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Virus Research 124 (2007) 237–244

Short communication

A single stem-loop structure in Tacaribe arenavirus intergenic region isessential for transcription termination but is not required for a correct

initiation of transcription and replication

Nora Lopez ∗, Marıa T. Franze-Fernandez 1

Centro de Virologıa Animal (CEVAN)2, Consejo Nacional de Investigaciones Cientıficas (CONICET), Serrano 669, C1414DEM Buenos Aires, Argentina

Received 2 May 2006; received in revised form 17 August 2006; accepted 20 October 2006Available online 27 November 2006

bstract

The genome of Tacaribe virus (TV), prototype of the New World arenaviruses, comprises two RNA segments each encoding two proteins in anmbisense orientation separated by an intergenic region (IGR). We used a TV minireplicon system to investigate the nature of the IGR structuresequired for transcription termination. We show that efficient generation of subgenomic (SG) RNAs is related to a single hairpin structure comprisingstem with variable numbers of uninterrupted base pairs and stabilized by high �G values. The low ability of highly stable hairpin structures

omprising bulged stems to support SG RNA synthesis suggested the importance of hairpin configuration for transcription termination. Neitherhe sequences downstream nor those upstream from the hairpin played a role in SG RNA accumulation. We also show that independently of the

′ ′

GR structure the unencapsidated mRNAs contained short stretches of nontemplated bases at their 5 ends which are capped, whereas the 5 endsf the nucleocapsid-associated antiminigenomes contained an uncapped extra residue. The results support the conclusions that: (i) transcriptionermination in TV is related to a structural element that is independent of sequence and (ii) the transcription termination signal is not required forcorrect initiation of transcription and replication.2006 Elsevier B.V. All rights reserved.

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eywords: Tacaribe arenavirus; Transcription termination

. Introduction

The family Arenaviridae comprises a unique genus (Are-avirus) which includes 23 viral species so far (Charrel etl., 2003). Tacaribe virus (TV) is the prototype of the Neworld group of arenaviruses. Within this group the viruses

an be discriminated into three monophyletic lineages, one ofhich includes TV together with the four known South Ameri-

an pathogens producers of severe hemorrhagic disease: Junin,achupo, Guanarito and Sabia viruses (Bowen et al., 1996;

∗ Corresponding author at: Centro de Virologıa Animal (CEVAN-CONICET),aladillo 2468, C1440FFX Buenos Aires, Argentina. Tel.: +54 11 4686 6225;ax: ++54 11 4686 6225.

E-mail address: noralopar@yahoo.com.ar (N. Lopez).1 Present address: Catedra de Genetica y Biologıa Molecular, Facultad dearmacia y Bioquımica, Universidad de Buenos Aires, Junin 956, C1113AADuenos Aires, Argentina.2 Now at: Saladillo 2468, C1440FFX Buenos Aires, Argentina.

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168-1702/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2006.10.007

harrel et al., 2003). TV, however, seems not to be a humanathogen.

TV genome comprises two single-stranded RNA segmentsalled S and L. Each genome segment encodes two proteins: theucleoprotein (N, 64 kDa) and the glycoprotein precursor (GPC,5 kDa) are encoded by the S RNA (Franze-Fernandez et al.,987), and the L RNA encodes the RNA-dependent RNA poly-erase (L protein, 240 kDa) (Iapalucci et al., 1989b) and a small

rotein with a RING finger motif called Z (11 kDa) (Iapaluccit al., 1989a). In both S and L RNAs, the genes are arranged inpposite orientation and are separated by a noncoding intergenicegion (IGR) with the potential to form stable secondary struc-ures (Franze-Fernandez et al., 1993). Although the 5′ region ofrenavirus genomes and antigenomes are positively-stranded,hey are not translated directly into proteins. Rather, genomesnd antigenomes are found only as nucleocapsids tightly bound

o N protein and the coding sequences are expressed from subge-omic (SG) RNAs transcribed from the 3′ region of the genomesr antigenomes (Bishop and Auperin, 1987; Franze-Fernandezt al., 1993; Salvato, 1993).

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38 N. Lopez, M.T. Franze-Fernandez

The 5′ ends of arenavirus genomes and antigenomes are notapped and contain one nontemplated G residue (Garcin andolakofsky, 1990; Raju et al., 1990). This unprecedented result

ed to postulate a novel mechanism for the initiation of arenavirusenome replication (Garcin and Kolakofsky, 1992; Kolakofskynd Garcin, 1993). Arenavirus mRNAs are nonpolyadenylatedt their 3′ ends and contain short stretches of additional bases atheir 5′ ends which are capped (Garcin and Kolakofsky, 1990;

eyer and Southern, 1993; Raju et al., 1990) suggesting thatrenaviruses, as influenza viruses (Bouloy et al., 1978; Krug,981) and bunyaviruses (Bishop et al., 1983; Kolakofsky andacker, 1991; Patterson and Kolakofsky, 1984; Jin and Elliott,993), cap-snatch to initiate mRNA synthesis.

In a previous report we have shown that the 3′ ends of TVRNAs derived from both the S and L RNA segments mappedithin the IGR in each segment, suggesting that IGRs provide

ranscription termination signals. We also found that the 3′ endequences of the four mRNAs can theoretically adopt the formf a hairpin structure. On these bases we suggested that theonserved secondary structure at the 3′ end of the transcripts,ather than specific sequences, could be the termination signalor TV polymerase and proposed a model for transcription ter-ination (Franze-Fernandez et al., 1993; Iapalucci et al., 1991).ater mappings of the 3′ end termini of lymphocytic chori-meningitis virus (LCMV) and Junin virus S-derived mRNAsMeyer and Southern, 1993; Tortorici et al., 2001) were con-istent with our results, suggesting a generalized scheme forrenavirus transcription–termination. A recent report using anCMV reverse genetic system with minigenomes (MGs) con-

aining or lacking the IGR showed that SG RNA was synthesizednly by those containing the IGR (Pinschewer et al., 2005). Thistudy confirmed the role of the IGR in RNA synthesis termina-ion but provided no direct evidence concerning the nature ofhe signals involved.

We here describe experiments investigating the structuralequirements of the IGR for transcription termination. For this,e used a reverse genetic system based on synthetic TV MGs

nd plasmid-supplied TV proteins (Lopez et al., 2001). Thisllowed for IGR sequence manipulation to examine for effectsn the generation of SG RNAs. Our results showed that effi-ient termination of RNA synthesis is related to a single, highlytable hairpin structure comprising a terminal loop and a stemith variable numbers of uninterrupted bp. We also analyzed

he MG-derived RNAs on the basis of their association to theprotein and the characteristics of their 5′ ends, showing that

he transcription termination signal is not required for a correctnitiation of transcription and replication.

This study represents the first report for a member ofhe Arenavidae family providing direct experimental evidenceupporting and extending the structure-dependent model forranscription–termination.

. Results and discussion

The reverse genetic system used in our previous studies wasompetent in genome amplification and reporter gene expres-

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s Research 124 (2007) 237–244

ion and consisted of TV MGs containing the S-segment 5′ and′ untranslated regions (UTRs) as cis-acting elements and Nnd L proteins as trans-acting factors (Lopez et al., 2001). Tonvestigate the structural requirements of the IGR for generat-ng SG RNAs we then engineered plasmids expressing MGshich comprised the S-wild type or modified IGR sequences

o that the MG-derived antigenomic-sense transcripts terminat-ng within the IGR and the full length antiminigenomes (aMGs)ould be distinguished by their sizes. We first generated expres-ion plasmid pWT (Fig. 1A), which directs the synthesis of aG containing (5′ to 3′) the entire S-genome 5′ UTR sequence,

ollowed by 171 nt of the 3′ terminal region of the GPC-ORFlus the complete S-genome IGR sequence (S-IGR), then thehloramphenicol acetyltransferase (CAT) ORF in an antisenserientation and the complete S-genome 3′ UTR sequence. The-IGR sequence is depicted in Fig. 1B in the antigenomic sensend is represented in the predicted double hairpin configura-ion (Iapalucci et al., 1991). From pWT a number of expressionlasmids harboring deletions in the S-IGR sequence were con-tructed. The restriction sites used to generate deletions arendicated in Fig. 1B and the resulting deleted sequences arehown in Fig. 1C. We then analyzed the predicted IGR secondarytructure of the antigenomic-sense transcripts finding that all, bututant transcript #52, exhibited as a particular feature a single

tem-loop comprising a 4 to 16-uninterrupted bp stem and a 3 to-nt terminal loop. Nucleotides committed to adopt this type ofairpin configuration are depicted in each sequence (Fig. 1C) andhe prototypical stem-loop configuration is shown in Fig. 1D, inhe structure of transcript #53-predicted hairpin. Regarding tran-cript #52, most of its sequence (indicated in brackets in Fig. 1C)as predicted to take up a hairpin configuration comprising a-nt terminal loop and a stem with short (2–4)-bp stretches sepa-ating two internal loops and a bulge loop (Fig. 1D). A schematicepresentation of the resulting IGR-predicted secondary struc-ure of the transcripts and the free energy stabilizing each hairpin�G) are indicated in Table 1.

We then used the reverse genetic system (Lopez et al., 2001)o study the RNAs generated upon transfection with the WT andGR-mutant plasmids. We also included p23 (former pGenCATLopez et al., 2001)) which is devoid of the IGR. Northern blotnalysis of MG-derived RNAs hybridized with a negative-senseAT probe (Fig. 2) showed that consistent with our previousndings (Lopez et al., 2001), when p23 was co-transfected

ogether with plasmids expressing the TV N protein (pN) and theV L protein (pL) (Lopez et al., 2001), a single band appeared

hat would comprise both, the aMG and the putative CAT mRNAA, lane 1). Upon co-transfection of pWT with support plasmidsL and pN, two bands were clearly detected: a slower band hav-ng the size and polarity predicted for the full length aMG and aaster one with the size expected for a SG RNA terminating intohe IGR (A, lane 3; B, lane 2), whereas no band was detectedhen the WT MG was coexpressed with N or L individually (A,

ane 2; B, lane 1). Two bands having the sizes expected for the

ntigenomic full length and SG RNA species also appeared upono-transfections with plasmids #s 72, 59, 64, 53 and 50 (A, lanes, 7–9 and 11) meanwhile with plasmids #s 70, 67 and 52, theand with the size expected for the corresponding SG RNA was

N. Lopez, M.T. Franze-Fernandez / Virus Research 124 (2007) 237–244 239

Fig. 1. Expression plasmid pWT and IGR sequences of WT and deletion-mutant transcripts. (A) Schematic representation (not to scale) showing plasmid pWT, whichwas engineered by inserting a DNA fragment comprising nucleotides (nt) 1350–1610 of genomic TV S RNA into the SnaBI site of pGenCAT (Lopez et al., 2001).Nucleotides were numbered considering as 1 the 5′ end of TV S genome. The dashed box represents the 3′ terminal region of TV GPC ORF. T7Pr: T7 polymerasepromoter; HDV Rz: Hepatitis delta virus ribozyme; T�: T7 RNA polymerase terminator. (B) Nucleotide sequence (in the antigenomic sense) and predicted secondarystructure of the WT transcript-IGR. The proximal (P) and distal (D) stem-loops are indicated. Arrows show key restriction endonuclease sites: Sa: SacII, Sm: SmaI,Sf: SfiI, Bs: BsaiI and Ac: AccI. (C) The multiple sequence alignment of the relevant IGR sequences (antigenomic sense) of WT and deletion-mutant transcripts wasperformed by using the Clustal X version 1.82 program (Thompson et al., 1997). The CAT (N) stop codon and the following 13 nt of the IGR have been omittedfrom the alignment. The gaps in the alignment (dashes) correspond to the deletions introduced after digestion with the following endonucleases: BsaAI-SmaI (p70),BsaAI-SfiI (p72), SacII-AccI (p67), SfiI-AccI (p59), SmaI-AccI (p64), SacII-SfiI (p53), SfiI-SmaI (p50) and SacII-SmaI (p52). Open horizontal boxes in the WTsequence show the P and D stem-loops. Nucleotides committed to adopt a hairpin configuration are indicated in bolded-italics in each IGR sequence (see D). Thecomplementary sequence to the stop codon of TV GPC gene is indicated with a vertical open box. Arrowheads in B and C indicate the previously mapped transcriptiontermination site for N mRNA (Iapalucci et al., 1991). (D) Predicted hairpin structures in the antigenomic sense-IGR sequence of representative mutant transcripts,generated by using mfold program (version 3.2; (Mathews et al., 1999; Zuker, 2003)). The 5′ and 3′ ends of the RNAs are indicated. A stem-loop configuration asthat represented for transcript #53, is predicted for transcripts #s 59, 64 and 50 with a 16 uninterrupted-bp stem. Transcripts #s 72, 70 and 67 exhibited 13, 5 and4 g into ′U includ2 t of D

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uninterrupted-bp stems, respectively. Mutant p49 was constructed by insertinTR, comprising nt 10 270 to 10 442 and 10 618 to 10 723. This last fragment005). The secondary structure represented in transcript #49, corresponds to tha

arely detected or undetectable (A, lanes 5, 6 and 10). The RNAands from 3 independent experiments were quantified and theeans are represented in Table 1 as the ratio of SG RNA to totalNA transcribed by each MG. The results showed that MGs #s9, 64, 50, 53 and 72 expressed levels of SG RNA comparableo that of the WT whereas MGs #s 52, 70 and 67 produced frombout 7-fold less (#52) to almost undetectable (#67) amountsf SG RNA. CAT activity, which represents antigenomic RNActive in translation, was detected for all MGs (not shown).

The ability of the WT and S-IGR deletion-mutant MGs to pro-uce SG RNA was then related to the IGR-predicted secondarytructure of the transcripts (Table 1). It was observed that effi-ient production of SG RNA by MGs #s 59, 64, 50, 53 and 72 was

ssociated to a transcript-predicted single hairpin comprising atem varying in length from 11 to 16 uninterrupted-bp, stabilizedy �G values ranging from −26 to −34 kcal/mol, whereas theery low or undetectable amounts of SG RNA generated by

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the SnaBI site of pGenCAT two DNA fragments from Dengue virus (DV) 3ed the highly structured DV 3′ terminal stem-loop (DV-3′ SL) (Alvarez et al.,V-3′SL.

Gs #70 and #67 was related to shorter hairpins comprising,espectively, a 5- and a 4-bp stem and weaker free energy val-es (see Fig. 1 and Table 1). The observation that the level ofG RNA expressed by mutant #52 was remarkably low as com-ared i.e. with that of #53 suggested that either the stability ofts hairpin would be insufficient or its configuration might play

role in the efficiency of SG RNA production (Table 1 andig. 1D). In order to get an insight into this point, we investi-ated whether the strong 3′ terminal stem-loop of Dengue virusDV-3′ SL) would be functional to generate SG RNA as it com-rises a stem exhibiting several interior loops separated by 3-o 7-bp stretches (Fig. 1D) and the stability of this structure−36 kcal/mol) is higher than any of those calculated for the

T and deletion mutant-predicted hairpins (Table 1). For this,e constructed plasmid p49 in which the S-IGR sequence was

ubstituted by DNA fragments from DV 3′ UTR in order toocate the DV-3′ SL sequence in the same position as the P hair-

240 N. Lopez, M.T. Franze-Fernandez / Virus Research 124 (2007) 237–244

Table 1Relationship between IGR secondary structure and SG RNA production

IGRa Transcriptb �Gc S/G total (%)d

WT −34, −33 49

#59 −34 49#64 −34 48#50 −34 51#53 −26 49#72 −29 34#70 −6 3#67 −3 <1

#52 −18 7

#49 −36 15

#10 −34, −33 44

a Schematic representation of the WT and mutant IGR secondary structures. The 61-nt spacer sequence (including positions 10–71 of genomic TV S RNA) intranscript #10 is indicated with a solid bar.

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b Transcript numbers were defined in Fig. 1.c �G values were estimated for the hairpin structure.d Ratio of SG over total (antigenome plus SG) RNA. The relative variability intra-s

ig. 2. Northern blot analysis of total intracellular RNA from cells transfected withhe recombinant vaccinia virus vTF7-3 which expresses the T7 RNA polymerase (Fubsequently transfected with the indicated MG-expression vector and, when indicantracellular RNA was purified following the procedure of Chomczynski and Sacchi (19egative-sense CAT riboprobe. RNA bands were visualized by autoradiography usingnd quantified with ImageQuant software (Amersham Biosciences, GE Healthcare, Faizes (in kb) of marker RNAs (Promega, Invitrogen) are indicated on the right side o

ample ranged from 8 to 17%.

expression plasmids. Subconfluent cultures of CV1 cells were infected withuerst et al., 1986), as previously described (Jacamo et al., 2003). Cells wereted by (+), with support plasmids pN and pL. At 24 h after transfection total87) and analyzed by Northern blotting. Blots were hybridized with a 32P-labeledBioMax Films (Kodak, Rochester, NY) or by exposure on a Phosphorimager

irfield, CT). Results in panels A and B correspond to independent experiments.f each panel.

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in loop relative to CAT stop codon (Table 1). We found thathe efficiency of MG #49 for generating SG RNA was abouthree-fold less compared to that of the WT and twice that of

utant #52 (Fig. 2B and Table 1), suggesting that in addition totability, hairpin configuration is important for the terminationf RNA synthesis.

The distance between the protein-stop codon and the pre-icted secondary structure within the S-IGR of differentrenaviruses is variable, ranging from 6 to no more than 27 nt.n order to examine whether the position of the P stem-loop rel-tive to CAT stop codon would affect SG RNA synthesis, wengineered MG #10 so that its derived transcript contained andditional 61-nt spacer sequence unrelated to the IGR sequence,mmediately upstream the hairpin. Then, the distance betweenAT stop codon and the predicted P stem-loop would be three

imes longer than in the WT (Table 1). Comparison of the amountf SG RNA expressed by the WT and #10 MGs (Fig. 2B andable 1) indicated that the level of SG RNA production was

ndependent from the distance between CAT stop codon andhe stem-loop as well as on the sequence context upstream theairpin.

From these results it can be concluded that efficient termi-ation of RNA synthesis is related to a single hairpin structureomprising a terminal loop and a stem with variable numbersf uninterrupted bp and stabilized by high �G values (Fig. 1Dnd Table 1). The low ability of highly stable hairpin structuresomprising bulged stems to support SG RNA synthesis stronglyuggested that hairpin configuration in addition to its strengths important for RNA synthesis termination (Table 1, mutants52 and #49). It was also found that the distance between CATtop codon and the stem-loop structure does not influence theevel of SG RNA produced (Table 1, mutant #10). Finally, itas shown that neither the sequences downstream nor those

pstream from the hairpin play a role in the level of SG RNAccumulation (Fig. 1C and Table 1). This is at variance withrokaryotic transcription termination or with metazoan histoneRNA 3′ processing, which require the presence of two cis-

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ig. 3. Analysis of encapsidated and unencapsidated RNAs. CV1 cells were either iransfected with the MG-expressing plasmids pWT, p67, p52 or p59 as indicated on tL and incubated for 24 h. Purified total intracellular RNA (lanes T) was obtained as inlanes Nc) as previously described (Lopez et al., 2001), or purified from nucleocapsidndicated before (Iapalucci et al., 1991). Unencapsidated RNA (lanes P) was recoverel., 1991). Purified RNA from 1 × 105 to 3 × 105 cells (lanes T, Nc and B) or from 1 ×els, blotted and hybridized to a 32P-labeled antisense N (lanes 1–3) or CAT (lanes 4xperiments. Arrowheads on the left panel show the positions of TV S-antigenome (S-NA bands were quantified as in Fig. 2. The amount of full length-size RNA recover

otal RNA (lanes 1 and 4).

s Research 124 (2007) 237–244 241

cting conserved sequence elements: a stem-loop structure andconserved downstream sequence (Gick et al., 1986; Marzluff

nd Duronio, 2002; Platt, 1986; Yarnell and Roberts, 1999). Ouresults support the conclusion that termination of RNA synthe-is in TV is related to a structural element that is independent ofequence.

To interpret results on RNA synthesis termination, thexpressed RNAs should be distinguished by their peculiarroperties. For this purpose, we determined the nature of theranscripts synthesized by the WT and some mutant MGs inerms of both, its association with N protein and the character-stics of their 5′ ends. We expected that to behave as authenticV RNAs: (i) the aMGs should be associated with the N pro-

ein forming nucleocapsids banding at a density of 1.31 g/ml onsCl density gradients and the putative CAT mRNAs should benencapsidated, sedimenting in the pellet fraction of the gra-ient (Iapalucci et al., 1991; Raju et al., 1990) and (ii) the 5′nds of CAT mRNAs should possess 1–5-nt extensions rela-ive to the genomic template which should be capped, whereashe nucleocapsid-associated RNAs should contain an additionalncapped G residue at their 5′ ends (Garcin and Kolakofsky,990, 1992; Raju et al., 1990).

We analyzed first the encapsidated and unencapsidated RNAsxpressed by the WT and mutants #s 59, 52 and 67 MGs. Mutantsere selected considering that #59 yielded similar levels ofG RNA as the WT whereas mutants #52 and #67 producedither very low (#52) or undetectable (#67) amounts of SGNA (Table 1). Fig. 3 shows Northern blots of total intracellularNA (lanes T), encapsidated RNA (lanes Nc and B) and unen-apsidated RNA (lanes P) obtained from plasmid-transfectedr TV-infected cell extracts. Blots were hybridized with anntisense-CAT probe to visualize the aMGs and putative CATRNAs (lanes 4–17) or with an antisense-N probe to detect S

ntigenome and N mRNA (lanes 1–3). The results indicated thatmmunoselection with anti-N antibodies is a reliable procedureor the analysis of nucleocapsids since only TV S-antigenome,hich is always encapsidated (Iapalucci et al., 1991; Kolakofsky

nfected with TV (lanes 1–3) as previously described (Jacamo et al., 2003), orop (lanes 4–17). All dishes were co-transfected with support plasmids pN andFig. 2. Encapsidated RNA was extracted from nucleocapsids immunoselected

s sedimenting (lanes B) at a density of 1.31 g/ml in a CsCl density gradient asd from the pellet fraction of the CsCl density gradient as described (Iapalucci et106 to 3 × 106 cells (lanes P) was analyzed in 1.5% denaturing agarose-MOPS–17) riboprobe. Results in central and right panels correspond to independent

aG) and N-mRNA. The sizes (in kb) of commercial RNA markers are indicated.ed from immunoselected nucleocapsids (lanes 2 and 5) was about 50% that of

2 / Virus Research 124 (2007) 237–244

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Fig. 4. Primer extension and selection of capped RNA-cDNA hybrids. Subcon-fluent cultures of CV1 cells were either infected with TV or co-transfected withMG-expressing plasmids pWT or p67 with (+) or without (−) the addition of pNand pL as indicated. After 24 h of incubation, cells were harvested and used forthe isolation of immunoselected nucleocapsid-associated RNA (Nc) or unencap-sidated RNA from the pellet of a CsCl density gradient (P). Purified RNA from0.5 × 106 to 2 × 106 transfected- or TV-infected cells was annealed to 10 pmolof a primer (prTVS3344) covering nt 3344–3361 of TV S-genome. Labeledprimer extensions were then carried out as described (Bouloy et al., 1990) usingSuperScript II RT (Invitrogen). Eighty-five percent of the total reaction volumewas used to select the capped-RNA/cDNA hybrids with monoclonal anti-capantibody H20 (Bochnig et al., 1987) coupled to Protein A Sepharose, followingthe procedure described before (Lopez et al., 2001). The reaction products wereloaded onto 8% sequencing gels either before (A, lanes 1–3 and 7; B, lanes 1and 2, indicated by a (−) on the bottom of each panel) or after immunoselectionwith anti-cap antibodies (A, lanes 4–6; B, lanes 3 and 4, indicated by a (+) onthe bottom of each panel) and electrophoresed along with the sequence ladder(lanes TGC) generated by the same primer on pWT. Positions +1 and −1, areiPl

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42 N. Lopez, M.T. Franze-Fernandez

nd Garcin, 1993), was present in the immunoprecipitate (lane) whereas the N mRNA, which pelleted by centrifugation in thesCl density gradient (lane 3) was excluded. Immunoselectionith anti-N antibodies of cytoplasmic extracts from cells co-

ransfected with pWT and support plasmids showed that onlyhe aMG-size RNA was associated with the N protein to formucleocapsids (lanes 5 and 12). Further analysis by centrifu-ation in a CsCl density gradient revealed that the assembleducleocapsids banded at a density of 1.31 g/ml (lane 7) likeuthentic TV nucleocapsids whereas the SG RNA sedimentedn the pellet fraction as the authentic N mRNA (lanes 6 and 13).imilar results were obtained with mutant #59-derived RNAs,

he aMG-size RNA being encapsidated (lane 16) and the SGNA sedimenting in the pellet fraction of the CsCl density gradi-nt (lane 17). When the RNAs produced by mutant MGs #67 and52 were similarly analyzed, we found full-length RNA asso-iated with the N protein (lanes 9 and 14), the nucleocapsidsedimenting as authentic TV-nucleocapsids in a CsCl densityradient (lane 11). Since mutants #67 and #52 synthesized veryow or undetectable amounts of SG RNA (Table 1) we searchedor an unencapsidated CAT-sense full length RNA sedimentingn the pellet of their respective CsCl density gradients. However,everal attempts to detect RNA species in the CsCl pellet fromhese mutants were not successful, the RNA appearing degradedlanes 10 and 15). The instability of these RNAs might be relatedo the absence of a proper secondary structure at their 3′ ends.

The encapsidated and unencapsidated RNAs were also char-cterized on the basis of their 5′ end features (Fig. 4). For this,NAs were annealed to a primer complementary to positions9–61 from the 5′ ends of the aMGs and TV S-antigenome,rimer was extended with reverse transcriptase and the prod-cts were examined in sequencing gels together with a sequenceadder generated by the same primer on pWT (lanes TGC; posi-ion +1 sets the 3′ end terminus of the MGs and TV S-genomeGarcin and Kolakofsky, 1990; Raju et al., 1990)). This protocolas applied to the nucleocapsid-associated RNA and the CsClellet-RNA obtained from the WT MG which contained, respec-ively, the aMG-size and the SG RNA species (see Fig. 3, lanesand 6; 12 and 13). A single product migrating to position −1as demonstrated with the nucleocapsid RNA template (A, lane). We then selected the primer-extended product with anti-capntibodies before its analysis in a sequencing gel. As shown inane 4, this product was not selected with anti-cap antibodies aso band could be detected even after long exposure of the filmnot shown). When the CsCl pellet RNA was used as template,ands at positions −1, −3 and −4 over the background were pre-ominant (lane 2) whereas no bands were detected on the controlNA (lane 3). Of the products made on the WT pellet RNA, only

hose migrating to positions −3 and −4 and in minor proportiono position −2 were immunoselected with anti-cap antibodieslane 5). A pattern of bands similar to that in lane 5 was observedhen the primer was extended on the TV N-mRNA sedimenting

n the CsCl pellet from TV-infected cell extracts and the extended

roducts were analyzed either before or after immunoselectionith anti-cap antibodies (Fig. 4A, lanes 6 and 7). These resultsere confirmed by RNA ligase-mediated rapid amplification of′ cDNA ends (using the GeneRacerTM kit, Invitrogen) fol-

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ndicated on the right side of the sequence ladder. Bands were quantified on ahosphorImager. Recovery of capped N mRNA relative to total N mRNA (A,

anes 6 and 7) was about 80%.

owed by sequencing (not shown), further demonstrating thatG expression of SG RNA represents the synthesis of bona

de arenavirus mRNA.We also synthesized primer extended products on the

ucleocapsid- and CsCl pellet-RNAs derived from mutant #67s this mutant, unlike the WT, produced no SG RNA (see Fig. 2).esults were similar to those with the WT extensions: primerxtended to a single product at −1 on the nucleocapsid RNAemplate (Fig. 4B, lane 2) of which only about 5% could beelected with antibodies to cap (lane 4). When the CsCl pelletNA was used as template, products extended predominantly toositions −1, −3 and −4 (lane 1). When the latter extensions

ere immunoselected with anti-cap antibodies, products at posi-

ions −3 and −4 and in minor proportion at −2 were recoveredut not the product migrating to −1 (lane 3). Thus, althoughe could not visualize RNA species in the CsCl pellet from

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utant #67 (see Fig. 3, lane 10), the RNA was sufficiently intactor primer extension leading to demonstrate for this mutant aattern of capped-RNA/cDNA hybrids similar to those derivedrom the WT CAT-mRNA and TV N-mRNA (in Fig. 4 compare, lane 3 with A, lanes 5 and 6).

In summary, the results in Figs. 3 and 4 demonstrated thatoth, the WT and mutant #67-derived aMG-size RNAs weressociated with the N protein forming nucleocapsids banding insCl density gradients at the expected density for TV nucleocap-

ids, their 5′ ends exhibiting, like the authentic TV antigenomes,n uncapped extra residue. Moreover, the unencapsidated RNAserived from both, the WT and #67 mutant MGs containedhort stretches of nontemplated bases at their 5′ ends whichre capped, as expected for authentic TV mRNAs. It can beoncluded then, that for a correct initiation of transcription andeplication the transcription–termination signal is not requirednd the viral proteins L and N are sufficient.

The results in this report provide experimental evidence touggest a model in which the virus polymerase in the tran-cription mode initiates RNA synthesis by using capped-shortligonucleotide primers (Raju et al., 1990) and elongates theNA chain just up to the downstream side of the duplex regionf a hairpin structure, whose formation stimulates pausingucleotide addition and consequent arrest and termination, asroposed for prokaryotes (Landick, 2001). In absence of the ter-ination signal, elongation would continue to the end of the

emplate. When the virus polymerase is in the replication mode,ynthesis starts with an uncapped RNA containing a 5′ extra GGarcin and Kolakofsky, 1990, 1992) and chain elongation pro-eeds with concurrent encapsidation of the nascent RNA. Thisould create the context for the polymerase to read-through theairpin structure and to continue elongation to the end of theemplate. It should be noticed that Junin virus N protein appearso play a role as a transcriptional antiterminator in vivo (Tortoricit al., 2001).

It is interesting that primer extensions on CsCl pellet RNAserived from MGs, contained a high proportion, relative to prod-cts extending to −3 and −4, of a product extending to −1hich was not selected with anti-cap antibodies (Fig. 4A, lanesand 5; B, lanes 1 and 3). This result cannot be explained by

ucleocapsid contamination of the pellet since, at least in theT-derived pellet, no aMG-size RNA could be detected (see in

ig. 3, lanes 5 and 6; 12 and 13). A possible explanation for thisnding is that the CsCl pellet fraction obtained from MGs con-

ained transcripts initiated in the replicative mode which, in thebsence of encapsidation either aborted chain elongation shortlyfter initiation as has been described in Sendai virus infectionsVidal and Kolakofsky, 1989) or continue elongation up to theermination signal. In any case, this observation might suggesthat arenavirus transcriptases and replicases might somehow beifferent independently of assembly, as recently reported foresicular stomatitis virus RNA polymerase (Qanungo et al.,004). It should be noticed that the reconstituted system used

n this study was settled for maximal reporter gene expressionLopez et al., 2001) and the amount of N could be a limitingactor for nucleocapsid assembly. This system will be useful toearn more about how arenavirus RNAs are initiated.

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cknowledgements

We thank Dr. Reinhard Luerhmann for kindly providing thenti-cap antibody H20 and to Dr. A.V. Gamarnik for the plasmidhich harbors the DV-3′ SL sequence. We are grateful to Dr.. Moss for providing the recombinant vaccinia virus vTF7-. Special thanks to Dr. M. Bouloy for critically reading theanuscript. We also thank Rodrigo Jacamo, Maximiliano Wilda

nd Juan Cruz Casabona for helpful discussions. The technicalssistance of J. Acevedo and S. Rojana is acknowledged. NLnd MTFF are research investigators of CONICET. This workas supported by Agencia Nacional de Promocion CientıficaTecnologica (ANPCyT) and a grant from the Institut Pasteur

Paris) through AMSUD/Pasteur.

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