Proc. Natl. Acad. Sci. USA Vol. 89, pp. 207-211, January 1992 Biochemistry Infectious enveloped RNA antigenic chimeras (/random mutagenesis/virus vectors/protein engineering/glycoproteins) STEVEN D. LONDON*, ALAN L. SCHMAUOHNt, JOEL M. DALRYMPLEt, AND CHARLES M. RICE*t *Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8230, St. Louis, MO 63110-1093; and tUnited States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011 Communicated by Edwin D. Kilbourne, October 7, 1991 (receivedfor review August 26, 1991)

ABSTRACT Random insertion mutagenesis has been used would allow recovery of infectious chimeric . The to construct infectious Sindbis virus structural protein chime- insertional mutagen was an oligonucleotide encoding an ras containing a neutralization epitope from a heterologous 11-amino acid neutralization epitope (4D4) derived from the virus, virus. Insertion sites, permissive for external G2 glycoprotein of Rift Valley fever virus (RVFV). recovery of chimeric viruses with growth properties similar to Both a polyclonal anti-peptide antiserum and a monoclonal the parental virus, were found in the virion E2 glycoprotein and antibody (mAb), mAb 4D4, that react with this epitope on the secreted E3 glycoprotein. For the E2 chimeras, the epitope RVFV virions and also with denatured and reduced RVFV was expressed on the virion surface and stimulated a partially G2 (13, 14) are available. Thus, chimeric viruses expressing protective immune response to Rift Valley fever virus infection this epitope can be identified by Western analysis. Further- in vivo. Besides providing a possible approach for developing more, the antigenic and immunogenic properties of Sindbis- live attenuated vaccine viruses, insertion ofpeptide ligands into 4D4 chimeras are of interest since 4D4 epitope-specific virion surface proteins may ultimately allow targeting of virus antibodies inhibit RVFV plaque formation in cell culture and infection to specific cell types. protect mice against lethal challenge with RVFV (13, 14). Sindbis virus, a relatively benign , has been used MATERIALS AND METHODS as a model enveloped RNA virus for inserting heterologous epitopes into virion structural glycoproteins. Alphaviruses Plasmid Constructs and Random Insertion Mutagenesis. are a diverse group of enveloped animal RNA viruses trans- Standard recombinant DNA techniques were used in all mitted in experiments (15). The plasmid used for random insertion nature to their vertebrate hosts by mosquitoes mutagenesis, pMTE2, consists of the Stu I (nt 8572)-BssHII (reviewed in ref. 1). Besides providing models for studying (nt 9804) Sindbis cDNA fragment (Sindbis nucleotide posi- virus entry and membrane protein biogenesis and function (1, tions refer to the full-length cDNA sequence reported in ref. 2), recent studies suggest that engineered alphaviruses may 5) from pTR2000 (16) cloned into pMT21 (obtained from also be used as live immunogens (3) and gene expression H. V. Huang, Washington University). The plasmid DNA vectors (ref. 4; C. S. Hahn, A. Diehl, C. Xiong, H. V. was linearized by treatment with DNase I in the presence of Huang, and C.M.R., unpublished results). Sindbis virus 1.5 mM MnCl2 (12) and repaired with T4 DNA polymerase contains a single-stranded, positive-sense RNA genome of and Escherichia coli DNA ligase (15). The resultant linear 11,703 nucleotides (nt) (5). Icosahedral nucleocapsids, com- molecules were isolated on a 0.8% low melting temperature posed of genomic RNA complexed with basic protein agarose gel and ligated to the nonphosphorylated double- molecules, bud from the surface of infected vertebrate cells stranded 45-mer encoding the RVFV 4D4 epitope (see Fig. and acquire a host-derived lipid bilayer containing two trans- 2A). Further details of the library construction are given in membrane viral glycoproteins, El and E2. The characteristic Results and Fig. 2B. The transcription vector used to regen- virion spikes visualized by electron microscopy (6, 7) are erate full-length Sindbis virus cDNA, pTR2002, has a 604-nt believed to be composed of trimers of an E1-E2 heterodimer lethal deletion [Ban I (8862)-Dra 1 (9470)] in E2. The parental (Fig. 1A; ref. 9). Although this heterodimer is likely a virus for these experiments, TR2001, was derived from functional unit, some properties have tentatively been as- transcripts of pTR2001. pTR2001 is a derivative of pTR2000 signed to individual glycoproteins. E2 is believed to be (16) in which the Sac I recognition site upstream from the SP6 involved in receptor binding (10), and E2-specific monoclonal promoter has been eliminated. antibodies often neutralize virus infectivity (11). El, the viral Generation of Chimeric Virus cDNA Clones. Double- hemagglutinin, probably mediates fusion of the virion enve- stranded cDNA was prepared from poly(A)+ RNA obtained lope and the endosomal membrane upon acidification, re- from chicken embryo fibroblasts (CEF) cells infected with sulting in delivery ofthe nucleocapsid to the cytoplasm (1, 2, twice plaque-purified virus isolates from the full-length ran- 11). dom insertion library (15, 17). The Stu I-BssHII cDNA Sindbis structural proteins are produced from a polypro- fragment was cloned into pTR2002 to generate cDNA clones tein by a complex series of processing events (Fig. 1B; ref. for each chimeric virus. The sequence of the Stu I-BssHII 2). PE2, the E2 precursor, was initially selected for insertion region was determined for each of the three clones used to of heterologous peptides since this precursor is cleaved, late generate virus stocks for further characterization. in virus maturation, to yield both secreted (E3) and virion- associated (E2) polypeptides. In addition, E2 is the less conserved of the two alphavirus virion glycoproteins (11). RESULTS Since high-resolution structures of the Sindbis virion glyco- Construction of the RVFV 4D4 Epitope-Sindbis Virus PE2 proteins are not yet available, random insertion mutagenesis Random Insertion Library. The scheme for PE2 mutagenesis, (12) was used to identify permissive insertion sites, which Abbreviations: mAb, monoclonal antibody; nt, nucleotide(s); pfu, The publication costs of this article were defrayed in part by page charge plaque-forming units; RVFV, Rift Valley fever virus; CEF, chicken payment. This article must therefore be hereby marked "advertisement" embryo fibroblasts. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 207 Downloaded by guest on September 30, 2021 208 Biochemistry: London et al. Proc. Natl. Acad. Sci. USA 89 (1992)

A. [vIn 45-mer oligonucleotide (Fig. 2A), encoding the 11-amino acid Ii 4D4 epitope and flanking restriction sites (5' Xba I and 3' Sac I) not present in Sindbis virus cDNA (or cloning vectors), was ligated to repaired linear molecules of a PE2 subclone (pMTE2), which had been produced by limited DNase I digestion (Fig. 2B). In the initial random insertion library, -7% of the colonies contained the RVFV 4D4 oligonucleo- tide, which corresponded to -8000 independent insertions in the Stu I-BssHII region (which spans from amino acid residue 46 of E3 to residue 391 of E2). To enrich for insertion mutations, plasmid DNA was digested with Sac I and the linear molecules were isolated, recircularized by ligation at 13 low DNA concentration, and used to transform E. coli. After the first Sac I selection, the Stu I-BssHII region from the

26)4 ;Lit 6-11 t 423 a >> iii 4t11.) diie random insertion library was subcloned into pMTE2 to I... ( aiipsal I S. 2 -_ ...... - eliminate insertions in the plasmid sequences, and two fur- -T--L-- - ther Sac I selections were performed. Finally, a library of A t insertion mutations in the context of a transcription vector FIG. 1. Organization and processing of the Sindbis structural containing the full-length Sindbis cDNA was constructed by proteins. (A) Schematic representation of the spike heterodimer. subcloning the mutagenized Stu I-BssHII fragment into Oligosaccharides (o-o), covalently attached fatty acids (-), and the pTR2002, followed by two additional Sac I selections. uncleaved signal sequence (stippled box) present in the N-terminal Recovery and Characterization of Infectious Chimeras. SP6 region of E3 are indicated. El and E2 each contain an N-terminal RNA transcripts obtained from the full-length random inser- ectodomain, a membrane anchor near the C terminus, and a short tion library were used to transfect CEF or BHK-21 cells (17, C-terminal cytoplasmic tail. Permissive insertion sites for the 4D4 19). Transcripts obtained from the insertion library were 70- peptide are indicated (A). The figure is not drawn to scale. (B) The Sindbis virus structural polyprotein and its cleavage sites. After to 77-fold less efficient at generating plaques at 37TC than autocatalytic cleavage of the capsid protein (at A), the N-terminal parental (pTR2001) RNA transcripts, suggesting that, as portion of PE2 (the E2 precursor) acts as an uncleaved signal expected, all sites in the mutagenized region were not per- sequence to initiate cotranslational translocation into the endoplas- missive for insertion of the 4D4 oligonucleotide. mic reticulum. A series of stop transfer and internal signal sequences Individual plaques, isolated from monolayers transfected result in the formation of a PE2-E1 oligomer in the infected cell with RNA obtained from the random insertion library and membrane. Cellular signalase catalyzes the cleavages flanking the incubated at 37°C, were used to generate first passage virus 6-kDa (6K) protein (*), and the delayed cleavage of PE2 into E3 and E2 ( t ) occurs in a post-Golgi compartment, possibly mediated by a stocks. Plaques morphologically similar to those of the pa- host protease (1, 2). Stippled areas represent hydrophobic regions rental strain of Sindbis virus were selected with the hope that (8), and the facing arrows indicate the mutagenized region ofthe PE2 they would have growth characteristics similar to that of the gene. The sizes of the individual proteins are indicated. aa, Amino parental virus. RNA obtained from these samples was ana- acids. lyzed for the presence of the RVFV 4D4 oligonucleotide by hybridization. Positive isolates were plaque purified for a using a full-length Sindbis virus cDNA clone from which second time and screened by hybridization, S1 nuclease infectious RNA can be transcribed in vitro using SP6 RNA mapping, and Western analysis. Fifty of 137 isolates were polymerase (17), is shown in Fig. 2. A double-stranded analyzed for the approximate location of the inserted oligo-

A Gln Tyr Lys Gly Thr Met Asp Ser Gly Gln Thr Lys Arg Glu Leu Ser Arg TCT AMA AAG GGA ACC ATG GAC TCA GGT CAG ACT AAG CGC GAG CTC AG&-CT TTC CCT TGG TAC CTG A= CC&..C Tg&.TTC GCG CTC GAG XbaI SacI FIG. 2. Construction of a random epi- B tope insertion library in Sindbis virus PE2. 13sI 1I11 (A) Sequence of the oligonucleotide encod- ing the RVFV 4D4 epitope. The top line indicates the authentic RVFV G2 glycopro-

I.L r _1kLI I ;'Mrl iCrslrTIIilI(I 8 J, -."': .1,2 :,! ". tein amino acid sequence. The second line Insertion RKepa r I S,,: .- .: I, %:-.,- SNi B "t indicates the deduced amino acid sequence l ibllrarx "' *['4 !)NA i. 1,-'T" .' -.'% .' of the oligonucleotide used for insertional :L,_;I mutagenesis, as shown below. Nucleotides that differ from the authentic RVFV se-

.\baI Sac quence (18) are shown in boldfaced type and " .1 :)( }li were incorporated to introduce unique re- striction enzyme sites (5' Xba I and 3' Sac I) - or termination codons (underlined) or to Hsd III1 optimize codon usage for Sindbis virus 26S ,tinton1 ii' RNA. Termination codons were engineered K i il1IlI l-2.ltltl (Ilk in four of the five alternative reading frames to enrich for viable mutants expressing the 4D4 epitope. (B) PE2 mutagenesis scheme. ~ ~ ~ ~ lS 5lliiiC ill1. [(Us, ) , t d The scheme used to generate the RVFV Characterization S-C1Ciim !wr k11 1c- iLrarv 4D4-Sindbis virus random insertion library Of multarnt viruses Mutnenmflecl)INAoriv o' MUMIlt -Viruss is described in the text. Details of the meth- r c~ in SeVlesl ;;th1 zIC il c odology and the Sindbis virus cDNA clones *-C.T.le ie3lrL' used are described in Materials and Meth- ;l k:,2st1"j s L'D[N A '1l ods. bp, Base pairs. Downloaded by guest on September 30, 2021 Biochemistry: London et al. Proc. Natl. Acad. Sci. USA 89 (1992) 209

nucleotide by S1 nuclease analysis (Table 1). A limited (58) E2 E3-62 TCG TCT QGC AGA AGC 4D4 OLIGO QGC AGA AGC AAA AGA AGC GTC ATT number of permissive insertion sites were found (which are Ser Ser Gly Arg Nor Gly Arg Nor Lys Arg Ser Val Ile not necessarily independent), with clustering in the C-termi- DUPLICATION (60) [ E2 nal region of E3, the N-terminal region of E2, and an internal GGC AGA AGC AAA AGA haC GTC ATT 4D4 OLIGO AGA AGC GTC ATT position in the region of E2 amino acid 240. In addition, one E2-3 Gly Arg Ser Lys Arg Ner Val Ile Arg Nor Vol Il- example of an insertion near E2 residue 88 was observed, DUPLICATION (241) CAA which was negative by Western analysis and has not been CCG TTG ATC [AGA CAT GAC GAC 4D4 OLIGO CAC ACG GTC studied further. Of the 50 isolates examined by S1 analysis, Pro Asp Leu Ile Arg His Asp Asp His Thr Val Gin only 29 were positive by Western analysis [with a high DELETED propensity for those being in the C-terminal region of E3 or FIG. 3. cDNA sequences at the E3-62, E2-3, and E2-244 4D4- the N-terminal region of E2 (Table 1)]. The apparent lack of permissive insertion sites. The region surrounding each insertion site immunoreactivity suggested that either the alternative read- is shown. In all cases the sequence ofthe RVFV 4D4 oligonucleotide was that shown in Fig. 2A. Duplicated nucleotides are shown in ing frame encoded by the inserted oligonucleotide was being boldfaced type. Deleted nucleotides are shown in brackets. Numbers expressed or that the epitope was present but not reactive refer to the position of the indicated amino acid residue from the N with a polyclonal rabbit anti-4D4 peptide antiserum. terminus of the respective protein (5, 20). The arrow indicates the Permissive Insertion Sites, Growth Properties, and Chimeric position of the PE2 cleavage site (20). Glycoproteins. One representative isolate from each of the three classes of insertions was selected for detailed analysis. immunoprecipitated from lysates ofcells infected with E3-62. Infectious, full-length cDNA clones were generated for each The E2 protein in purified virions of both E2 insertion isolate. The sequence surrounding the insertion mutation is mutants was also immunoprecipitated by mAb 4D4, as was shown for each clone in Fig. 3. One insertion was in the secreted E3 present in the supernatant from cells infected C-terminal region of the E3 protein after amino acid 62 with E3-62 (data not shown). Additional analyses revealed (E3-62). Two permissive insertion sites were also identified in that the kinetics of host protein synthesis shutoff and PE2 the E2 glycoprotein: one in the N-terminal region after E2 cleavage were similar to those observed with parental residue 3 (E2-3) and another after residue 244 (E2-244). The TR2001. Analysis of radiolabeled virus did not reveal evi- oligonucleotide was present in-frame and without mutation in dence for the incorporation of PE2 or E3 into virions for any all clones. However, there were either duplications or dele- of these three isolates (data not shown; but see below). tions of the flanking parental Sindbis virus sequences at the Binding and Neutralization of Chimeras by RVFV and site of insertion in all cases. The alterations in the DNA Sindbis Virus-Specific mAbs. Reactivity of the chimeric vi- surrounding the insertion sites are consistent with the obser- ruses with RVFV mAb 4D4 and Sindbis-specific mAbs was vation that DNase I treatment of covalently closed super- also examined using neutralization, ELISA, and solution helical plasmid DNA, in the presence ofMn2+ ions, produces binding assays. Plaque reduction neutralization tests were blunt-ended double-stranded breaks and staggered, single- performed by infection of Vero or CEF cell monolayers with stranded nicks (21). Thus, the repair of plasmid DNA with control (TR2001) and chimeric viruses after incubation with staggered nicks on opposing strands results in the duplica- a 1:100 dilution of mAb 4D4 or control mAb. Only E2-3 was tions or deletions that we, and others (12, 22, 23), have consistently neutralized by the mAb 4D4 with plaque reduc- observed using this methodology. tion neutralization titers ranging from 50%o reduction in Vero First passage virus stocks were generated from each cDNA cells to 88-97% in CEF cells. By ELISA (Table 2) and clone. All viruses exhibited growth kinetics similar to paren- solution binding assays (Table 3), the RVFV 4D4 mAb was tal virus (TR2001), grew to similar titers in CEF cells after low able to bind to the E2-3 and E2-244 chimeras but less well to [0.1 plaque-forming units (pfu) per cell] or high (10 pfu per the E3-62 chimera. Taken together, these results show that cell) multiplicity passage, and had plaquing efficiencies sim- the 4D4 epitope is exposed on the virion surface in the E2 ilar to that observed with TR2001 on CEF cells at 30°C, 37°C, chimeras. The low, but detectable, reactivity of mAb 4D4 or 40°C and on C7-10 cells at 34.5°C (data not shown). E3-62 E2-3 E2-244 TR2001 Viral proteins present in infected cell lysates, the super- Lv V Ly V Lv v Lv %V natant of infected cells, and purified virions were analyzed. N - N ALN A mAb 4D4 (anti-RVFV G2 4D4 epitope) immunoprecipitated -p > t > > t > _~ te PE2 and E2 from lysates prepared from cells infected with 200 -- E2-3 or E2-244 (Fig. 4). As expected, PE2 but not E2 was 100- --*- B Table 1. Localization of insertion sites and 4D4 reactivity of 69 - _ -PE2 viable insertion mutants urnm -w El Insertion location* Number 4D4-positivet E3 (C-terminal region) 23 20 FIG. 4. Immunoprecipitation of chimeric Sindbis virus proteins. E2 (N-terminal region) 10 6 mAb 4D4 (mouse IgGl) (13, 14) or an isotype-matched, vaccinia E2 (near residue 88) 1 0 virus-specific control mAb (V207) was used to immunoprecipitate E2 residue 16 3 "S-labeled Sindbis virus proteins with protein A immobilized on (near 240) Trisacryl GF-2000 (Pierce). Immunoprecipitates were analyzed on The approximate insertion sites for 50 randomly selected (twice 12.5% N,N'-diallyltartardiamide/acrylamide gels. For the produc- plaque-purified) virus isolates were mapped by S1 nuclease protec- tion of "5S-labeled proteins, BHK cell monolayers were infected with tion analyses (15). Cytoplasmic RNA was obtained from CEF cells second passage virus stocks at a multiplicity of infection of20-30 for at 10 hr postinfection; the probe was a 3' end-labeled Aha II fragment 1 hr at 37rC. After 4 hr, the cells were labeled with [35S]methionine (nt 7997-9822) derived from pTR2001. at 50 ,uCi/ml (1 Ci = 37 GBq) for an additional 4 hr. For each virus, *Estimated location of the RVFV 4D4 oligonucleotide insertions the first two lanes are cell lysates (Ly) and the second two lanes are corresponding to nucleotide coordinates of the Sindbis genome purified virions (V). Virus was isolated from the supernatants by sequence (5): E3 (C-terminal region), nt 8597-8622; E2 (N-terminal pelleting through a 20o sucrose cushion for 2.5 hr at 30,000 rpm in region), nt 8622-8647; E2 (area of aa 88), nt 8875-8915; E2 (area of an SW41 rotor (Beckman). The positions of virus-specific proteins aa 240), nt 9297-9397. are indicated. The B protein, a PE2-6K-E1 polyprotein, is present in tReactivity with polyclonal rabbit anti-4D4 peptide antiserum was infected BHK cells. Molecular size markers (in kDa) are indicated at determined by Western blot analysis. left. Downloaded by guest on September 30, 2021 210 Biochemistry: London et al. Proc. Natl. Acad. Sci. USA 89 (1992) Table 2. ELISA analysis for the expression of the 4D4 epitope on chimeric viruses Virus mAb 4D4 (OD450)* -80- TR2001 0.22 ± 0.02 260