10.1128/JVI.75.6.2646-2652.2001.

2001, 75(6):2646. DOI:J. Virol. William E. Severson, Xiaolin Xu and Colleen B. Jonsson RNA by Its N ProteinHantaan Virus S-Segment Viral Genomic

-Acting Signals in Encapsidation ofcis

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JOURNAL OF VIROLOGY,0022-538X/01/$04.0010 DOI: 10.1128/JVI.75.6.2646–2652.2001

Mar. 2001, p. 2646–2652 Vol. 75, No. 6

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

cis-Acting Signals in Encapsidation of Hantaan Virus S-SegmentViral Genomic RNA by Its N Protein

WILLIAM E. SEVERSON,1 XIAOLIN XU,1 AND COLLEEN B. JONSSON1,2*

Graduate Program in Molecular Biology1 and Department of Chemistry and Biochemistry,2

New Mexico State University, Las Cruces, New Mexico

Received 17 October 2000/Accepted 16 December 2000

The nucleocapsid (N) protein encapsidates both viral genomic RNA (vRNA) and the antigenomic RNA(cRNA), but not viral mRNA. Previous work has shown that the N protein has preference for vRNA, and thissuggested the possibility of a cis-acting signal that could be used to initiate encapsidation for the S segment.To map the cis-acting determinants, several deletion RNA derivatives and synthetic oligoribonucleotides wereconstructed from the S segment of the Hantaan virus (HTNV) vRNA. N protein-RNA interactions wereexamined by UV cross-linking studies, filter-binding assays, and gel electrophoresis mobility shift assays todefine the ability of each to bind HTNV N protein. The 5* end of the S-segment vRNA was observed to benecessary and sufficient for the binding reaction. Modeling of the 5* end of the vRNA revealed a possiblestem-loop structure (SL) with a large single-stranded loop. We suggest that a specific interaction occursbetween the N protein and sequences within this region to initiate encapsidation of the vRNAs.

Hantaviruses are tripartite, negative-stranded viruses har-bored by a variety of rodents in the Muridae family, which aredistributed throughout the world. Transmission of these vi-ruses to humans occurs through inhalation of rodent excretaand can result in one of two illnesses depending on the virus,hantavirus pulmonary syndrome (HPS) or hemorrhagic feverwith renal syndrome (HFRS) (10). Because of the geographi-cal distribution of the rodent reservoirs of these viruses, HPShas emerged as a significant illness throughout the Americas,while HFRS is limited to the Old World. One of the moresevere HFRS illnesses is caused by Hantaan virus (HTNV),which causes death in 5 to 15% of the cases (5, 6).

The hantavirus genome encodes an RNA-dependent RNApolymerase (RdRp) (L segment), a nucleocapsid (N) protein(S segment), and two glycoproteins, G1 and G2 (M segment)(11, 12). Minimally, the components of replication include theRdRp, the N protein, and the virus genomic and antigenomicRNA templates. Following entry of the virion into the cyto-plasm, the RdRp initiates viral cRNA synthesis from the L, M,and S viral genomic RNAs (vRNAs). Additional vRNA issynthesized from the cRNA. Both vRNA and cRNA are com-plexed with the N protein throughout transcription and repli-cation, but mRNA is not (3). The cis-acting viral sequenceswhich promote the specific interaction of N with vRNA andcRNA during viral replication are unknown. It is likely thatsequences or structures present in the 59 ends of the RNAmolecules would provide a point of nucleation for subsequentencapsidation of the entire genomic or antigenomic segment(8).

Interaction of the HTNV N protein with its vRNA showslittle ionic strength dependence (13). However, increase in theionic strength of the binding reaction greatly reduced the

HTNV N protein’s affinity for nonviral RNA. In the samestudy, filter-binding assays demonstrated a moderate bindingpreference of the HTNV N for the S-segment vRNA com-pared to an RNA comprising only the open reading frame(ORF) of the S segment and a strong preference for vRNAcompared to nonspecific RNA. These studies suggested thatthe HTNV N protein may specifically recognize its vRNA at aunique structure and/or sequence. Herein, we explore the hy-pothesis that the HTNV N protein recognizes this signal forencapsidation and assembly of the nucleocapsid. To map thecis-acting RNA sequences required for HTNV N protein in-teractions, in vitro-transcribed RNA derivatives of the HTNVS segment and synthetic oligoribonucleotides based on the 39vRNA end and the 59 vRNA end were explored. Our resultssuggest a model for encapsidation of the vRNA that entailsboth specific and nonspecific interactions with the N protein.We propose that a specific interaction occurs between the Nprotein and sequences in the 59 end of the nascent vRNA.

MATERIALS AND METHODS

Preparation of hantavirus RNA substrates. HTNV vRNA was transcribedfrom the vector pGEM1-HTNVS (Connie Schmaljohn, Virology Division,USAMRIID) as described previously (13). A construct containing the 59- and39-terminal untranslated sequences of the S segment, or minipan (pGEM1-MP),was made by partial restriction digestion of pGEM1-HTNVS with BamHI. Afterdigestion, the linearized fragment was gel purified and ligated, resulting in aplasmid that contains 37 bp of the 59 end and 454 bp of the 39 end of the gene.A 1,684-bp DNA fragment lacking 12 bp of the 59 sequences of the S-segmentvRNA (pTAR-12) was constructed by PCR amplification of the plasmidpGEM1-HTNVS and cloned into the pTARGET cloning vector (Promega) toproduce a deletion construct referred to as D12. An ORF RNA was generated bytranscription from the HTNV N cDNA (pHTNV-N) cloned into the NdeI andXhoI sites of pET23b. A nonspecific 67-nucleotide (nt) control RNA was tran-scribed from pGEM7Zf1 as described previously (13). Plasmid DNAs wereprepared for the in vitro transcription reactions by digesting pGEM1-HTNV Swith XbaI, pHTNV-N with XhoI, pGEM1-MP with XbaI, pTAR-12 with SmaI,and pGEM7Zf1 with SmaI. [32P]UMP-radiolabeled transcripts were producedand purified from linearized plasmids using the MaxiScript SP6/T7 RNA tran-scription kit (Ambion) as described previously (13).

Oligoribonucleotides. Oligoribonucleotides were synthesized and high-pres-sure liquid chromatography purified by Integrated DNA Technologies, Inc.

* Corresponding author. Mailing address: Department of Chemistryand Biochemistry, MSC 3C, P.O. Box 30001, New Mexico State Uni-versity, Las Cruces, NM 88003. Phone: (505) 646-3346. Fax: (505)646-2649. E-mail: cjonsson@nmsu.edu.

2646

(Coralville, Iowa). Synthesis was performed on a 1 mM scale. The oligoribonu-cleotides used in the present study are described in Table 1. The synthetic RNAswere labeled at the 59 terminus with [g-32P]ATP and T4 polynucleotide kinase(New England Biolabs) and purified on quick-spin columns (Roche).

UV cross-linking assay. UV cross-linking assays were performed as previouslydescribed (13). Briefly, in standard reactions, 175 ng of HTNV N protein/ml wasadded to reaction buffer, and reaction mixtures were incubated for 10 min at37°C. RNA-protein complexes were covalently cross-linked by exposing to 1.8kJ of UV light in a UV cross-linker (UVC500; Hoefer). Unbound RNA wasdigested by adding 1 U of RNase V1 (Amersham Pharmacia Biotech) or 50 U ofRNase T1 (Ambion) and incubating for 30 min at 37°C. Reaction products wereseparated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis.Signals were quantified using ImageQuaNT version 4.2 software (MolecularDynamics).

Filter-binding assay. The filter-binding assays were done as described previ-ously (13). RNAs were prepared by in vitro transcription in the presence of[a-32P]UTP or synthesized as described above. HTNV N protein was purified asdescribed previously (13). Briefly, HTNV N protein was serially diluted in bind-ing buffer (40 mM HEPES [pH 7.4], 40 mM NaCl, 20 mM KCl, and 1.5 mMdithiothreitol) to give a final concentration range of 3.5 3 1029 to 3.5 3 1026 M.Apparent dissociation constants (Kd) were calculated by fitting a nonlinear bind-ing curve to the empirical data using the Origin program (MicroCal). Theapparent Kd corresponds to the concentration of N protein required to obtainhalf-saturation, assuming that the complex obeys a simple binding bimolecularequilibrium. We assumed that the plateau in the percent binding of the RNArepresents complete binding of the RNA, to allow the calculation at half-satu-ration.

Competition experiments. Competition experiments were performed by filter-binding assays. A constant concentration of the HTNV N protein (3.5 3 102 7

M) was incubated with 1 ng of [a-32P]UTP-labeled RNA (0.05 nM) for 10 minat 37°C. Various concentrations (0.5 to 500 nM) of unlabeled RNA were addedto the binding interactions, and a further 10-min incubation followed. The reac-tion mixtures were slot blotted onto nitrocellulose filters as described previously(13). Analyses of competition assays were performed by a nonlinear fit of thedata using the Origin program (MicroCal).

Gel electrophoresis mobility shift assay (GEMSA). One nanogram of 32P-radiolabeled vRNA S segment, prepared as described above, was incubated witha 22.7 mM concentration of purified protein in binding buffer (20 mM HEPES[pH 7.4], 100 mM NaCl, 1 mM MgCl2, 0.2 mM dithiothreitol, 5% glycerol, and20 U of RNase inhibitor [Ambion]) in a final reaction volume of 20 ml andincubated at 37°C for 20 min. Two microliters of sample buffer (80% glycerol and0.2% bromophenol blue) was added to each reaction mixture, and the reactionproducts were loaded onto a 1% agarose gel, separated by electrophoresis in0.53 Tris-borate-EDTA at 120 V (constant voltage) for 1.5 h, and visualized byautoradiography.

Computer-predicted secondary structures of HTNV S-segment RNAs. Theoptimal and suboptimal secondary structures of the HTNV S-segment RNAswere predicted with the RNA secondary structure prediction program mfold,version 3.0 (14).

RESULTS

Determination of the region(s) within the S-segment vRNAthat interacts with the HTNV N protein specifically. An en-capsidation signal had not been previously defined for any ofthe hantaviral RNAs. Therefore, we were interested in firstdetermining whether any such signal was present in the full-

length S-segment RNA. Three deletion mutants were con-structed from the HTNV S-segment vRNA: an ORF RNA,which contains no 59 or 39 flanking untranslated sequences; aminipan RNA, which represents the region flanking the ORFRNA; and a D12 vRNA, which contains a deletion of theterminal 12 nt from the 59 end of the noncoding region. Eachof the RNAs was examined for RNA binding affinity to theHTNV N protein with a UV cross-linking assay (Fig. 1). Thebinding of the HTNV N protein to its vRNA was greater thanthat demonstrated with the ORF RNA, which showed a 4.5-fold reduction in band intensity compared to the wild-typevRNA (Fig. 1, compare lanes 1 and 2). The minipan RNA hada 3.0-fold reduction in binding in comparison to the vRNA(Fig. 1, compare lanes 1 and 3). The D12 RNA showed asimilar decrease in signal intensity compared to the full-lengthvRNA (Fig. 1, compare lanes 1 and 4).

TABLE 1. Synthetic oligoribonucleotides

RNA Sequencea

HTNV vRNA(1–39)...........................................................................................59-UAgUAgUAUgCUCCCUAAAAAgACAAUCAAggAgCAAUC-39HTNV vRNA(1661–1696).................................................................................59-CgUUgUUCUAgUAgCUCUUUAgggAgUCUACUACUA-39HTNV vRNA(1–22)...........................................................................................59-UAgUAgUAUgCUCCCUAAAAAg-39HTNV vRNA(1675–1696).................................................................................59-CUCUUUAgggAgUCUACUACUA-39HTNV cRNA(1–36)...........................................................................................59-UAgUAgUAgACUCCCUAAAgAgCUACUAgAACAACg-39HTNV cRNA(1658–1696).................................................................................59-gAUUgCUCCUUgAUUgUCUUUUUAgggAgCAUACUACUA-39Random RNA.....................................................................................................59-ACCAACAAAgAUgAgUgUUACAgCUCUUgC-39

a Lowercase letters were used solely for ease of reading.

FIG. 1. Analysis of the complex formed between HTNV N proteinand deletion RNAs by UV cross-linking analysis. The concentration ofN protein used in each binding reaction was 3.5 3 1026 M. Reactionmixtures were assembled in 100 mM NaCl with 5 mM MgCl2 inaddition to standard reaction components as described in Material andMethods. Binding reactions were separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis, and unbound RNA was di-gested by adding 1 U of RNase V1. Signals were imaged with theMolecular Dynamics Storm PhosphorImager and quantified using Im-ageQuaNT version 4.2 software (Molecular Dynamics). The RNAsused to form the complexes are HTNV S-segment vRNA (lane 1),ORF RNA (lane 2), minipan RNA (lane 3), and D12 RNA (lane 4).

VOL. 75, 2001 ENCAPSIDATION ACTIVITY OF THE HTNV N PROTEIN 2647

To corroborate the UV cross-linking results, filter-bindingexperiments were performed with increasing amounts ofHTNV N protein and a constant amount of the various dele-tion RNAs. Data was analyzed using the Klotz plot, and anapparent Kd for each complex was calculated from each bind-ing curve (Fig. 2A to E). The HTNV vRNA showed the great-est affinity for the HTNV N protein with a Kd of 53 nM (Fig.2A). The Kd of the ORF RNA-HTNV N complex was 270 nM,indicating that this interaction was 5-fold weaker than thevRNA-HTNV N protein interaction (Fig. 2B). The minipanRNA-HTNV N complex and the D12 RNA-HTNV N com-plexes had apparent Kds of 72 and 94 nM, respectively (Fig. 2Cand D). Compared to the HTNV N protein-vRNA complexes,the minipan RNA showed a 1.4-fold reduction in binding,while the D12 RNA showed a 1.8-fold reduction. The controlRNA showed very little affinity for the HTNV N protein (Fig.2E). A summary of the binding isotherms for each data set(Fig. 2A to E) is shown in Fig. 2F. According to these results,the vRNA was the preferred substrate, followed by the D12RNA, the minipan RNA, and ORF RNA. The UV cross-linking and filter-binding experiments suggested that there areregions in the vRNA that are preferred by the HTNV N pro-tein and that these regions may lie in the noncoding region.

Competition of the HTNV N protein-vRNA complex withviral RNAs. To further examine the relative contribution ofthese flanking sequences, we investigated the ability of unla-beled in vitro-transcribed D12 RNA, minipan RNA, and ORFRNA to compete with 32P-labeled HTNV vRNA in a filter-binding assay (Fig. 3). A constant concentration of the HTNVN protein (3.5 3 102 7 M) was incubated with [a-32P]UTP-labeled vRNA (0.05 nM) and increasing concentrations (0.5 to500 nM) of unlabeled competitor RNA (Fig. 3 and Table 2).As expected, the ORF RNA was not an effective competitor ofthe HTNV N-vRNA complex. The minipan RNA was not aneffective competitor of the HTNV N protein-vRNA complex.However, the D12 RNA at 500 nM concentration reducedbinding by approximately 60%, which suggested that the re-moval of nucleotides that have been reported to form a pan-handle structure in the 59 noncoding region are not importantdeterminants in the HTNV N protein-vRNA interaction. Thisalso suggests that the cis-acting determinants are singlestranded, since the D12 RNA was predicted by mfold to notform a panhandle or double-stranded structure like that ob-served with the minipan RNA (data not shown). In the UVcross-linking and filter-binding studies, the N protein showedsimilar affinities for the D12 RNA and the minipan RNA. Thecompetition experiments suggest a fundamental difference,however, in how the N protein recognizes each of these RNAs.We suggest that the competition experiments revealed theadded affinity of single-stranded versus double-stranded struc-

tures in competing for N protein binding, which was not dis-cernible in the UV and filter-binding studies.

Binding isotherms of HTNV N protein with synthetic HTNVvRNA and cRNA oligoribonucleotides. The experiments per-formed indicated that sequences in the untranslated regionswere preferred sites of interaction for the HTNV N protein. Todetermine if a specific binding affinity could be mapped tothese sequences, binding isotherms for six distinct hantaviralsingle-stranded RNA substrates and a random RNA substratewere analyzed (Table 1). Three duplex RNAs that were exam-ined were vRNA(1–39)/vRNA(1661–1696), vRNA(1–22)/vRNA(1675–1696), and cRNA(1–36)/cRNA(1658–1696). The disso-ciation constants for each binding isotherm are summarized inTable 3. The apparent Kds for full-length and the other RNAstested earlier are also summarized. The HTNV vRNA(1–39)showed the greatest preference for the N protein, with anapparent Kd of 132 nM. cRNA(1–39)/cRNA(1658–1696), vRNA(1–39)/vRNA(1661–1696), cRNA(1–39), cRNA(1658–1696),vRNA(1–22), random RNA, and vRNA(1675–1696) showed anapproximate 2- to 3-fold reduction in binding compared toHTNV vRNA(1–39). The Kd of vRNA(1661–1696) was 624 nM,or 4.7-fold greater than the constant for the vRNA(1–39)-HTNVN protein interaction (Table 3). The vRNA(1–22)/vRNA(1675–1696)-HTNV N complexes had an apparent Kd of 1,138 nM.Compared to the HTNV N protein-vRNA(1–39) complexes,vRNA(1–22)/vRNA(1675–1696) showed an 8.6-fold reduction inthe apparent dissociation constant.

Gel electrophoretic mobility analysis of synthetic vRNA oli-goribonucleotide mimics and HTNV N protein. The interac-tion between the HTNV N protein and the vRNA was alsoinvestigated with a GEMSA. Protein-RNA complex formationwith the HTNV N protein was examined for three oligoribo-nucleotide substrates: the 59 vRNA, a duplex of the 59 and 39ends, and the 39 vRNA (Table 1). The weakest protein-RNAcomplex was that comprising the N protein and the duplexsubstrate (Fig. 4, lane 3). The strongest interaction was notedwith the 59 vRNA substrate (Fig. 4, lane 2). The interaction ofthe 39 vRNA was weaker than that of the 59 vRNA substrate.These results are in agreement with those observed in thefilter-binding assay and suggest that the major determinant ofbinding of the S-segment vRNA by the N protein lies in the 59end and is not a predominantly double-stranded RNA.

DISCUSSION

The biological role of the hantavirus N protein in the viruslife cycle requires differential interactions with the three typesof viral RNAs, the vRNA, cRNA, and mRNA. During theassembly of the virion, the vRNA is packaged preferentially,which suggests an operational mechanism for selection of the

FIG. 2. Saturation binding curves of various RNA substrates and the HTNV N protein. Binding isotherms were constructed followingmeasurement of the binding of HTNV N protein with full-length S-segment vRNA (A), ORF vRNA (B), minipan RNA (C), D12 vRNA (D), andcontrol RNA (E); panel F shows a compilation of data for all RNAs. Binding reaction mixtures were assembled in 100 mM NaCl and 1 mM MgCl2in addition to standard reaction components as described in Materials and Methods. 32P-labeled RNA was incubated with the indicated molarconcentrations of N protein. The amount of radioactively labeled N protein retained on the filter was calculated relative to maximum radioactivityretained in each experiment. Apparent dissociation constants were calculated by nonlinear curve fitting and correspond to the amount of proteinnecessary to obtain 50% saturation, assuming that the concentration of free protein is equivalent to the concentration of total protein.

2648 SEVERSON ET AL. J. VIROL.

VOL. 75, 2001 ENCAPSIDATION ACTIVITY OF THE HTNV N PROTEIN 2649

vRNA that may require the N protein. The N protein willencapsidate vRNA and cRNA, but not mRNA, in a virus-infected cell. In vitro, the HTNV N protein shows a preferencefor vRNA compared to ORF vRNA. These observations arguestrongly for the presence of a unique signal for encapsidationand assembly. To determine whether the vRNA contained asignal for encapsidation within the vRNA, we used UV cross-linking, filter binding, and GEMSA to probe the interaction ofthe HTNV N protein with a panel of vRNA and cRNA sub-strates. The binding data generated herein suggest that a spe-cific binding interaction takes place in the 59 end of the S-segment vRNA.

Several deletions were constructed in the HTNV S segmentto determine whether an encapsidation signal was present inthe vRNA. Filter-binding and UV cross-linking experimentssuggested a signal in the untranslated regions of the vRNA. Tofurther confirm the relative contribution of sequences or theeffect of the absence of sequences of the minipan, D12, and

ORF vRNAs in binding the N protein, we investigated theability of unlabeled in vitro transcribed D12 RNA, minipanRNA, and ORF RNA to compete with 32P-labeled HTNVvRNA in a filter-binding assay. Neither minipan RNA norORF RNA was an effective competitor of the HTNV N pro-tein-vRNA interaction (Table 2). However, D12 RNA moder-ately reduced binding by 60%, suggesting that the nucleic acidbinding site was located in the 59-terminal sequences of thevRNA. To determine whether the 59 end of the vRNA wassufficient for the interaction, a panel of oligoribonucleotidesthat represent viral terminal sequences were synthesized and

FIG. 3. Competition experiments. Competition experiments werecarried out as described in Materials and Methods by filter-bindingassays. HTNV N protein was bound to 1 ng of [a-32P]UTP-labeledRNA (0.05 nM) in the presence of variable concentrations (0.5 to 500nM) of vRNA, ORF RNA, D12 RNA, and minipan RNA. Analyses ofcompetition assays were performed by fitting a binding curve to theempirical data using the Origin program (MicroCal).

FIG. 4. GEMSA of the HTNV N protein with 59- and 39-endvRNAs. HTNV N protein was incubated with 59-, 32P-labeled S-seg-ment vRNA oligoribonucleotide derivatives: 59-end vRNA 1–39 (lane2), a duplex RNA composed of 59-end vRNA(1–39) and 39-endvRNA(1661–1696) (lane 4), and 39-end vRNA(1661–1696) (lane 6).The reaction products were loaded onto a 1% agarose gel after anincubation period of 20 min, and the protein-RNA complexes wereseparated from free RNA by gel electrophoresis in 0.53 Tris-borate-EDTA buffer. Odd-numbered lanes show results of reactions run with-out N protein.

TABLE 2. Competition analysis of HTNV vRNA-Nprotein interactions

Competitor RNA

% Binding in presence of molar excess (fold)of competitor RNAa

None 10 100 1,000

vRNA 100 86 30 13ORF RNA 100 84 78 65Minipan RNA 100 93 76 62D12 RNA 100 80 64 41

a Values were calculated from the percentage of the labeled vRNA bound inthe presence of the unlabeled competitor RNA.

TABLE 3. Apparent dissociation constants for HTNV N proteinwith various RNAs

RNA Dissociation constant (nM)

vRNA ............................................................................ 53 6 8ORF RNA.................................................................... 270 6 32Minipan RNA .............................................................. 72 6 5D12 RNA...................................................................... 94 6 16Control RNA ............................................................... 260 6 15vRNA(1–39) ................................................................. 132 6 9vRNA(1–22) ................................................................. 350 6 0vRNA(1675–1696) ....................................................... 417 6 9cRNA(1–36) ................................................................. 298 6 53vRNA(1661–1696) ....................................................... 624 6 76cRNA(1658–1696) ....................................................... 332 6 34Random RNA.............................................................. 367 6 87vRNA(1–39)/vRNA(1661–1696)................................ 266 6 143vRNA(1–22)/vRNA(1675–1696)................................ 1,138 6 88cRNA(1–39)/cRNA(1658–1696) ................................ 245 6 35

2650 SEVERSON ET AL. J. VIROL.

FIG. 5. Secondary structures of HTNV RNAs predicted using the mfold program. The parameters for running the mfold program (version 3.0)were as follows: linear structure of the sequence, 37°C folding temperature, no limit for the maximum distance between nucleotide pairs, and abatch processing option. (A) vRNA(1–39); (B) vRNA(1661–1669); (C) cRNA(1–36); (D) cRNA(1658–1669); (E) vRNA(1–39) complexed withvRNA(1661–1669); (F) cRNA(1–36) complexed with cRNA(1658–1696).

VOL. 75, 2001 ENCAPSIDATION ACTIVITY OF THE HTNV N PROTEIN 2651

tested for their ability to bind the N protein by filter bindingand GEMSA. The greatest binding was observed with the59-end vRNA(1–39), with an apparent Kd of 132 nM, while theother viral RNAs showed a twofold or greater reduction inbinding affinity. As far as we are aware, the dissociation con-stants have not been reported for many other virus encapsida-tion complexes. Recently, a Kd of 110 (SEM, 650) was re-ported for the human immunodeficiency virus type 1nucleocapsid (NC)-SL2 complex (1). The NC-SL2 interactionwas proposed to play a direct role in specific recognition andpackaging of the full-length retroviral RNA genome. We sug-gest that the region spanning nucleotides 1 to 39 in the 59 endof the vRNA contains the major cis-acting element for specificrecognition and encapsidation. Additional sequences down-stream of this region may increase binding activity, but onlyslightly, i.e., twofold. Only one other study (2) has attempted toexamine the substrate preference of a hantavirus N protein. Incontrast to our findings for the HTNV N, Gott et al. (2)reported that the Puumala virus N protein had a twofold-higher affinity for double-stranded viral RNA over single-stranded viral RNA. Unfortunately, it is difficult to directlycompare the substrates used in our studies with those reportedfor Puumala virus N protein.

RNA sequences can form complex secondary and tertiarystructures, and we were interested in the possible secondarystructures in the putative encapsidation signal. For these rea-sons, we used mfold-generated algorithms to predict secondarystructures of the HTNV viral RNA sequences (14). The se-quence of the vRNA modeled by mfold was from position 11to 39 of the HTNV S segment (Fig. 5A). The vRNA structurefolded into stem loop structure. Inspection of this structureshows one double-helical tract (stem) that gives rise to a largeloop of unpaired nucleotides (Fig. 5A). Additional secondarystructure modeling of the untranslated regions of this segment(1 to 60, 1 to 130, and 1 to 370 nt) preserves the presence of thelarge single-stranded loop (C. B. Jonsson, data not shown).Models were also generated for the 39 end of the vRNA(1661–1696) (Fig. 5B) and the 59 and 39 ends of the cRNA (Fig. 5Cand D). In contrast to the large single-stranded region pre-dicted for the 59-end vRNA stem-loop (SL) structure, theseRNAs showed large, stable SLs with a greater amount of dou-ble-stranded RNA and greater values of DG (data not shown).Modeling of the duplex hybrids of the 59 and 39 ends of thevRNA (Fig. 5E) examined by GEMSA as well as the cRNAend (Fig. 5F) also revealed a very stable SL, which was mainlydouble stranded. Previous work in our laboratory has demon-strated that the HTNV N protein when complexed with vRNAis readily digested with RNase V1, a double-stranded nuclease,but not RNase T1, a single-stranded nuclease (13). Further,the D12 vRNA, which is predicted by mfold to be unable toform a panhandle or the SL structure in the 59 end (data notshown), competed effectively with vRNA. This suggests thatthe N protein may interact with single-stranded regions in theSL or, alternatively, a more complex structure which also hassingle-stranded regions. We propose a two-step model for en-capsidation of the viral genome (vRNA) and antigenome(cRNA) that entails both specific and nonspecific interactionswith the N protein. We hypothesize that initially, a specificinteraction occurs between the N protein and the sequences in

the single-stranded region of the predicted SL structure (Fig.5A) in the 59 end of the nascent vRNA. This is similar to whathas been observed and proposed for vesicular stomatitis virus(7) and rabies virus (4). For the second part of the model, wesuggest that the initial binding may be followed by N protein-Nprotein interactions, which could drive the nonspecific bindingof the remaining vRNA template.

In viruses, secondary structures such as consecutive hairpinsand internal loops serve as assembly sites for numerous bio-logical activities associated with the life cycles (9). We haveshown that the 59 end of the S-segment vRNA can fold into aunique secondary structure by modeling. Modeling of theHTNV M segment showed smaller SLs in the same region,although modeling of the HTNV L-segment 59 end predictedthe region to be single stranded (data not shown). At a mini-mum, this secondary structure prediction, along with deletionmapping of the RNA, suggests sequence and/or structural mo-tifs in the vRNA that allow specific interaction with the Nprotein. Future experiments will focus on how the HTNVS-segment RNA folds and on the nucleotide contacts that arecritical for protein-RNA interactions. In addition, it will beimportant to define the location of encapsidation signals forthe other two genomic segments, M and L.

ACKNOWLEDGMENTS

We thank Connie Schmaljohn for plasmids and RNAs used in thesestudies.

This research was supported by NIH grant 1RO3AI41114 to C.B.J.

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