Journal of Virological Methods 130 (2005) 154–156

Short communication

Improved rapid amplification of cDNA ends (RACE) for mapping boththe 5′ and 3′ terminal sequences of paramyxovirus genomes

Zhuo Lia,b, Meng Yub, Hong Zhanga, Hai-Yan Wanga, Lin-Fa Wangb,∗a Renal Division and Institute of Nephrology, Peking University First Hospital, Beijing, China

b CSIRO Livestock Industries, Australian Animal Health Laboratory, 3220 Geelong, Vic., Australia

Received 25 April 2005; received in revised form 16 June 2005; accepted 23 June 2005Available online 1 August 2005

Abstract

Rapid amplification of cDNA ends (RACE) is a powerful PCR-based technique for determination of RNA terminal sequences. However,most of the RACE methods reported in the literature are developed specifically for the mapping of eukaryotic transcripts with 3′ poly-Atail and 5′ cap structure. In this study, an improved RACE strategy was developed which allows both 5′ and 3′ RACE of paramyxovirus

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genomic RNA using the same set of common molecular biology reagents without having to rely on expensive RACE kits. MaRNA genome terminal sequences is an essential part of characterizing novel paramyxoviruses since these sequences contasignals for genome replication and transcription, and are important molecular markers for studying virus evolution. The usefulnstrategy was demonstrated by rapid characterization of both genome ends for a novel paramyxovirus recently isolated from humprimary cells. The RACE strategy described in this paper is simple, cost-effective and can be used to map genome ends oviruses.© 2005 Elsevier B.V. All rights reserved.

Rapid amplification of cDNA ends (RACE) is a pow-erful PCR-based technique for determination of RNA ter-minal sequences (Shaefer, 1995). Since the first descriptionof RACE, there have been many modified RACE strategiesreported (Shaefer, 1995; Edwards et al., 1991; Trout et al.,1992; Fromont-Racine et al., 1993; Liu and Gorovsky, 1993).However, all of these methods were developed specificallyfor the mapping of eukaryotic transcripts.Tillett et al. (2000)reported a simple 5′ RACE method for the mapping of bac-terial mRNA transcripts, which does not rely on the 5′ capstructure of eukaryotic mRNA. Here we report an improvedstrategy which enables both 5′ and 3′ RACE of viral genomicRNA using the same set of reagents as described byTillettet al. (2000).

Viruses in the family Paramyxoviridae are composedof a non-segmented, negative sense, single-stranded RNAgenome of approximately 15–19 kb in length (Lamb and

∗ Corresponding author. Tel.: +61 3 5227 5121; fax: +61 3 5227 5555.E-mail address: Linfa.wang@csiro.au (L.-F. Wang).

Kolakofsky, 2001; Wang et al., 2003). The genome terminsequences are highly conserved and there is complemity between the 3′ and 5′ termini. These conserved terminsequences, especially the first 12–13-nt, are believed totain the genome and anti-genome promoters essentireplication and transcription (Lamb and Kolakofsky, 2001),and are useful markers for classification of new virusesstudying virus evolution in the family (Wang et al., 2003).Hence, the determination or mapping of virus genomemini, both the 5′ and 3′ terminal sequences, is an essentialof the molecular characterization of novel paramyxoviru

During the study of angiotensin II-regulated geneshuman kidney mesangial cell line, two cDNA sequencesisolated which were up-regulated (Liang et al., 2003). Laterit was discovered that these cDNAs were homologouparamyxovirus genes coding for the P, M and F proteins,highest homology to those proteins ofJ-virus (Miller, 2004;Jack et al., 2005). Subsequently, a novel paramyxovirtentatively namedBeilong virus (BeV), has been isolatefrom this cell line in our group, and the full-length geno

0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.jviromet.2005.06.022

Z. Li et al. / Journal of Virological Methods 130 (2005) 154–156 155

sequence determined (Li et al., manuscript in preparation).Using a similar strategy as described inWang et al. (2003), thesequences of the 3′ leader and 5′ trailor regions were deter-mined using a genome-end primer of closely related virusand a BeV-specific primer. However, exact sequence of theterminal 13-nt on each end was not known due to the factthat they were derived from the primer sequence, rather thanthe native BeV sequence. The RACE method ofTillett et al.(2000)was employed to map these 13-nt terminal sequences.In the past it has been shown that paramyxoviruses containtrace amounts of the anti-genome (Kolakofsky and Bruschi,1975), which is an exact complementary copy of the negativesense genome, and mapping the 5′ end of the anti-genome canbe used to deduce the 3′ end sequence of the genome (Tidonaet al., 1999; Bowden et al., 2001; Harcourt et al., 2001; Milleret al., 2004). However, due to the low abundance of the anti-genome, this approach does not always work. For BeV, the 5′RACE worked well for the genome RNA, but failed for theanti-genome RNA despite multiple attempts using differentvirus preparations and primer sets.

To overcome this problem, we tried a modified RACEstrategy for direct mapping of the 3′ end of the genomic RNA.As shown inFig. 1, the 3′ RACE method uses the identicalset of reagents as for the 5′ RACE and the two RACE meth-ods differ only in the order of adaptor ligation and cDNAsynthesis. In the 5′ RACE, adaptor ligation was carried out

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after cDNA synthesis whereas in the 3′ RACE the order wasreversed. Our improved protocols are as follows:

5′ RACE: An aliquot of approximately 1�g RNA, puri-fied from virus pellet using the RNeasy Mini Kit (Qiagen,Germany), was incubated at 65◦C for 5 min to removethe secondary structure, followed by cDNA synthesis usingthe Thermoscript RT-PCR System Kit (Invitrogen, USA)and a virus-specific primer VSP5-1, located 400 bp down-stream of the 5′ end. The reaction mixture was incubatedat 65◦C for 60 min followed by inactivation at 85◦C for5 min and treatment with two units of RNaseH for 20 minat 37◦C. After purification using the QIAquick PCR Purifi-cation Kit (Qiagen, Germany), the cDNA was ligated with a 3′end cordecypin-blocked adaptor DT88 (5′-GAAGA GAAGGTGGAA ATGGC GTTTT GG-3′) using T4 RNA ligase (NewEngland Biolabs, USA) following procedures as described(Tillett et al., 2000). The resulting adaptor-ligated cDNA wasthen amplified using a second virus-specific primer VSP5-2, located internal to VSP5-1, and the 27-nt primer DT89which is complementary to the adaptor (5′-CCAAA ACGCCATTTC CACCT TCTCT TC-3′). The PCR amplification wasconducted in a 25-�l reaction using the Platinum PCR Super-mix Kit (Invitrogen, USA), 20 pmol of each primer and 1�l ofa 1:10 diluted cDNA mix using the GeneAmp 2400 thermo-cycler (Applied Biosystems, USA) with the following cycling

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ig. 1. Schematic diagram for comparison of the two RACE methodsegative sense RNA genome (dotted line) is represented in 3′–5′ orientationhe solid line represents cDNA. For 5′ RACE, cDNA synthesis was carriut first using a virus-specific primer-1 (VSP5-1), followed by RNa

reatment, then ligation of adaptor (DT88). In 3′ RACE, adaptor ligation waarried out first directly with the viral RNA, followed by cDNA synthesing the DT89 primer complementary to adaptor DT88. The rema

teps of the primary and hemi-nested PCRs are the same for both RACEethods with the exception of the primers used. For primary PCR, DT89as used with either VSP5-2 in 5′ RACE or VSP3-1 in 3′ RACE. For hemi-ested PCR DT89 was used with either VSP5-3 in 5′ RACE or VSP3-2 in′ RACE.

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parameters: 94C for 4 min, followed by 35 cycles of 94Cfor 20 s, 55◦C for 20 s, 72◦C for 30 s, and a final 7 min exten-sion at 72◦C. A secondary PCR (i.e. a hemi-nested PCR) wcarried out using DT89 and VSP5-3, located 200 bp dowstream of the 5′ end, using essentially the same conditionsthe primary PCR and replacing the cDNA template with 1�lof a 1:100 diluted primary PCR product.

3′ RACE: Adaptor ligation was carried out first beforecDNA synthesis as follows: 1�g of RNA, 20 pmol of DT88,2�l of 10× RNA ligase buffer, 20 U of T4 RNA ligase, andRNase-free water to a final volume of 20�l were mixed andincubated at 37◦C for 1 h. A 2�l-aliquot was taken out tomake cDNA using 20 pmol of primer DT89 and the Thermoscript RT-PCR System Kit (Invitrogen). The reaction waconducted at 65◦C for 60 min followed by inactivation at85◦C for 5 min and RNase treatment as above. PCR wperformed as for the 5′ RACE above using 1�l of 1:10diluted cDNA, 20 pmol each of primer DT89 and VSP3-1The hemi-nested PCR was performed using 1�l of 1:100diluted primary PCR products as template, 20 pmol eachDT89 and a nested gene-specific primer VSP3-2, locat100 bp from the 3′ end.

PCR products of expected size were purified usinthe QIAquick Gel Purification Kit (Qiagen, Germany)sequenced directly using VSPs or, if the PCR band is weacloned into pCR-Blunt-II TOPO vector (Invitrogen, USA) forsequencing using vector-specific primers. Using this stratewe were able to directly determine the 3′ terminal sequenceof the genome RNA (Fig. 2). Interestingly, using the same 3′RACE approach, we also succeeded in directly mapping t3′ end of the anti-genome and confirmed that it had the ex

156 Z. Li et al. / Journal of Virological Methods 130 (2005) 154–156

Fig. 2. Sequences of the genome termini ofBeilong virus. (A) Trace fileillustrating the 3′ end region of the viral RNA genome, given as cDNA in theanti-genome sense (5′–3′). Nucleotides 1–27 were derived from the adaptorprimer DT89, whereas nucleotide 28 represents the last nucleotide of thegenome (3′) or the first nucleotide of the anti-genome (5′) and (B) alignmentof the genome terminal sequences derived using the two different RACEmethods. The 3′ terminal sequence is presented on top as the anti-genome(5′–3′) so that it can be directly compared to the 5′ genome terminal sequence,also presented in 5′–3′ orientation. The first 13-nt of both sequences areshaded, and the lower case letters indicate nucleotides in the first 13-nt ofthe 5′ genome terminal region that differ from those in the 3′ genome end.

complementary sequence of the 5′ end of the genome (datanot shown). This would suggest that the 3′ RACE strategy ismore sensitive than the 5′ RACE strategy, at least in this case,and is better suited for mapping viral RNA of low abundance.

The method byTillett et al. (2000)is simple and does notrequire an expensive kit to achieve 5′ RACE. In this study, wehave further improved this method by changing conditions forRNA purification, cDNA synthesis and purification, and byincreasing the adaptor concentration. Most importantly, bychanging the order of adaptor ligation and cDNA synthesis,we have shown that the same set of reagents can now be useto conduct both 5′ and 3′ RACE. It is now possible to usethis combined RACE strategy to map the two ends of boththe genome and anti-genome RNA molecules of any newparamyxovirus. This will undoubtedly enhance the reliabilityand success rate of mapping paramyxovirus genome endsThis same cost-effective RACE strategy should be equallyapplicable to the mapping of termini for other RNA virusgenomes, including those from positive sense RNA virusesor double-stranded RNA viruses.

Acknowledgements

This work is supported in part by National Natural ScienceFund of PR China (Grant No. 30370651) and the NationalH m of

China (863 Program, Grant No. 2002BA711A01-18). Wethank Eric Hansson and Tony Pye for technical assistancewith cell culture and DNA sequencing.

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