JOURNAL OF VIROLOGY, Nov. 2006, p. 11283–11292
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 22
Hantavirus N Protein Exhibits Genus-Specific Recognition
of the Viral RNA Panhandle?
M. A. Mir,1,3B. Brown,1,3B. Hjelle,2,3W. A. Duran,2,3and A. T. Panganiban1,3*
Department of Molecular Genetics and Microbiology,1Department of Pathology,2and Infectious Disease and Inflammation Program,3
University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
Received 20 April 2006/Accepted 30 August 2006
A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the
presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic
segments into “panhandle” hairpin structures. The hantavirus nucleocapsid protein (N protein), which is
encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro
trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by com-
plementary base sequence of 5? and 3? ends of viral genomic RNA. N protein trimers from the Andes, Puumala,
Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as
other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly
reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus.
Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by
the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23
nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational
change while interacting with panhandles from members of the genus Hantavirus but not while interacting with
panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity
interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.
Hantaviruses are classified as emerging viruses which cause
two often fatal diseases that arise by infection of endothelial
cells: hemorrhagic fever with renal syndrome and hantavirus
cardiopulmonary syndrome (30–33). Each hantavirus is carried
by one or a limited number of wild rodent species and trans-
mitted to humans through the aerosol route. The two diseases
associated with hantaviruses both cause striking increases in
vascular permeability and are elicited by viruses such as Han-
taan virus and Sin Nombre virus (SNV), respectively. Hemor-
rhagic fever with renal syndrome and hantavirus cardiopulmo-
nary syndrome are generally restricted to the Old World and
New World, respectively (34). Hantaviruses comprise a genus
in the family Bunyaviridae. Members of this virus family have
genomes composed of three minus-strand viral RNA (vRNA)
segments whose mRNAs encode an RNA-dependent RNA
polymerase (RdRp) (L segment), the nucleocapsid protein (N
protein; S segment) and G1 and G2 glycoproteins (M seg-
ment). The G1 and G2 proteins are posttranslationally pro-
cessed through the endoplasmic reticulum and Golgi appara-
tus and ultimately presented on the viral surface. These
proteins enable viruses to enter new host cells via their attach-
ment to integrin receptors (7, 8). In the virion the three
genomic RNA molecules form a complex with N protein and
presumably with RdRp.
During replication, assembly is initiated with the binding of
nucleocapsid protein at a unique encapsidation signal on the
genomic RNA. The specific recognition of genomic RNA pro-
motes the oligomerization of N protein. The interaction with
other viral proteins subsequently results in the formation of
virions. For hantaviruses it has been suggested that sequences
at the 5? end of the genomic RNA provide the nucleation point
for encapsidation by N protein (25). Studies with Bunyamwera
virus indicate that N protein specifically encapsidates vRNA
and cRNA but not mRNA or nonviral RNA molecules (13). In
vitro studies indicate that nucleocapsid protein preferentially
binds vRNA over cRNA and nonviral RNA (9, 23, 27, 35, 37).
A cis-acting element that shows a high binding affinity for
Hantaan and Bunyamwera N proteins has been mapped to the
5? end of the viral RNA (23, 35, 37). The RNA binding domain
for Hantaan virus N protein has been mapped to the central
conserved region corresponding to amino acids 175 to 217
(39). N protein has also been found to undergo trimerization
under in vivo (1, 2) and in vitro (14, 15) conditions.
The vRNAs of the Bunyaviridae family each feature a higher-
order “panhandle” formed from the hydrogen bonding of
the nucleotides that comprise the 5? and 3? ends of the RNA.
We have recently shown that SNV N protein, in trimeric form,
specifically recognizes the panhandle structure of the genomic
RNA, and this specific interaction is postulated to have a role
in either encapsidation or viral genome replication initiation
(19–21). Here we show that trimeric nucleocapsid proteins
from other members of the genus Hantavirus, including Andes,
Puumala, Prospect Hill, and Seoul viruses, also specifically
recognize the panhandle of their viral genome. N proteins
from these members of the genus Hantavirus bind viral RNA
panhandles with intragenus specificity and affinity. In addi-
tion, interaction between N and both homologous and het-
erologous hantavirus panhandles results in a conformational
change in N.
* Corresponding author. Mailing address: Department of Molecular
Genetics and Microbiology, University of New Mexico Health Sciences
Center, Albuquerque, NM 87131. Phone: (505) 272-4214. Fax: (505)
272-9912. E-mail: email@example.com.
?Published ahead of print on 13 September 2006.
MATERIALS AND METHODS
Oligonucleotides and enzymes. PCR primers were from Sigma Genosys. All
restriction enzymes were from New England Biolabs. Hot Mastertaq polymerase
was purchased from Eppendorf. DNase I was from Invitrogen, and T7 transcrip-
tion reagents were from Fermentase. All other chemicals were purchased from
Sigma. RNA purification kits were from QIAGEN.
Cloning of bacterial N expression constructs. As reported previously (26, 38),
hantavirus N genes were subcloned from the S genomic segment of the P360
strain of Puumala virus (a generous gift of C. Schmaljohn) or directly from viral
RNA prepared from the following strains of virus (all grown in Vero E6 cells).
Prospect Hill virus strain PHV was provided by R. Yanagihara, Seoul virus strain
80/39 was from H. W. Lee, and Sin Nombre virus strain SN77734 and Andes virus
strain CHI-7913 were from H. Galeno. Briefly, in each case, except for that of
SNV, primers were designed to enable the in-frame insertion of each N gene in
the pET21b (Novagen) backbone so that the initiating ATG immediately follows
a HindIII restriction site, and the termination codon was replaced by a site for
XhoI. The SNV N gene was cloned in pTri.Ex1.1 vector using the NcoI and
HindIII sites. N proteins were expressed as C-terminal histidine-tagged proteins
in Escherichia coli. Histidine tags were not removed after purification. Protein
expression was carried out as recommended by Novagen. In each case, produc-
tion of the N proteins was verified by Western analysis. Each N protein was
purified using Ni2?chelating columns under denaturing conditions as recom-
mended by QIAGEN. The bacterial pellet was lysed in lysis buffer (100 mM
NaH2PO4, 10 mM Tris-Cl, 8 M urea, pH 8.0) and centrifuged at 3,000 rpm. The
resulting supernatant was incubated with N-NTA beads for 15 min. Beads were
washed twice with wash buffer A (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea,
10 mM imidazole, pH 8.0) followed by two additional washes with wash buffer C
(100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea, 100 mM imidazole, pH 8.0).
Bound N protein was eluted with elution buffer (100 mM NaH2PO4, 10 mM
Tris-Cl, 8 M urea, 500 mM imidazole, pH 8.0). Proteins were renatured by
stepwise dialysis to remove the urea and imidazole from the purified N protein.
Protein purity was assessed and confirmed by Coomassie and Western blot
analysis. N proteins from these viruses were all assembled into stable trimers at
4°C (Fig. 1). Trimeric N proteins were purified by sucrose density gradient
centrifugation as described previously (21).
Preparation of RNA substrates. The “minipanhandle” RNAs used in this
study contained the 32 nucleotides from both the 5? and 3? ends of S segment
viral RNA, separated by a loop composed of six uracil residues. This RNA was
synthesized by in vitro transcription with T7 RNA polymerase. A single-stranded
template sequence with a T7 promoter juxtaposed to the minipanhandle se-
quences was amplified by PCR using two opposing primers. For the synthesis of
mutant panhandles, primers containing the designed mutation or the single-
stranded template sequence were used in PCR. All PCR-generated templates
were gel purified and used directly in T7 transcription reactions. [?-32P]CTP-
radiolabeled transcripts were produced from different templates using a T7
transcription kit (MBI Fermentas). After transcription the reaction mixture was
digested with DNase I to remove the DNA template. Purification of the RNA
was performed using Trizol (Invitrogen). Purified RNA was stored at ?20°C in
20-?l aliquots for up to 2 weeks.
Sucrose density gradient centrifugation. Sucrose gradients were used to ex-
amine the oligomeric forms of N proteins from Andes, Puumala, Seoul, and
Prospect Hill viruses using methodology described previously (21). One hun-
dred-microliter samples containing 100 ?M purified N protein were layered onto
linear gradients containing 10 to 45% (wt/vol) sucrose in RNA binding buffer
(21) and centrifuged at 30,000 rpm in an SW40 rotor at 40°C for 22 h. Fractions
of 0.6 ml were collected from the bottom of the gradient. The protein molecular
mass markers in the form of bovine serum albumin (82 kDa) and alcohol
dehydrogenase (150 kDa) were fractionated in parallel. A 100-?l sample of each
fraction was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel elec-
trophoresis. N proteins were detected by Western blot analysis with anti-SNV N
RNA secondary structure analysis. We examined multiple suboptimal second-
ary structure plots to identify alternative structures that might be nearly as stable
as those shown in Fig. 2. In addition, we determined the P-num values for each
of the nucleotides in the RNAs. The P-num value of a nucleotide is a represen-
tation of the number of potentially stable pairing partners for that nucleotide
elsewhere in the same RNA. In particular, P-num is a predictive measure of
pairing fidelity at suboptimal free energy values (?G), and bases with low values
can be used parametrically to identify motifs with the highest probability of
assuming similar configurations among a series of energetically related struc-
FIG. 1. (a) N proteins from Andes, Puumala, Prospect Hill, and Seoul viruses were expressed in E. coli with C-terminal histidine fusion
proteins. Each protein was purified by using Ni-NTA beads according to the manufacturer’s protocol. Proteins were analyzed by SDS-polyacryl-
amide gel electrophoresis and visualized by Coomassie staining. (b) N proteins from Andes, Puumala, Prospect Hill, and Seoul viruses were
sedimented through 10 to 45% sucrose gradients, and N was detected using Western blot analysis with anti-N antibody. Gradients A, C, E, and
G correspond to N proteins from Andes, Puumala, Prospect Hill, and Seoul viruses, respectively. Gradients B, D, F, and H are trimeric N proteins
that have been resedimented to examine the relative stability of trimeric N. Protein standards (bovine serum albumin [BSA] and alcohol
dehydrogenase [ADH]) were run in parallel with the N samples, and arrows indicate the migration of those standards.
11284MIR ET AL.J. VIROL.
Uukuniemi virion RNA: an electron microscopic study. J. Virol. 21:1085–
12. Ikegami, T., C. J. Peters, and S. Makino. 2005. Rift valley fever virus non-
structural protein NSs promotes viral RNA replication and transcription in
a minigenome system. J. Virol. 79:5606–5615.
13. Jin, H., and R. M. Elliott. 1993. Characterization of Bunyamwera virus S
RNA that is transcribed and replicated by the L protein expressed from
recombinant vaccinia virus. J. Virol. 67:1396–1404.
14. Kaukinen, P., V. Koistinen, O. Vapalahti, A. Vaheri, and A. Plyusnin. 2001.
Interaction between molecules of hantavirus nucleocapsid protein. J. Gen.
15. Kaukinen, P., A. Vaheri, and A. Plyusnin. 2003. Mapping of the regions
involved in homotypic interactions of Tula hantavirus N protein. J. Virol.
16. Kohl, A., T. J. Hart, C. Noonan, E. Royall, L. O. Roberts, and R. M. Elliott.
2004. A Bunyamwera virus minireplicon system in mosquito cells. J. Virol.
17. Lakowicz, J. R. 1999. Principles of fluorescence spectroscopy, p. 237–265.
Plenum Press, New York, N.Y.
18. Li, D., A. L. Schmaljohn, K. Anderson, and C. S. Schmaljohn. 1995. Com-
plete nucleotide sequences of the M and S segments of two hantavirus
isolates from California: evidence for reassortment in nature among viruses
related to hantavirus pulmonary syndrome. Virology 206:973–983.
19. Mir, M. A., and A. T. Panganiban. 2006. The bunyavirus nucleocapsid pro-
tein is an RNA chaperone: possible roles in viral RNA panhandle formation
and genome replication. RNA 12:272–282.
20. Mir, M. A., and A. T. Panganiban. 2005. The hantavirus nucleocapsid pro-
tein recognizes specific features of the viral RNA panhandle and is altered in
conformation upon RNA binding. J. Virol. 79:1824–1835.
21. Mir, M. A., and A. T. Panganiban. 2004. Trimeric hantavirus nucleocapsid
protein binds specifically to the viral RNA panhandle. J. Virol. 78:8281–
22. Obijeski, J. F., D. H. Bishop, F. A. Murphy, and E. L. Palmer. 1976. Struc-
tural proteins of La Crosse virus. J. Virol. 19:985–997.
23. Osborne, J. C., and R. M. Elliott. 2000. RNA binding properties of
Bunyamwera virus nucleocapsid protein and selective binding to an element
in the 5? terminus of the negative-sense S segment. J. Virol. 74:9946–9952.
24. Pettersson, R. F., and C. H. von Bonsdorff. 1975. Ribonucleoproteins of
Uukuniemi virus are circular. J. Virol. 15:386–392.
25. Raju, R., and D. Kolakofsky. 1989. The ends of La Crosse virus genome and
antigenome RNAs within nucleocapsids are base paired. J. Virol. 63:122–
26. Rawlings, J. A., N. Torrez-Martinez, S. U. Neill, G. M. Moore, B. N. Hicks,
S. Pichuantes, A. Nguyen, M. Bharadwaj, and B. Hjelle. 1996. Cocirculation
of multiple hantaviruses in Texas, with characterization of the small (S)
genome of a previously undescribed virus of cotton rats (Sigmodon hispidus).
Am. J. Trop. Med. Hyg. 55:672–679.
27. Richmond, K. E., K. Chenault, J. L. Sherwood, and T. L. German. 1998.
Characterization of the nucleic acid binding properties of tomato spotted
wilt virus nucleocapsid protein. Virology 248:6–11.
28. Rizvanov, A. A., S. F. Khaiboullina, and S. St. Jeor. 2004. Development of
reassortant viruses between pathogenic hantavirus strains. Virology 327:225–
29. Rodriguez, L. L., J. H. Owens, C. J. Peters, and S. T. Nichol. 1998. Genetic
reassortment among viruses causing hantavirus pulmonary syndrome. Virol-
30. Schmaljohn, C., and B. Hjelle. 1997. Hantaviruses: a global disease problem.
Emerg. Infect. Dis. 3:95–104.
31. Schmaljohn, C. M. 1996. Molecular biology of hantaviruses. Plenum Press,
New York, N.Y.
32. Schmaljohn, C. S., and J. W. Hooper. 2001. Bunyaviridae: the viruses and
their replication, p. 1581–1602. In K. A. Howley (ed.), Fields virology, vol. 2.
Lippincott, Williams, and Wilkins, Philadelphia, Pa.
33. Schmaljohn, C. S., and C. B. Jonsson. 2001. Replication of hantaviruses, p.
15–32. In S. A. Nichol (ed.), Hantaviruses. Springer-Verlag, Berlin, Ger-
34. Schmaljohn, C. S., A. L. Schmaljohn, and J. M. Dalrymple. 1987. Hantaan
virus M RNA: coding strategy, nucleotide sequence, and gene order. Virol-
35. Severson, W., L. Partin, C. S. Schmaljohn, and C. B. Jonsson. 1999. Char-
acterization of the Hantaan nucleocapsid protein-ribonucleic acid interac-
tion. J. Biol. Chem. 274:33732–33739.
36. Severson, W., X. Xu, M. Kuhn, N. Senutovitch, M. Thokala, F. Ferron, S.
Longhi, B. Canard, and C. B. Jonsson. 2005. Essential amino acids of the
Hantaan virus N protein in its interaction with RNA. J. Virol. 79:10032–
37. Severson, W. E., X. Xu, and C. B. Jonsson. 2001. cis-acting signals in encap-
sidation of Hantaan virus S-segment viral genomic RNA by its N protein.
J. Virol. 75:2646–2652.
38. Torrez-Martinez, N., M. Bharadwaj, D. Goade, J. Delury, P. Moran, B.
Hicks, B. Nix, J. L. Davis, and B. Hjelle. 1998. Bayou virus-associated
hantavirus pulmonary syndrome in Eastern Texas: identification of the rice
rat, Oryzomys palustris, as reservoir host. Emerg. Infect. Dis. 4:105–111.
39. Xu, X., W. Severson, N. Villegas, C. S. Schmaljohn, and C. B. Jonsson. 2002.
The RNA binding domain of the Hantaan virus N protein maps to a central,
conserved region. J. Virol. 76:3301–3308.
11292MIR ET AL. J. VIROL.