Role of Ebola virus VP30 in transcription reinitiation.
ABSTRACT VP30 is a phosphoprotein essential for the initiation of Ebola virus transcription. In this work, we have studied the effect of mutations in VP30 phosphorylation sites on the ebolavirus replication cycle by using a reverse genetics system. We demonstrate that VP30 is involved in reinitiation of gene transcription and that this activity is affected by mutations at the phosphorylation sites.
- SourceAvailable from: Peter J Myler[Show abstract] [Hide abstract]
ABSTRACT: The ebolaviruses can cause severe hemorrhagic fever. Essential to the ebolavirus life cycle is the protein VP30, which serves as a transcriptional cofactor. Here, the crystal structure of the C-terminal, NP-binding domain of VP30 from Reston ebolavirus is presented. Reston VP30 and Ebola VP30 both form homodimers, but the dimeric interfaces are rotated relative to each other, suggesting subtle inherent differences or flexibility in the dimeric interface.Acta crystallographica. Section F, Structural biology communications. 04/2014; 70(Pt 4):457-60.
- [Show abstract] [Hide abstract]
ABSTRACT: The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional, but not replication activity of EBOV polymerase; to support transcription, VP30 must be in a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously, but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation as expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03 that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome, and completely suppressed replication of EBOV in cultured cells. Finally, mutations of VP30's Thr143 and Thr146 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29-46 suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for development of antivirals against EBOV and other filovirus species.Journal of Biological Chemistry 06/2014; · 4.65 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Reverse genetics allows the generation of recombinant viruses entirely from cDNA. One application of this technology is the creation of reporter-expressing viruses, which greatly increase the detail and ease with which these viruses can be studied. However, there are a number of challenges when working with reporter-expressing viruses. Both the reporter protein itself as well as the genetic manipulations within the viral genome required for expression of this reporter can result in altered biological properties of the recombinant virus, and lead to attenuation in vitro and/or in vivo. Further, instability of reporter expression and purging of the genetic information encoding for the reporter from the viral genome can be an issue. Finally, a practical challenge for in vivo studies lies in the attenuation of light signals when traversing tissues. Novel expression strategies and the continued development of brighter, red and far-red shifted reporters and the increased use of bioluminescent reporters for in vivo applications promise to overcome some of these limitations in future. However, a "one size fits all" approach to the design of reporter-expressing viruses has thus far not been possible. Rather, a reporter suited to the intended application must be selected and an appropriate expression strategy and location for the reporter in the viral genome chosen. Still, attenuating effects of the reporter on viral fitness are difficult to predict and have to be carefully assessed with respect to the intended application. Despite these limitations the generation of suitable reporter-expressing viruses will become more common as technology and our understanding of the intricacies of viral gene expression and regulation improves, allowing deeper insight into virus biology both in living cells and in animals.Antiviral research 01/2014; · 3.61 Impact Factor
JOURNAL OF VIROLOGY, Dec. 2008, p. 12569–12573
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 24
Role of Ebola Virus VP30 in Transcription Reinitiation?
Miguel J. Martínez,1,2† Nadine Biedenkopf,3,4† Valentina Volchkova,1,2,5,6Bettina Hartlieb,3,4
Nathalie Alazard-Dany,1,2‡ Olivier Reynard,1,2Stephan Becker,3,4* and Viktor Volchkov1,2,5,6*
INSERM, U758, Filovirus Laboratory, 21 Av. Tony Garnier, Lyon F-69007, France1; IFR 128, Lyon F-69007, France2; Robert-Koch-Institut,
ZBS5 Berlin, Germany3; Institut fu ¨r Virologie, Philipps Universita ¨t Marburg, Marburg, Germany4; Universite ´ de Lyon,
Lyon F-69007, France5; and Universite ´ de Lyon 1, Villeurbanne F-69622, France6
Received 3 July 2008/Accepted 19 September 2008
VP30 is a phosphoprotein essential for the initiation of Ebola virus transcription. In this work, we have
studied the effect of mutations in VP30 phosphorylation sites on the ebolavirus replication cycle by using a
reverse genetics system. We demonstrate that VP30 is involved in reinitiation of gene transcription and that
this activity is affected by mutations at the phosphorylation sites.
Ebola virus (EBOV) causes a severe hemorrhagic fever syn-
drome, characterized by high mortality rates, in humans and
nonhuman primates (7, 13, 14). The EBOV replication cycle
takes place in the cytoplasm of infected cells, where inclusion
bodies are formed (4). The inclusions contain viral ribonucleo-
protein (RNP) complexes consisting of viral RNA and four
nucleocapsid proteins: the nucleoprotein (NP), VP35, the
polymerase (L), and VP30 (15, 16). Using an EBOV mini-
genome system, it was demonstrated that NP, VP35, and L are
sufficient for RNA replication, whereas the addition of VP30 is
required for transcription initiation (12). Recently, it has been
shown that VP30 provided in trans supports transcription of
the minigenome delivered by infectious virus-like particles
(VLPs) and replication of a recombinant EBOV lacking the
VP30 gene (5). VP30 is phosphorylated at two serine clusters
(amino acids 29 to 31 and 42 to 46), each containing three
serine residues (Fig. 1A). Previously, it has been shown that
mutants of VP30 with both serine clusters replaced by alanine
residues supported transcription of an EBOV minigenome but
did not accumulate in the NP-induced inclusions. In contrast,
when all serine residues at the phosphorylation sites were
replaced by aspartate residues, VP30 was unable to support
transcription (10). While a Ser3Ala substitution mimics non-
phosphorylated serine, a Ser3Asp substitution mimics con-
stantly phosphorylated serine.
In this study, we investigated the effect of mutations simu-
lating a constantly phosphorylated state of VP30 during a
Zaire EBOV infection (Fig. 1A). VP30 mutants with nonphos-
phorylated and highly phosphorylated states are represented
by VP30-AA and VP30-DD, respectively. Two other con-
structs, VP30-AD and VP30-DA, have one of the two serine
clusters replaced by an alanine cluster and the other by an
In an attempt to generate recombinant viruses containing
the designated mutations, we used a reverse genetics system
for EBOV (17). Only the wild-type EBOV genome was res-
cued into infectious virus (Fig. 1B). Failure to recover a virus
containing VP30-DD is likely explained by the lack of tran-
scription initiation activity of VP30-DD (10). To assess
whether the ability of VP30 to support viral transcription was
altered with the other three mutants, we quantified their ac-
tivities in an EBOV-specific minigenome system (8) (Fig. 2A
and B). VP30-AA was 30% more active than the VP30 wild
type (VP30-WT) in supporting EBOV transcription, and both
VP30-AD and VP30-DA supported transcription of the re-
porter gene in the same range as VP30-WT. Thus, the exper-
iments with minigenomes did not provide a plausible explana-
tion for the failure to recover recombinant viruses containing
the AA, AD, or DA mutations.
In addition to transcription initiation activity, VP30 is a
structural protein (3, 6, 14). Since it has been shown that
mutations at the phosphorylation site alter VP30’s association
with NP-induced inclusions (10), we presumed that mutations
at the phosphorylation site would impact the incorporation of
VP30 into viral particles and thus affect the efficiency of virus
recovery. To assess the ability of VP30 mutants to support viral
morphogenesis, we used an EBOV-specific VLP system (8).
We found that all mutants of VP30 were incorporated into
VLPs and were protease protected (Fig. 2C). This indicated
that VP30 mutants were localized inside the particles. To con-
firm these results, we tested whether the mutants of VP30 were
also incorporated into authentic EBOV. Subconfluent 75-cm2
flasks of Vero cells were transfected with 8 ?g of plasmids
encoding myc-tagged mutants of VP30 and were then infected
with EBOV at a multiplicity of infection (MOI) of 1 at 6 hours
posttransfection. Virions released into the culture medium
during 24 h of infection were collected by ultracentrifugation
through a 20% sucrose cushion and then analyzed by Western
blotting using anti-VP24 and anti-myc antibodies. Both
VP30-AA and VP30-DD mutants were found to be associated
with viral particles (Fig. 2D). These data are in agreement with
a recent study (6) showing that recruitment of VP30 into VLPs
is dependent on the C-terminal part of the protein, while our
* Corresponding author. Mailing address for Viktor Volchkov:
INSERM, U758, Filovirus Laboratory, 21 Av. Tony Garnier, 69365
Lyon, Cedex 07, France. Phone: 33 437 282450. Fax: 33 437 282459.
E-mail: email@example.com. Mailing address for Stephan
Becker: Institut fu ¨r Virologie, Philipps-Universita ¨t Marburg, 35037
Marburg, Germany. Phone: 49 6421 2866253. Fax: 49 6421 2868899.
‡ Present address: INSERM, U758, Ecole Normale Supe ´rieure, 46
alle ´e d’Italie, 69364 Lyon, Cedex 07, France.
† These authors contributed equally to this work.
?Published ahead of print on 1 October 2008.
mutations affected the N terminus. Moreover, our results sug-
gest that the intracellular colocalization of VP30 and NP de-
scribed in an earlier publication (10) does not reflect the ability
of VP30 to be incorporated into budding VLPs. It should be
mentioned that the smaller amount of VP30-DD in pelleted
virions compared with that of VP30-WT and VP30-AA was not
expected based on our results with VLPs. However, this is
explained by the negative effect of VP30-DD on viral replica-
tion. Since only approximately 10 to 15% of cells were trans-
fected and expressed mutated VP30, this effect is not seen at
the level of the VP24 expressed in all virus-infected cells. To
confirm the effect of VP30-DD on viral replication, 293T cells,
which have much higher transfection efficiency, were used
Taken together, our results showed that VP30-WT, VP30-
AA, VP30-AD, and VP30-DA were similar in terms of their
association with the nucleocapsid and transcription of EBOV
minigenomes, but they differed in their capacities to rescue
recombinant viruses. One possible explanation of their differ-
ences is that in the minigenome system, a monocistronic tem-
plate is transcribed, whereas rescue of full-length viral RNA
needs transcription of seven individual genes in a consecutive
manner. At every gene end, transcription stops and is reiniti-
ated at the start of the following gene (11). We therefore
analyzed the capacities of the VP30 mutants to reinitiate tran-
scription. The plasmid encoding VP30-WT was replaced by
others encoding VP30-DD, VP30-AA, VP30-AD, or VP30-DA
in the full-length rescue system in combination with the wild-
type EBOV genome (Fig. 3). If the mutants of VP30 are able
to support transcription of all viral genes, VP30, which is en-
coded by the fifth gene of the viral genome, will be synthesized
and will compensate for putative missing functions of the mu-
tant VP30. Here, we demonstrate that VP30-DD, VP30-AD,
and VP30-DA do not support the recovery of the wild-type
FIG. 1. (A) Nomenclature of VP30 mutants. Two clusters of serine residues (S29, S30, and S31; S42, S44, and S46) constitute the protein
phosphorylation sites. Several cellular protein kinases (e.g., PKA, CKII, and cAMKII) are predicted to recognize VP30 phosphorylation sites.
Amino acid substitutions and the nomenclature of the constructed mutants are indicated. (B) Recovery of recombinant viruses. (Left) Schematic
representation of the reverse genetics system. Mutations in the VP30 gene are introduced in a plasmid carrying a full-length cDNA of Zaire EBOV.
The recovery of the recombinant virus is then supported by four plasmids encoding wild-type nucleocapsid proteins (NP, VP35, VP30, and L).
(Right) Results of the recovery experiment. Successful recovery of the virus is indicated by a plus. Lack of virus recovery is indicated by a minus.
12570NOTES J. VIROL.
EBOV. In contrast, VP30-AA supported the recovery of the
wild-type EBOV genome, indicating that the nonphosphory-
lated form of VP30 activates transcription of all viral genes,
including the gene encoding VP30. Since VP30-AD and
VP30-DA showed an activity comparable to that of VP30-AA
in supporting transcription of the reporter gene, their failure to
recover the wild-type virus pointed to a difference in support-
ing transcription of downstream genes.
Next, we assessed the abilities of VP30-AD and VP30-DA to
support transcription reinitiation. We generated a plasmid car-
rying a full-length cDNA of EBOV containing the green fluo-
rescent protein (GFP) gene as an additional transcription unit
between the genes encoding VP35 and VP40 (the second and
third genes). When VP30-AA was used to rescue this virus,
GFP-expressing cells appeared 2 days posttransfection and
gradually increased in number, corresponding to the spread of
the recombinant virus (Fig. 4A). In contrast, no GFP-express-
ing cells were observed in the presence of VP30-AD or VP30-
DA, suggesting that these mutants did not support transcrip-
tion of the GFP gene. These results strongly support the idea
FIG. 2. Influence of permanent charges at the phosphorylation sites of VP30 on viral transcription and incorporation of VP30 into virus
particles. (A) Schematic representation of the minigenome and VLP assay. HEK293 cells were transfected with plasmids encoding all viral
proteins, the T7 polymerase, and the EBOV-specific minigenome (MG) containing a Renilla luciferase reporter gene. Additionally, the vector
pGL4 carrying the firefly luciferase reporter gene was transfected and used for normalization of the results. Nucleocapsid proteins provided in trans
direct transcription/replication of the EBOV minigenome, which is subsequently encapsidated into VLPs.
minigenome containing the luciferase reporter. (B) Transcription activation by VP30-WT and VP30 mutants. At 72 h posttransfection, cells were
harvested and analyzed for reporter activity using the Dual-Luciferase assay (Promega). Renilla luciferase activity (R-Luc), reflecting minigenome
replication and transcription, was normalized by firefly luciferase activity. Transcription activation by VP30-WT was set to 100%. Replacement of
VP30-WT by the respective mutants is indicated. Rel., relative. (C) Incorporation of flag-tagged VP30 into VLPs. VLPs from culture supernatants
collected at 72 h posttransfection were purified by ultracentrifugation through a 20% sucrose cushion. Incorporation of VP30-WT and mutants of
VP30 was analyzed by a protease protection assay. Samples were treated with proteinase K in the presence or absence of Triton X-100, separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then detected by Western blotting using antiflag antibodies. Proteins inside
VLPs were protected from proteinase K digestion and are visible in the middle columns. (D) Incorporation of myc-tagged VP30 proteins in EBOV
particles. Vero E6 cells were transfected with plasmids encoding myc-tagged VP30 (VP30-WT [WT], VP30-AA [AA], or VP30-DD [DD]) and then
infected with EBOV at an MOI of 1 PFU/cell. Virus particles from culture supernatants were purified by ultracentrifugation through a 20% sucrose
cushion. NT, nontransfected cells. (E) Expression of VP30-DD negatively affects EBOV replication. 293T cells were transfected with plasmids
expressing either VP30-WT or VP30-DD and then infected with EBOV at an MOI of 1 PFU per cell. Samples of culture supernatants (SN) and
lysed cells were analyzed by Western blotting using anti-VP24, anti-NP, and antiactin antibodies.
, transcription initiation of the
VOL. 82, 2008NOTES 12571
that the AD and DA substitutions in VP30 affected the ability
of the polymerase complex to reinitiate transcription of viral
genes downstream of the second gene.
To confirm that VP30-AA was able to reinitiate transcrip-
tion without the expression of VP30-WT, we mutated the full-
length cDNA of EBOV containing the GFP gene by introduc-
ing the AA mutation in the VP30 gene and employed this
construct in the rescue system by expressing the different mu-
tants of VP30 in trans. Individual cells expressing GFP were
observed when VP30-AA was employed, whereas no expres-
sion of GFP was detected when VP30-AD or VP30-DA was
employed (Fig. 4B). This result confirmed that VP30-AA was
able to support viral transcription of the first gene as well as
consecutive genes. In contrast, the mutants VP30-AD and
VP30-DA, while capable of initiating transcription of the first
gene, were unable to reinitiate transcription of downstream
genes. Besides, these data suggest that a cluster of negative
charges mimicking phosphorylation could inhibit VP30’s tran-
scription reinitiation function.
Our experiments demonstrate that the serine clusters at the
N terminus of VP30 represent a critical region for the activity
of the protein. A combination of alanine and aspartate resi-
dues (VP30-AD and VP30-DA) resulted in a VP30 protein
that was able to support transcription of an EBOV-specific
monocistronic minigenome but unable to reinitiate transcrip-
tion in the full-length genome. Permanently uncharged amino
acids at the phosphorylated region (VP30-AA) resulted in
VP30 molecules that were able to activate transcription of a
minigenome and all the genes of the EBOV genome in a
consecutive manner. However, a recombinant EBOV encoding
VP30-AA could not be generated, indicating that this mutant
lacks another function needed to produce fully infectious viri-
ons. The step at which phosphorylated VP30 is essential in the
viral life cycle is currently unknown. Since VP30-DD blocks
EBOV transcription, the phosphorylation of VP30 might play
a role in switching off the transcription favoring replication of
the viral RNA. In this regard, the permanently nonphosphor-
ylated VP30-AA would impair the regulation of the transcrip-
tion/replication processes. In addition, since a phosphorylated
form of VP30 is present in EBOV virions (3), phosphorylated
VP30 may be necessary for a very early step in the viral life
cycle. Our results thus suggest that the dynamic phosphoryla-
tion of VP30 is essential for the function of the protein in
It is presumed that transcription in viruses from the order
Mononegavirales follows a sequential stop-start mechanism in
which transcription of downstream genes is dependent on ter-
mination of the synthesis of the upstream gene. While the
majority of studies have focused on the influence of cis-acting
sequences in transcription regulation (1, 2, 19), in our work, we
provide experimental evidence of the participation of a struc-
tural protein other than the viral polymerase in transcription
reinitiation. The function of VP30 in reinitiation of transcrip-
tion expands on results from a previous report showing that a
specific stem-loop structure at the beginning of the NP gene is
involved in VP30-dependent transcription and that only tran-
scription initiation of the NP gene requires VP30 (18). In the
artificial bicistronic EBOV minigenome, transcription of the
second gene did not require the activity of VP30. Using a
full-length EBOV genome, we obtained results that partially
FIG. 3. Rescue of wild-type EBOV using mutants of VP30. (Left)
Schematic representation of the experimental setup. The recovery of a
wild-type recombinant EBOV is supported by plasmids expressing the
nucleocapsid proteins NP, VP35, and L together with mutants of
VP30. (Right) Results of the recovery experiment. Successful recovery
of the virus is indicated by a plus. Lack of virus recovery is indicated by
FIG. 4. Recovery of recombinant EBOV expressing GFP. The GFP gene was introduced into the full-length cDNA of either wild-type EBOV
(A) or EBOV/VP30-AA (B). Recombinant virus recovery was performed in BHK T7 cells using standard protocols and different VP30-expressing
plasmids as indicated. The presence of groups of green cells indicated recovery and replication of the recombinant viruses. The appearance of
single green cells indicates the ability of mutated VP30 to support both transcription initiation and reinitiation but with subsequent failure of virus
replication. , transcription initiation;, transcription reinitiation.
12572 NOTESJ. VIROL.
contradict this earlier report. This, however, only highlights the
need to complement minigenome studies with the analysis of
full-length genome expression systems. Recently, EBOV VP30
was shown to be an RNA-binding protein (9). Notably, VP30
binds to the sequence in the NP gene’s stem-loop structure that
includes the transcriptional start signal and its complementary
sequence. Sequences complementary to the respective tran-
scription start signal are present in close proximity to the start
signal of all EBOV genes, suggesting that the stem-loop struc-
tures also play a role in the transcription of other genes (11).
Interestingly, analysis of these structures using the mfold pro-
gram (20) showed that their energetic stability (?G) is in-
creased in a 3?35? order (NP, ?7.40; VP35, ?8.6; VP40,
?17.6; GP, ?14.6; VP30, ?19.1; VP24, ?27.9; L, ?23.9).
Given that VP30 is involved in transcription reinitiation, it is
reasonable to speculate that there is a gradient along the viral
genome that controls the efficiency of transcription, especially
at the 5? genome end.
In conclusion, our data indicate, for the first time, that ac-
tivity of VP30 is required for reinitiation of gene transcription
and that mutations at the phosphorylation sites of VP30 affect
this activity. While the mechanism of VP30 action is not yet
clearly understood, our results suggest that VP30 could be an
important target for antiviral therapy.
All experiments involving live EBOV were carried out in the
INSERM BSL-4 laboratory Jean Merieux in Lyon, France. We thank
the biosafety team members for their support and assistance in con-
ducting experiments and Ulla Thiesen for expert technical assistance.
We are also grateful to Robin Buckland for his helpful comments on
This work was supported by INSERM, the French Ministe `re de la
Recherche (grant 04G537), the Agence National de la Recherche
(ANR), the National Institutes of Health (grant AI059536), and the
Deutsche Forschungsgemeinschaft (SFB 593 to V.V. and SFB 535 to
S.B.). M.J.M. was partially supported by the Sociedad Espan ˜ola de
Enfermedades Infecciosas y Microbiologia Clinica (SEIMC). N.B. is
supported by the Ernst Schering Foundation.
1. Conzelmann, K. K. 1998. Nonsegmented negative-strand RNA viruses: ge-
netics and manipulation of viral genomes. Annu. Rev. Genet. 32:123–162.
2. Conzelmann, K. K. 2004. Reverse genetics of mononegavirales. Curr. Top.
Microbiol. Immunol. 283:1–41.
3. Elliott, L. H., M. P. Kiley, and J. B. McCormick. 1985. Descriptive analysis
of Ebola virus proteins. Virology 147:169–176.
4. Geisbert, T. W., and P. B. Jahrling. 1995. Differentiation of filoviruses by
electron microscopy. Virus Res. 39:129–150.
5. Halfmann, P., J. H. Kim, H. Ebihara, T. Noda, G. Neumann, H. Feldmann,
and Y. Kawaoka. 2008. Generation of biologically contained Ebola viruses.
Proc. Natl. Acad. Sci. USA 105:1129–1133.
6. Hartlieb, B., T. Muziol, W. Weissenhorn, and S. Becker. 2007. Crystal struc-
ture of the C-terminal domain of Ebola virus VP30 reveals a role in tran-
scription and nucleocapsid association. Proc. Natl. Acad. Sci. USA 104:624–
7. Hoenen, T., A. Groseth, D. Falzarano, and H. Feldmann. 2006. Ebola virus:
unravelling pathogenesis to combat a deadly disease. Trends Mol. Med.
8. Hoenen, T., A. Groseth, L. Kolesnikova, S. Theriault, H. Ebihara, B. Hart-
lieb, S. Bamberg, H. Feldmann, U. Stro ¨her, and S. Becker. 2006. Infection of
naïve target cells with virus-like particles: implications for the function of
ebola virus VP24. J. Virol. 80:7260–7264.
9. John, S. P., T. Wang, S. Steffen, S. Longhi, C. S. Schmaljohn, and C. B.
Jonsson. 2007. Ebola virus VP30 is an RNA binding protein. J. Virol.
10. Modrof, J., E. Muhlberger, H. D. Klenk, and S. Becker. 2002. Phosphoryla-
tion of VP30 impairs ebola virus transcription. J. Biol. Chem. 277:33099–
11. Muhlberger, E., S. Trommer, C. Funke, V. Volchkov, H. D. Klenk, and S.
Becker. 1996. Termini of all mRNA species of Marburg virus: sequence and
secondary structure. Virology 223:376–380.
12. Mu ¨hlberger, E., M. Weik, V. E. Volchkov, H.-D. Klenk, and S. Becker. 1999.
Comparison of the transcription and replication strategies of Marburg virus
and Ebola virus by using artificial replication systems. J. Virol. 73:2333–2342.
13. Peters, C. J., and J. W. LeDuc. 1999. An introduction to Ebola: the virus and
the disease. J. Infect. Dis. 179(Suppl. 1):ix–xvi.
14. Sanchez, A., T. W. Geisbert, and H. Feldmann. 2007. Filoviridae: Marburg
and Ebola viruses. In D. M. Knipe, P. M. Howley, and D. E. Griffin (ed.),
Fields virology, 5th ed. Wolters Kluwer/Lippincott Williams & Wilkins, Hag-
15. Sanchez, A., M. P. Kiley, B. P. Holloway, and D. D. Auperin. 1993. Sequence
analysis of the Ebola virus genome: organization, genetic elements, and
comparison with the genome of Marburg virus. Virus Res. 29:215–240.
16. Volchkov, V. E., V. A. Volchkova, A. A. Chepurnov, V. M. Blinov, O. Dolnik,
S. V. Netesov, and H. Feldmann. 1999. Characterization of the L gene and 5?
trailer region of Ebola virus. J. Gen. Virol. 80:355–362.
17. Volchkov, V. E., V. A. Volchkova, E. Muhlberger, L. V. Kolesnikova, M.
Weik, O. Dolnik, and H. D. Klenk. 2001. Recovery of infectious Ebola virus
from complementary DNA: RNA editing of the GP gene and viral cytotox-
icity. Science 291:1965–1969.
18. Weik, M., J. Modrof, H.-D. Klenk, S. Becker, and E. Mu ¨hlberger. 2002.
Ebola virus VP30-mediated transcription is regulated by RNA secondary
structure formation. J. Virol. 76:8532–8539.
19. Whelan, S. P., J. N. Barr, and G. W. Wertz. 2004. Transcription and repli-
cation of nonsegmented negative-strand RNA viruses. Curr. Top. Microbiol.
20. Zuker, M. 2003. Mfold web server for nucleic acid folding and hybridization
prediction. Nucleic Acids Res. 31:3406–3415.
VOL. 82, 2008NOTES12573