhage, and reduced activity. Blood samples for virus titration, hematology, and serum biochemistry were collected prior to infection and on days 3, 4, 6, and 7 postinfection. Animals that showed severe signs of disease were euthanized in compliance with approved protocols. Animal studies were performed under biosafety level 4 biocontainment conditions at the U.S. Medical Research Institute of Infectious Diseases and approved by the U.S. Medical Research Institute of Infectious Diseases Laboratory Animal Care and Use Committee. Animal research was conducted in compliance with the Federal statues and regulations relating to animals and experiments involving animals, adhering to the principles in the Guide for the Care and Use of Laboratory Animals (7a). The facility used is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

All infected animals were asymptomatic until day 4 postinfection, when all four animals became febrile (temperature > 39.7°C). By day 5, all four had developed characteristic macular cutaneous rashes (Fig. 1A). On day 7, one animal infected with ZEBOVCleav expired, while the remaining three monkeys became terminally ill and were euthanized. These findings demonstrate that wild-type ZEBOV generated by reverse genetics is as virulent as the original ZEBOV (4) and that Ebola virus lacking the consensus furin recognition motif in GP is as capable as wild-type virus of causing fatal disease in nonhuman primates.

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FIG. 1. Comparison of monkeys infected with ZEBOV (left panels) or ZEBOVCleav (right panels). (A) Macular cutaneous rashes on day 6 postinfection. (B) Phosphotungstic acid hematoxylin-positive fibrin in spleen. Staining was carried out as described in reference 5. Note that there is no apparent difference in the amount or distribution of polymerized fibrin (see arrows that point to fibrin-stained regions). Original magnification, x40. (C) Immunostaining of inguinal lymph nodes. Staining was carried out as described in reference 4. Note that positive immunostaining of monocytes-macrophages for Ebola virus (red) is evident in both animals. Also, lymphoid depletion and lymphocytolysis are prominent in both animals. Original magnification, x20.

No appreciable differences in hematology and serum biochemistry (measured as described previously; see reference 4) were detected between monkeys infected with ZEBOV virus and those infected with ZEBOVCleav virus. In similarity to results previously obtained for Ebola virus infections in cynomolgus macaques (4, 6), all animals developed lymphopenia and thrombocytopenia with elevated liver enzyme levels during late infection. The most pronounced increases were in alanine aminotransferase and aspartate aminotransferase levels on days 6 and 7 postinfection, indicative of liver failure. In addition, monkeys infected with ZEBOV or ZEBOVCleav virus revealed similar levels of fibrin deposits (Fig. 1B) and similar numbers of infected monocytes-macrophages (Fig. 1C).

The clinical signs and blood biochemistry of rhesus macaques infected with ZEBOVCleav virus suggested replication of the virus in monkeys; this growth may, however, have been attenuated due to the lack of the consensus furin recognition motif in GP. We, therefore, determined virus titers in serum and organs. When plaque assays were performed, virus titers in serum were undetectable or low on day 3 postinfection but reached high levels on days 6 and 7 postinfection (6.7 to 7.5 log₁₀ PFU/ml) (Fig. 2) for ZEBOV-infected animals. The two animals infected with ZEBOVCleav reached similar titers (6.8 to 8.2 log₁₀ PFU/ml) on day 6 and 7 postinfection, respectively. Note that one animal infected with ZEBOVCleav expired on day 7 prior to blood sampling. We also conducted quantitative real-time reverse transcription-PCR and found low numbers of genomic copies on day 3 postinfection for animals infected with ZEBOV and no viral genomic copies in animals infected with ZEBOVCleav (note that the detection limit of the assay is 140 genomic copies), suggesting mild attenuation of the mutant virus. Quantitative reverse transcription-PCR analysis on day 6 postinfection revealed no differences in the numbers of genomic copies between the two groups.

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FIG. 2. Viremia in rhesus macaques infected with wild-type Ebola virus (ZEBOV) generated by reverse genetics and in a mutant lacking the conserved recognition sequence for furin (ZEBOVCleav). Note that one animal infected with ZEBOVCleav expired on day 7 postinfection (); therefore, no fresh serum sample for that animal could be obtained for virus titration.

ZEBOV and ZEBOVCleav also replicated to comparable titers in various organs (Table 1), although viral titers were low in the spleen and undetectable in the pancreas of the animal infected with ZEBOVCleav that died on day 7. Virus titers in the pancreas and brain of one ZEBOV-infected monkey were also low, suggesting normal variability among these infected animals. Our findings establish that Ebola virus replication in nonhuman primates is not significantly attenuated by the loss of the furin recognition motif in GP.

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TABLE 1. Virus titers in various organs of rhesus macaques infected with wild-type Ebola virus (ZEBOV) or with an Ebola virus lacking the consensus furin recognition motif in GP (ZEBOVCleav)

To confirm that the amino acid changes introduced into the furin recognition motif of ZEBOVCleav were retained during replication, we sequenced the GP genes of viruses from plasma and spleen samples of ZEBOVCleav-infected animals on day 6 and day 7 postinfection, respectively. For all samples, the introduced mutations were retained. Moreover, no other nucleotide replacements were found in the GP gene.

GP cleavage by host cell proteases is critical for the virulence of many viruses, and the conservation of the furin cleavage motif among Ebola virus GP sequences suggested a similar role in Ebola virus replication. Our previous and present findings, however, establish that the furin recognition sequence in Ebola virus GP is dispensable in cell culture (8) and in nonhuman primates (this study), indicating that GP cleavage is dispensable for Ebola virus replication in these systems. However, GP cleavage by furin or a furin-like endoprotease may be required for Ebola virus replication in its natural host. GP1 degradation by endosomal cathepsins is important for the initiation of Ebola virus uncoating (1, 11). Our findings indicate that GP cleavage by furin into GP1 and GP2 is not a prerequisite for cathepsin digestion. Whether proteolytic processing by other proteases in endosomes is required prior to cathepsin digestion remains unknown.

 
We thank Susan Watson for editing the manuscript, Denise Braun for technical assistance with animal studies, Tom Larsen for pathology support, and Elizabeth Fritz and Leslie Wachter for assistance with the quantitative PCR assays. We also thank Lisa Hensley for helpful discussions.

This work was sponsored in part by Public Health Service research grants from the National Institute of Allergy and Infectious Diseases and by the National Institutes of Health-National Institute of Allergy and Infectious Diseases Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research Program. We acknowledge membership within and support from the Region V "Great Lakes" Regional Center of Excellence (National Institutes of Health award 1-U54-AI-057153). This study was also supported in part by financial support from the Public Health Agency of Canada and a grant from the Canadian Institute of Health Research (MOP-43921) and the Medical Chemical/Biological Defense Research Program and Military Infectious Diseases Research Program, U.S. Army Medical Research and Material Command (project number 02-4-4J-081).

Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.

 
* Corresponding author. Mailing address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, 2015 Linden Drive, Madison, WI 53706. Phone: (608) 265-4925. Fax: (608) 265-5622. E-mail: kawaokay{at}svm.vetmed.wisc.edu.

Published ahead of print on 17 January 2007.

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Journal of Virology, March 2007, p. 2995-2998, Vol. 81, No. 6
0022-538X/07/$08.00+0     doi:10.1128/JVI.02486-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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