Generation and characterization of recombinant vaccinia viruses expressing a hepatitis C virus Core protein, genotype 1b, individually or as a polyprotein

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Generation and characterization of recombinant vaccinia viruses expressing a hepatitis C virus Core protein, genotype 1b, individually or as a polyprotein
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  Generation and characterization of a recombinantRift Valley fever virus expressing a V5 epitope-tagged RNA-dependent RNA polymerase Benjamin Brennan, Ping Li and Richard M. Elliott Correspondence Richard M. Elliottrme1@st-andrews.ac.ukReceived 27 July 2011Accepted 2 September 2011 Biomedical Sciences Research Centre, University of St Andrews, North Haugh, St Andrews,Fife KY16 9ST, UK The viral RNA-dependent RNA polymerase (RdRp; L protein) of Rift Valley fever virus (RVFV;family  Bunyaviridae ) is a 238 kDa protein that is crucial for the life cycle of the virus, as it catalysesboth transcription of viral mRNAs and replication of the tripartite genome. Despite its importance,little is known about the intracellular distribution of the polymerase or its other roles duringinfection, primarily because of lack of specific antibodies that recognize L protein. To begin toaddress these questions we investigated whether the RVFV (MP12 strain) polymerase couldtolerate insertion of the V5 epitope, as has been previously demonstrated for the Bunyamweravirus L protein. Insertion of the 14 aa epitope into the polymerase sequence at aa 1852 resultedin a polymerase that retained functionality in a minigenome assay, and we were able to rescuerecombinant viruses that expressed the modified L protein by reverse genetics. The L proteincould be detected in infected cells by Western blotting with anti-V5 antibodies. Examination ofrecombinant virus-infected cells by immunofluorescence revealed a punctate perinuclear orcytoplasmic distribution of the polymerase that co-localized with the nucleocapsid protein. Thegeneration of RVFV expressing a tagged RdRp will allow detailed examination of the role of theviral polymerase in the virus life cycle. INTRODUCTION Rift Valley fever virus (RVFV) is a mosquito-bornepathogen of both livestock and humans that is foundprimarily in sub-Saharan Africa and the Arabian Peninsula.In ruminants, particularly sheep and cattle, RVFV disease ischaracterized by fetal deformities, abortion and high ratesof mortality among young animals (Smithburn, 1949).RVFV is predominantly transmitted by mosquitoes and thevirus has been isolated from more than 40 species innature, while under laboratory conditions many differentarthropods are capable of transmitting the virus (Turell et al. , 2008 and references therein). RVFV is a member of the genus  Phlebovirus   in the family   Bunyaviridae  . The viruscontains a tripartite ssRNA genome comprising twonegative-sense segments and one ambisense segment(Bouloy & Weber, 2010). The small (S) segment (approx.1.7 kb) encodes the nucleocapsid (N) protein and a non-structural protein (NSs) in an ambisense manner. The Nprotein is translated from a subgenomic mRNA that istranscribed from the genomic RNA, while NSs is translatedfrom a subgenomic mRNA that is transcribed from theantigenomic (replicative-intermediate) RNA. The medium(M) segment (approx. 3.8 kb) encodes four proteins in asingle ORF: the virion envelope glycoproteins Gn and Gc,and two other proteins called NSm1 and NSm2 (Gerrard et al. , 2007; Won  et al. , 2006). The large (L) segment(approx. 6.4 kb) encodes the viral RNA-dependent RNApolymerase (RdRp or L protein). The RNA segments areencapsidated by N protein to form ribonucleoprotein(RNP) complexes, the functional templates for viral RNAsynthesis (Ortı´n & Parra, 2006).The RVFV L protein is 2092 aa long (237.7 kDa) andassociates with the RNP to catalyse viral transcription andreplication (Lopez  et al. , 1995). Comparison of bunyavirusL protein sequences with those of other RNA polymerasesenabled the definition of four amino acid motifs, called Ato D, that comprise the ‘polymerase module’ that isconserved in all RNA polymerases (Delarue  et al. , 1990;Poch  et al. , 1989). These are located in the middle of the bunyavirus polymerase. Subsequently, a fifth motif upstream of motif A, termed pre-motif A, was identifiedand shown to be a feature of all RdRp, while a motif downstream of motif D, termed E, was found to beconserved in all segmented virus RdRp (Muller  et al. ,1994). A further conserved region downstream of motif Ehas been described that is specific for bunyavirus RdRp(Aquino  et al. , 2003). Conserved motif C contains theactual catalytic domain for nucleotide polymerization, andfor all segmented negative-sense RNA viruses this ischaracterized by the conserved tripeptide sequence SDD(Jin & Elliott, 1992). Bunyavirus mRNA synthesis is primedby oligonucleotides cleaved from the 5 9  ends of host Journal of General Virology   (2011),  92,  2906–2913  DOI  10.1099/vir.0.036749-02906 036749 G 2011 SGM  Printed in Great Britain  mRNAs in a process known as cap-snatching (Patterson et al. , 1984) and the N-terminal domain of bunyavirus Lproteins contains a conserved PD-(D/E)xK nuclease motif (Reguera  et al. , 2010).Through the use of transient transfection of cDNAsexpressing N-terminal or C-terminal externally taggedRVFV L proteins, Zamoto-Niikura  et al.  (2009) reportedthat L forms oligomers, and they also identified regions of the protein responsible for both inter- and intramolecularinteractions. Furthermore, the importance of L oligomer-ization for viral RNA synthesis was demonstrated in aminigenome assay (Zamoto-Niikura  et al. , 2009).However, these authors were unable to produce aninfectious virus expressing a tagged L protein.Previously we utilized reverse-genetic technology (Bridgen& Elliott, 1996; Lowen  et al. , 2004) to generate recombin-ant Bunyamwera virus expressing L protein taggedinternally within the C-terminal domain with the 14 aaV5 epitope derived from parainfluenza virus 5 (Shi & Elliott, 2009; Southern  et al. , 1991). Here we demonstratethat RVFV RdRp can also tolerate introduction of theepitope into its C terminus and retain functionality in aminigenome assay, and furthermore that infectious virusexpressing a tagged L protein can be produced. We provideinsight into the intracellular location of the L protein ininfected cells and describe its co-localization with thenucleocapsid protein in the cytoplasm. RESULTS Generation of a V5-tagged RVFV RdRp Comparison of the amino acid sequences of bunyavirus Lproteins from viruses in all five genera of the family  Bunyaviridae   reveals little sequence similarity, apart fromthe short motifs described above. In particular, there is noobvious similarity between the C-terminal domain of Bunyamwera virus L, where the V5 epitope was successfully inserted, and the C-terminal end of RVFV L protein.Therefore, we compared the amino acid sequences of threephlebovirus L proteins (RVFV, Uukuniemi virus andToscana virus) and, along with bioinformatic predictionsof the RVFV L protein structure using criteria previously described (Shi & Elliott, 2009), we identified three potentialinsertion sites at aa 1750, 1794 and 1852. Accordingly, thesequence encoding the V5 epitope was inserted into the Lsegment cDNA, by using a PCR mutagenesis approach, inpTM1- or pTVT7-based plasmids for viral proteinexpression and genomic RNA expression, respectively (Fig. 1).The functionality of the recombinant L proteins wasassessed by using a minigenome assay comprising an S-segment reporter RNA in which the coding sequence of theNSs protein had been replaced with that of   Renilla  luciferase (Ren). The construct pTVT7-GSdelNSs:Rencontains anti-genomic sense cDNA to the modified Ssegment under the control of the T7 promoter, and hencethe reporter gene would be in the negative-sense orienta-tion in the T7-driven RNA transcript.  Renilla   luciferaseactivity will only be detected if a functional viralpolymerase is present in the system to transcribe theRNA and generate the appropriate subgenomic mRNA.Our minigenome system is thus similar to those previously reported for RVFV (Gauliard  et al. , 2006; Habjan  et al. ,2009; Ikegami  et al. , 2005; Na¨slund  et al. , 2009; Zamoto-Niikura  et al. , 2009).The minigenome-encoding plasmid was transfected intoBSR-T7/5 cells along with plasmids expressing N protein(pTM1-N) and either wild-type (wt) (pTM1-L) or mutant(pTM1-L1V5 to pTM1-L3V5) polymerases. As shown inFig. 2(a), insertion of the V5-epitope tag at aa 1750 (L1V5)or aa 1794 (L2V5) within the C terminus of the polymeraseabrogated activity in the minigenome assay. However, Lprotein with the epitope inserted further towards the Cterminus of the protein at aa 1852 (L3V5) was stillfunctional, showing 24% of the activity of the wtpolymerase. Varying the amounts of L-expressing plasmidDNA transfected (from 50 ng to 2  m g) did not result indetectable  Renilla   luciferase activity in pTM1-L1V5- orpTM1-L2V5-transfected cells, while the luciferase activity produced in wt L- or L3V5-transfected cells was maximalwith 100 ng of DNA (data not shown). None of theinsertions seemed to affect expression of the mutantpolymerases, as similar-sized bands were detected by Western blotting with anti-V5 antibodies (Fig. 2b). Effect of MP12 NSs protein on polymerase activity Work by  Ikegami  et al.  (2005) showed that expression of the RVFV ZH501 strain NSs protein in a minigenome assay enhanced RNA replication and transcription, as measuredby an increase in reporter gene activity. Therefore, in an EndonucleasedomainPolymerasemodule MP12 L protein    L   1   V   5   (   1   7   5   0   )   L   2   V   5   (   1   7   9   4   )   L   3   V   5   (   1   8   5   2   ) 2092 aa1 Fig. 1.  Schematic representation of the Rift Valley fever virus Lprotein and V5-epitope insertion sites. The 14 aa V5 epitope(GKPIPNPLLGLDST) was inserted into the L protein codingsequence in both pTM1-L and pTVT7-GL by using PCRmutagenesis at the sites indicated (  m ). The modified proteinswere designated L1V5–L3V5. The grey area represents theputative endonuclease domain ( Reguera  et al. , 2010) and thediagonally shaded box the polymerase module defined by Muller et al.  (1994). V5-epitope tagged RVFVhttp://vir.sgmjournals.org 2907  attempt to improve the activity of the L3V5 polymerase inour assay, varying amounts of the expression plasmidpTM1-NSs MP12  (from 1 ng to 1  m g) were transfected intoBSR-T7/5 cells along with the other constructs, asdescribed above. In contrast to the data reported by Ikegami  et al.  (2005), our results showed that increasingthe amount of NSs protein decreased the level of reportergene activity in cells expressing either wt or L3V5 mutantpolymerases. At the highest amount of co-transfectedpTM1-NSs MP12  (1  m g),  Renilla   luciferase was inhibited by 98% and 91% in wt L- or L3V5-transfected cells,respectively (Fig. 3). Rescue of recombinant RVFV expressing a V5-tagged polymerase The three pTVT7-based constructs expressing tagged Lproteins were then used in the reverse genetics system(Billecocq  et al. , 2008; Brennan  et al. , 2011) to determinewhether viable viruses could be rescued. In agreement withthe results seen in the minigenome assay, no viruses withRNA segments derived from the constructs pTVT7-GL1V5or pTVT7-GL2V5 were recovered (data not shown).However, a virus designated rMP12L3V5 was successfully rescued by using the construct pTVT7-GL3V5. The passage1 (p1) stock of this virus had a titre of 2.68 6 10 7 p.f.u.ml 2 1 but showed a smaller plaque phenotype than that of the parental virus, which was also generated by reversegenetics and designated rMP12 (Fig. 4a). We were also ableto rescue a recombinant two-segmented genome virus,designated r2segMP12L3V5, which expresses epitope-tagged L protein. This is based on the recently describedtwo-segmented virus (r2segMP12) in which the NSs gene isreplaced by that of the glycoproteins, and consequently thevirus lacks an M segment (Brennan  et al. , 2011). Bothviruses expressed L protein, which was clearly detected by Western blotting with anti-V5 antibodies (Fig. 4b).r2segMP12L3V5 was severely attenuated with respect torMP12, r2segMP12 or rMP12L3V5, and produced extre-mely small pinpoint plaques (Fig. 4c). Therefore, todetermine the titre of the virus, a focus-forming assay was performed by using an anti-N antibody. A p1 stock of r2segMP12L3V5 was generated and had a titre of 7.5 6 10 5 focus-forming units ml 2 1 . 15 000    A  r   b   i   t  a  r  y   l   i  g   h   t  u  n   i   t  s  10 0005000    N  o    L  w  t    L   L  1   V   5   L  2   V   5   L  3   V   5 a -V5 (a)(b) Fig. 2. Activity of V5-tagged L protein in the minigenome assay. (a)Luciferase activity. BSR-T7/5 cells were transfected with pTVT7-GSdelNSs:Ren, pTM1-N and either wt pTM1-L or V5-taggedvariants.  Renilla  luciferase activity was measured 24 h post-transfection. No L, Negative control without L cDNA (empty pTM1vector used); wt L, pTM1-L; L1V5, pTM1-L1V5; L2V5, pTM1-L2V5; L3V5, pTM1-L3V5. (b) Western blot analysis of transfectedBSR-T7/5 cells. Cell extracts were prepared in parallel with thosefor the minigenome assay, and proteins fractionated on a 4–12%NuPage gel (Invitrogen). After transfer to a nylon membrane, theblot was probed with an anti-V5 antibody to detect L protein. 25 00020 000    A  r   b   i   t  r  a  r  y   l   i  g   h   t  u  n   i   t  s 15 00010 000MP12 LMP12 LV55000    N  o    L  1  0  0  0  1  0  0  1  01   N  o    N  S  s   N  o    L  1  0  0  0  1  0  0  1  01   N  o    N  S  s NSs conc. (ng) NSs conc. (ng)0 Fig. 3.  Effect of MP12 NSs protein on polymerase activity in theminigenome assay. BSR-T7/5 cells were transfected with pTVT7-GSdelNSs:Ren, pTM1-N, either wt pTM1-L or pTM1-L3V5, andthe indicated amount of pTM1-NSs MP12 .  Renilla  luciferase activitywas measured 24 h post-transfection. Black and white barsrepresent experiments with wt L or L3V5 protein, respectively. NoL, Negative control without wt L cDNA (empty pTM1 vector used);No NSs, positive control reaction. B. Brennan, P. Li and R. M. Elliott2908  Journal of General Virology   92  Growth properties of rMP12L3V5 The replication of the three-segmented genome tagged viruswas compared with rMP12 in BHK-21, Vero E6 and  Aedes albopictus   C6/36 cells, each infected at an m.o.i. of 1 (Fig. 5).At different times post-infection (p.i.) the tissue-culturesupernatants were harvested and virus titres determined by plaque assay in BHK-21 cells. In all cell lines the replicationof the tagged virus, rMP12L3V5, was attenuated by 10- to100-fold compared with that of rMP12. Peak titres of 2.95 6 10 7 and 2.3 6 10 8 p.f.u. ml 2 1 in BHK-21 cells (Fig.5a), 8 6 10 6 and 1 6 10 8 p.f.u. ml 2 1 in Vero E6 cells (Fig. 5b),and 1.5 6 10 7 and 3.95 6 10 8 p.f.u. ml 2 1 in C6/36 cells (Fig.5c) were recorded for rMP12L3V5 and rMP12, respectively.The cell monolayers were also harvested to monitor theexpression of the N, NSs and L proteins by Western blotting.In all cell lines infected with rMP12 or rMP12L3V5, N andNSs expression was first detectable by 12 h p.i., except forNSs in C6/36 cells, which was delayed until 20 h p.i. Thelevels of N and NSs proteins synthesized in rMP12L3V5-infected cells were less than those seen in rMP12-infectedcells, reflecting the reduced growth observed for the taggedvirus. The L protein was readily detected by the anti-V5antibody in rMP12L3V5-infected cells: by 12 h p.i. in bothmammalian cell lines and by 20 h p.i. in C6/36 cells. Theoverall accumulation of L protein was greatest in infectedBHK-21 cells (Fig. 5). In addition, rMP12L3V5 appearedgenetically stable, in that tagged L protein was detected by Western blotting in cells infected with virus that had beenserially passaged three times. Cellular localization of N protein and polymeraseduring infection The intracellular localization of the RVFV polymerasewas determined by immunofluorescence imaging. Cellson coverslips were infected with rMP12L3V5 at an m.o.i.of 1, fixed at 24 h p.i., and stained with anti-N and anti-V5 antibodies. Both proteins were detected in thecytoplasm of infected cells and showed an overallpunctate pattern of staining (Fig. 6), with some areasof intense staining in the perinuclear region. These may represent the viral factories, as reported for Bunyamweravirus (Salanueva  et al. , 2003). There was a high degree of co-localization of the two immunofluorescent signals,which is indicative of interaction between L and Nproteins. No obvious co-localization of L with the Golgimarker protein GM130 was apparent when infected cellswere co-stained with anti-V5 and anti-GM130 antibodies(data not shown). DISCUSSION The RdRp of negative-sense RNA viruses plays a crucialrole in the virus life cycle, including replication of the viralgenome and transcription of viral mRNAs (Ortı´n & Parra,2006). As with other bunyaviruses, detailed study of theRVFV L protein has been impeded by the lack of antibodiesthat can detect the authentic L protein in infected cells by techniques such as Western blotting or immunofluores-cence. In addition, the lack of marked shut-off of host cellprotein synthesis during RVFV infection renders visualiza-tion of the polymerase in infected cell lysates extremely difficult. Previously, we showed that lack of monospecificantibodies could be overcome by inserting the V5 epitope,against which high-affinity antibodies are available, intothe Bunyamwera virus L protein, thus allowing convenientdetection of the protein in infected cells (Shi & Elliott,2009). Here, we investigated whether a virus in a differentgenus of the family   Bunyaviridae   could also be modified by introduction of the same tag. rMP12r2segMP12 r2segMP12L3V5   r   2  s  e  g   M   P   1   2   L   3   V   5 rMP12L3V5   r   M   P   1   2   L   3   V   5   M  o  c   k LTubulinNSsN (a) (b)(c) Fig. 4.  Characterizationofrecombinantviruses.(a) Comparison of plaque sizes of rMP12 andrMP12L3V5 on BHK-21 cells. Cell monolayerswere fixed at 96 h p.i. with 4% paraformalde-hyde and stained with Giemsa solution. (b)Western blot analysis of S and L segment-encoded proteins from rMP12L3V5- andr2segMP12L3V5-infected cells. Cell extractswere fractionated on 4–12% NuPage gels andblots were probed with anti-V5, anti-N, anti-NSs and anti-tubulin antibodies as indicated.(c) Comparison of plaque sizes of r2segMP12and r2segMP12L3V5 on BHK-21 cells. Cellmonolayers were fixed at 96 h p.i. with 4%paraformaldehyde and stained with Giemsasolution. Immunostaining, with anti-N antibody,of viral foci produced by r2segMP12L3V5 onBHK-21 cells is shown on the right. V5-epitope tagged RVFVhttp://vir.sgmjournals.org 2909  Little is known about the domain structure of bunyaviruspolymerases, and overall the L proteins of viruses indifferent genera show only limited regions of amino acididentity. Muller  et al.  (1994) described a region in thecentre of the RVFV L protein, aa 918–1193, to contain thepolymerase module composed of six motifs, Pre-A and A–E. These motifs are conserved across both bunyavirusesand arenaviruses, and mutation of certain conservedresidues in these motifs in the Bunyamwera virus L proteinwas shown to abolish polymerase activity (Jin & Elliott,1992). A motif downstream of E was reported as beingconserved among bunyaviruses, though no function can yet be ascribed to it (Aquino  et al. , 2003). An ovariantumour protease domain is found at the N terminus of Crimean–Congo hemorrhagic fever virus L protein (Honig et al. , 2004; Kinsella  et al. , 2004) and appears to be a unique rMP12rMP12rMP12L3V5rMP12L3V5rMP12rMP12L3V5rMP12rMP12L3V5LTubulinNSsN0 6 12 20 28 48 0 6 12 20 28 48rMP12 rMP12L3V5LTubulinNSsN0 6 12 20 28 48 0 6 12 20 28 480 20Time p.i. (h)    V   i  r  u  s   t   i   t  r  e   [   l  o  g    1   0    (  p .   f .  u .  m   l _   1    )   ] 40 600 20 40 600 20 40 60rMP12 rMP12L3V5LTubulinNSsN0 6 12 20 28 48 0 6 12 20 28 48    B   H   K  -   2   1   V  e  r  o   E   6   C   6   /   3   6 10 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 10 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 10 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 (a)(b)(c) Fig. 5.  Growth properties of recombinantviruses. Cells were infected with rMP12 orrMP12L3V5 (m.o.i. of 1). Western blot analysisof viral proteins and corresponding growthcurves from infected BHK-21 (a), Vero E6 (b)and C6/36 (c) cells are shown. Cell extractswere prepared at the time points indicated,proteins were fractionated on 4–12% NuPagegels and blots were probed with anti-N, anti-NSs, anti-V5 to detect L, and anti-tubulinantibodies, as indicated. Viral growth-curvesamples were titrated by plaque assay onBHK-21 cells. Anti-N Anti-V5 Merge Fig. 6.  Intracellular localization of viral N and V5-tagged L proteins in rMP12L3V5-infected cells. BSR-T7/5 cells were infectedwith rMP12L3V5 (m.o.i. of 1) and at 24 h p.i. cells were fixed with 4% paraformaldehyde and co-stained with anti-N and anti-V5antibodies, while nuclei were stained with DAPI. Cells were examined with a Zeiss LSM confocal microscope. B. Brennan, P. Li and R. M. Elliott2910  Journal of General Virology   92
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