Highly Stable Expression of a Foreign Gene from Rabies Virus Vectors TESHOME MEBATSION, MATTHIAS J

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7310-7314, July 1996 Microbiology

Highly stable expression of a foreign gene from rabies virus vectors TESHOME MEBATSION, MATTHIAS J. SCHNELL*, JAMES H. Cox, STEFAN FINKE, AND KARL-KLAUS CONZELMANNt Department of Clinical Virology, Federal Research Centre for Virus Diseases of Animals, Paul-Ehrlich-Strasse 28, D-72076 Tubingen, Germany Communicated by Hilary Koprowski, Thomas Jefferson University, Philadelphia, PA, April 1, 1996 (received for review January 30, 1996)

ABSTRACT A reverse genetics approach was applied to surface epitopes, altered nonsurface proteins, and bicistronic generate a chimeric nonsegmented negative strand RNA virus, segments expressing foreign genes could be generated (4, 5). rabies virus (RV) of the Rhabdoviridae family, that expresses Recently, we reported the recovery of infectious rabies virus a foreign protein. DNA constructs containing the entire open (RV) of the rhabdovirus family from a full-length cDNA clone, reading frame of the bacterial chloramphenicol acetyltrans- rendering the entire genome of a negative strand RNA virus ferase (CAT) gene and an upstream RV cistron border se- accessible to genetic manipulation (6). The approach, which quence were inserted either into the nontranslated pseudo- proved successful for recovery of recombinant vesicular stoma- gene region of a full-length cDNA copy of the RV genome or titis virus (VSV), another rhabdovirus, and also of paramyxo- exchanged with the pseudogene region. After intracellular T7 viruses (reviewed in ref. 3), involved intracellular expression of RNA polymerase-driven expression of full-length antigenome antigenomic RNA in cells also expressing the viral proteins RNA transcripts and RV nucleoprotein, phosphoprotein and needed for formation of a transcriptionally active RNP complex, polymerase from transfected plasmids, RVs transcribing namely, the nucleoprotein (N), phosphoprotein (P), and the viral novel monocistronic mRNAs and expressing CAT at high polymerase (L). The recovery of novel RVs with extensive levels, were recovered. The chimeric viruses possessed the alterations within the 3' noncoding region (pseudogene) of the growth characteristics of standard RV and were genetically glycoprotein (G) gene indicated that the RV genome is highly stable upon serial cell culture passages. CAT activity was still flexible. Deletion of the entire pseudogene region as well as the observed in cell cultures infected with viruses passaged for introduction of a functional transcriptional signal sequence did more than 25 times. Based on the unprecedented stability of the not affect the growth of the recombinant viruses in cell culture chimeric RNA genomes, which is most likely due to the structure (6). In the present study, we evaluated whether it was possible to of the rhabdoviral ribonucleoprotein complex, we predict the express a heterologous protein from genetically engineered RV. successful future use of recombinant rhabdovirus vectors for We report the recovery of RV possessing a complete additional displaying foreign antigens or delivering therapeutic genes. transcription unit encoding bacterial chloramphenicol acetyl- transferase (CAT). Remarkably, the foreign gene was maintained Successful recovery of infectious RNA viruses from cDNA stably in the absence of selective pressure and was expressed intermediates facilitates analysis of virus genetics and viral pro- during a high number of serial passages in cell culture. The tein functions in any step of the viral life cycle. This technology potential to stably retain nonessential sequences in the RV has also permitted the use of RNA virus replication machineries genome and the possibility of generating rhabdoviruses with for expression ofheterologous sequences in eukaryotic cells. Both altered surface proteins (7) make rhabdoviruses attractive vectors the small size of RNA virus genomes, which facilitates engineer- for a wide range of biomedical applications. ing of cDNA clones, and their cytoplasmic amplification, which eliminates possible deleterious effects of nuclear splicing, have MATERIALS AND made RNA viruses attractive candidates for development of METHODS RNA-based expression systems. Recovery of chimeric viruses Plasmid Construction. For construction of full-length RV expressing foreign antigens or antigenic structures has been cDNAs encoding CAT, pSAD L16 (6) expressing full-length reported for a range of positive strand RNA viruses, including positive strand (antigenome) RNA of the attenuated RV strain alphaviruses (Semliki Forest virus, Sindbis virus) and picornavi- Street Alabama Dufferin (SAD) B19 (8) was used as a basis. The ruses (polio virus) (for review, see ref. 1). Hindlll fragment of pCM7 (Pharmacia) was first subcloned into Until recently, these applications were restricted to positive pBluescript (Stratagene), re-excised with EcoRI and XhoI, and strand RNA viruses, whose genome RNAs may initiate an inserted downstream of the RV N/P cistron border cloned in infectious cycle when introduced into or generated from cDNA pNigP (6). An XbaI-XhoI fragment encompassing the cistron in an appropriate host cell. In contrast, neither full-length border and the CAT frame was then inserted into the Styl site genomic nor antigenomic RNAs of negative strand RNA viruses (SAD L16 position 4, 942) of the RV pseudogene cDNA present are infectious. To recover an infectious negative strand virus from in the plasmid pPsiX8 (6) after generation of blunt ends with cDNA transcripts, it is necessary first to assemble the ribonucle- Klenow enzyme. A StuI fragment spanning the altered pseudo- oprotein (RNP) complex that serves as the template for the viral gene and flanking parts of the G and L sequences was used to RNA-dependent RNA polymerase (2). In recent years, develop- replace the corresponding fragment in the full-length cDNA ments in several virus systems have permitted the expression and clone SAD L16 (positions 4014-6364) (see Fig. 1). The resulting manipulation of small model genomes and defective RNAs (for pSAD XCAT encoded a RV genome of 12,967 nucleotides. review, see ref. 3) and also of single genome segments of influenza virus (for review, see ref. 4). In the latter case, recom- Abbreviations: RNP, ribonucleoprotein; RV, rabies virus; VSV, ve- binant genome segments were incorporated into infectious in- sicular stomatitis virus; CAT, chloramphenicol acetytransferase; SAD, fluenza virus by reassortment and virions carrying heterologous Street Alabama Dufferin strain of RV; moi, multiplicity of infection; ffu, focus-forming units; N, nucleoprotein; P, phosphoprotein; L, viral polymerase; G, glycoprotein. The publication costs of this article were defrayed in part by page charge *Present address: Departments of Pathology, Cell Biology, and Biol- payment. This article must therefore be hereby marked "advertisement" in ogy, Yale University School of Medicine, New Haven, CT 06510. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 7310 Downloaded by guest on October 2, 2021 Microbiology: Mebatsion et aL Proc. Natl. Acad. Sci. USA 93 (1996) 7311 For construction ofpSAD VCAT, first, the N/P cistron border inger Mannheim) was used according to the supplier's instruc- cDNA was introduced into the BstXI site (SAD B19 position tions. 4991) of the pseudogene sequence of pPsiX8 after generation of Analysis of RNA. Denaturing agarose gel electrophoresis and blunt ends. The majority of the pseudogene sequence was re- Northern hybridization were performed as described (12). cDNA moved by digestion with AsuII (recognition site located in the fragments encompassing part of the RV G gene, pseudogene, or cistron border cDNA) and Hindlll (SAD B19 position 5337), and CAT gene were used as probes after labeling with deoxycytidine replaced after Klenow fill-in with the HindIII fragment contain- 5'-[a-32P]triphosphate (3,000 Ci/mMol) by nick translation (nick- ing the CAT open reading frame. As for SAD XCAT, the translation kit; Amersham). modified region was introduced into the full-length clone by Mice Inoculation. Groups of3- to 4-week-old NMRI mice were exchange of the StuI fragments. The encoded RV genome injected intracerebrally with 3000 focus-forming units (ffu) of the comprised 12,567 nucleotides. recombinant viruses in 30 ,ul volume. This dose killed all mice in Recovery and Propagation ofRecombinant Viruses. Transfec- 7-8 days. Brain samples were collected from dead or moribund tion experiments and recovery of recombinant virus were carried mice and ground in 0.5 ml ofPBS. Part (30 ,ul) ofthe brain extract out as described (6). Approximately 106 BSR cells were grown in was used either for direct CAT assays or for reisolation of virus 3.2-cm diameter dishes and infected with the recombinant vac- in cell culture. cinia virus vT`F7-3 (9) expressing T7 RNA polymerase (kindly provided by T. Fuerst and B. Moss, National Institute of Allergy RESULTS and Infectious Diseases, Bethesda). After 1 h, cells were trans- with a mixture of plasmids encoding RV N, P, and L The 12-kb genome of RV serves as a template for sequential fected transcription of a leader RNA and five nonoverlapping mono- proteins [5 jig of pT7T-N, 2.5 ,ug of pTTI-P, and 2.5 jig of cistronic mRNAs, the start and stop/polyadenylylation sites of pTI7T-L, respectively (10)] and with a plasmid encoding the which are defined by internal signal sequences. This modular full-length antigenomic RNA (4 ,tg of pSAD XCAT or pSAD organization of the genome should allow expression of an VCAT) by using the Stratagene mammalian transfection kit additional protein by the insertion of the corresponding gene (CaPO4 protocol). Two days after transfection, cells were sus- in the form of an extra transcription unit. We recently found pended in the culture supernatant and subjected to three cycles that correct transcription termination, polyadenylylation, and of freezing and thawing. Samples were clarified by centrifugation release of mRNA, and restart of transcription is directed by a (10,000 x g in a microfuge) and used to inoculate fresh BSR cell copy of the N/P gene border sequence after introduction into monolayers. After 2 days, supernatants were harvested, vaccinia the 3' nontranslated region of the G cistron (pseudogene). This virus was removed by filtration as described (6), and the recom- modification, as well as the deletion of the entire 0.4-kb binant RVs were further passaged until infection of the entire pseudogene region, did not affect replication or final titers of monolayer was observed. The resulting virus stocks were titrated recombinant RVs in cell culture (6), suggesting the feasibility by end point dilution. Infection of cells was monitored by direct of replacing the pseudogene region with a complete foreign immunofluorescence with an anti-RV nucleoprotein conjugate gene. Therefore, this region was selected for introduction of (Centocor). Serial passages in BSR cell cultures were carried out the CAT gene into the RV genome. at a multiplicity of infection (moi) of 0.01, and cultures were Two different RV full-length cDNAs were constructed as incubated for 2 or 3 days. detailed in Materials and Methods on the basis of pSAD L16, CAT Assays. BSR cells were infected at an moi of 1 with the which encodes the antigenomic RNA of the attenuated RV SAD recombinant RVs and incubated for 48 h. Cells were washed with B19 (Fig. 1). In one case (pSAD XCAT), a DNA construct PBS, scraped from the culture dishes in 100 ,ul of0.25 M Tris (pH composed of the coding region ofthe CAT gene and an upstream 7.5), and lysed by three cycles of freezing and thawing. Equal SAD B19 N/P cistron border sequence was inserted into the amounts of proteins were adjusted to a volume of 30 ,ul and pseudogene region at the Styl site (position 4931). According to incubated with 10 ,ul [0.25 ,uCi (1 Ci = 37 GBq)] of [14C]chlor- the location of the transcriptional signal sequences, the resulting amphenicol (53 mCi/mmol; Amersham) and 5 ,ul of 4 mM G cistron possesses a chimeric 3' noncoding region terminating acetylcoenzyme A (Boehringer Mannheim) for 1 h at 37°C (11). with the transcriptional stop/polyadenylylation signal from the N For quantitation of CAT protein, the CAT-ELISA Kit (Boehr- cistron. The novel CAT cistron is composed of the transcriptional A N P M G vP L 3' 5, B Sty I SAD VCAT CAT I-

Sty I BstX I Hinid III SAD Ll66

BstX I Hind III SAD XCAT { FIG. 1. (A) Organization of the 12-kb genome of RV. Filled boxes represent the open reading frames of the N, P, M (matrixprotein), G, and L genes. 4, represents the nontranslated 3' sequence of the G cistron (pseudogene). (B) Organization of chimeric viruses. Open reading frames are drawn as shaded boxes, and transcriptional stop/polyadenylylation signals are represented by flags. In SAD VCAT, a DNA fragment composed of the N/P cistron border and the CAT open reading frame was used to replace the major part of the pseudogene sequence of standard RV SAD L16; for construction of SAD XCAT, the fragment was inserted into the Styl site of the pseudogene. The former coding part of the N gene is shown as an open box. Downloaded by guest on October 2, 2021 7312 Microbiology: Mebatsion et al. Proc. Natl. Acad. Sci. USA 93 (1996)

start sequence derived from the P gene, the CAT open reading G-probe T1'-probe CAT-probe frame, and a 3' nontranslated sequence of 530 nucleotides containing the 3' terminal three quarters of the former pseudo- ! gene. In comparison to pSAD L16, the virus genome encoded by co R !R co !R !R CD the plasmid pSAD XCAT is enlarged by 1037 nucleotides to a total of 12,967 nucleotides. In the other full-length clone, pSAD VCAT, the N/P-CAT DNA construct was used to replace the V - - major part of the pseudogene region, encompassing nucleotides - 9.5 4992 (BstXI) to 5337 (HindIll). In this case, the novel CAT cistron - 7.5 possesses a short 3' noncoding region of 109 nucleotides. The resulting SAD VCAT RV genome encompasses 12,567 nucleo- - 4.4 tides. Virus Recovery. To recover virus from the full-length cDNAs, cells were infected with the recombinant vaccinia virus vTF7-3 G~55 - 2.4 (9), providing cytoplasmic T7 RNA polymerase, and were sub- 30 sequently transfected with pSAD XCAT or pSAD VCAT en- CATT - 1.4 coding the RV antigenome transcripts. The viral N, P, and L CAT U8 proteins necessary for encapsidation of the full-length antigenome transcript into nucleoprotein, replication of the antigenome, and transcription of mRNAs from the resulting genomic RNP were also expressed from transfected plasmids, pT7T-N, - 0.24 pT7T-P, and pT7T-L. Two days after transfection, cells were kb suspended in the culture medium and lysed by three cycles of FIG. 2. Northern blot analysis of viral transcripts. Total RNA from freezing and thawing. Half of the clarified lysates was used for cells infected with SAD L16 (L16), SAD XCAT (XCAT), and SAD inoculation of fresh cell cultures. After an incubation of 2 days, VCAT (VCAT) was isolated 2 days after infection and hybridized with culture supernatants were harvested, and cell monolayers were a probe specific for the RV G gene (G), the pseudogene (i/), or the examined by immunofluorescence with a conjugate directed CAT gene (CAT). The G mRNA of standard SAD L16 is composed against RV N protein. Fluorescent foci indicating the presence of of G and pseudogene sequences (Gqi), whereas in SAD XCAT and recombinant infectious virus were observed in 3 out of 10 SAD VCAT infected cells, truncated G mRNAs lacking pseudogene experiments for pSAD VCAT and in 4 out of 10 for pSAD sequences are transcribed (G). The different sizes of the CAT mRNAs XCAT. also reflect the presence (CAT+p) or absence of pseudogene sequences. The respective cell culture supernatants were passed through No hybridization with a pseudogene-specific probe was observed for 0.1-,um filters to remove vaccinia virus and used for further SAD VCAT RNAs, illustrating the pseudogene deficiency of this virus. passage to generate virus stocks. Infection of the entire cell v, viral genome RNA. monolayers was observed after two passages and resulted in titers infected at an moi of 1, and CAT assays were performed 2 days of 5 x 107 ffu/ml for both recombinant viruses, as determined by after infection. Both recombinant viruses directed efficient CAT end point dilution of supernatants. Under similar passage con- expression. CAT activity was correlated with the presence of ditions, the recombinant standard RV SAD L16 gave rise to a virus, as determined by infection of cells with serial dilutions of titer of 1 x 108 ffu/ml. No differences in growth kinetics and final supernatants and subsequent parallel analyses of CAT and im- titers were observed after infection of cell cultures with SAD munofluorescence (not shown). As anticipated from the different XCAT, SAD VCAT, and SAD L16, respectively, at an moi of 1 amounts of mRNAs and confirmed SAD VCAT (data not shown). by ELISA, The presence of the CAT gene in the genomes of recombinant produced -4-fold the amount of CAT protein compared with RVs was verified by reverse transcription-PCR amplification with SAD XCAT (800 ng and 210 ng of CAT protein per 106 infected the primers G3P and L4M located in the coding regions ofthe RV cells, respectively). G and Lgenes, respectively. Terminal sequencing ofthe amplified To evaluate the genetic stability of the additional gene in the DNA fragments did not reveal differences to the sequence recombinant RV genomes, successive cell culture passages were present in the plasmids pSAD VCAT or pSAD XCAT (data not performed. Initially, cells were infected at an moi of 1 and shown). incubated for 2 days. For subsequent passages, the supernatants Transcription Analysis of Viral RNA. To investigate whether were diluted (1:8000), and infected cultures were incubated for 2 the introduced signal sequences are able to direct transcription of or 3 days. On average, the resulting moi was approximately 0.01 an additional monocistronic mRNA, total RNA from cells in- in each passage. After every fifth passage, supernatants were fected with SAD XCAT and SAD VCAT, respectively, was titrated by end point dilution, and CAT activity of the cells was analyzed by Northern hybridization. For both viruses, truncated determined. Most remarkably, CAT expression by SAD VCAT G mRNAs lacking pseudogene sequences were demonstrated and SAD XCAT was maintained through the 25th cell culture with a G-specific probe (Fig. 2). After hybridization with a passage and correlated to the observed titers of infectious virus CAT-specific probe, RNAs of 1.5 and 0.8 kb were detected in cells (Fig. 3). After applying reverse transcription-PCR on viral RNA, infected with SAD XCAT and SAD VCAT, respectively. The exclusively fragments corresponding in length to those obtained presence of pseudogene sequences in the 1.5-kb CAT mRNA of from the original cDNA constructs were identified (data not SAD XCAT and their absence in the genome of SAD VCAT was shown). This result indicated that no total or partial loss of the demonstrated by hybridization with a pseudogene-specific probe CAT gene occurred during the cell culture passages. (Fig. 2). Interestingly, the 0.8-kb CAT mRNA of SAD VCAT Expression ofCAT in the Brain ofInfected Mice. To determine lacking the pseudogene trailer was more abundant than the 1.5-kb whether the incorporation of the foreign gene into the RV RNA of SAD XCAT. Since similar amounts of L mRNA were genomes markedly affected viral pathogenicity and, in addition, present (data not shown) and similar virus titers were obtained to look for expression of the reporter gene in the central nervous for the two viruses, it appears that the presence ofthe pseudogene system, animal experiments were carried out. As a control, the sequences affects the stability of the chimeric RNA rather than "parental" recombinant RV SAD L16 was used; this RV strain's the level of transcription. nucleotide sequence corresponds to the published sequence of Expression ofCAT and Stability ofthe Foreign Gene. To verify the attenuated RV strain SAD B19 (8), which is being used as a the expression of CAT protein from the novel cistrons, cells were live vaccine for oral immunization of foxes in Europe. As ob- Downloaded by guest on October 2, 2021 Microbiology: Mebatsion et al. Proc. Natl. Acad. Sci. USA 93 (1996) 7313

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**..@ ....e SAD XCAT ENII E0.

A t w tY 000 J~ u. mice 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 virus SAD XCAT SAD VCAT SAD L16 virus dilution (log1,) -3 -4 -5 -6 -7 -8 -3 -4 -5 -6 -7 -8 FIG. 4. CAT expression in the brains of mice infected intracere- virus titer ffu/1 00 gl 8x1or 5x1 O brally with 3000 ffu of SAD XCAT, SAD VCAT, and SAD L16, passage number 3 25 respectively. Brain samples were collected at 7 and 8 day after infection from dead or moribund animals and processed directly for CAT assay. the one hand, successful expression ofchimeric RV minigenomes containing sequences of various origin, such as 13-galactosidase, CAT, or firefly luciferase (10, 13), indicated that major restric- tions in terms of sequence composition do not exist. The other SAD VCAT was the finding that a putative transcriptional signal sequence directed correct transcription-stop and -polyadenylylation and transcription-restart when introduced into another sequence con- 46d * __t0; text (6). Furthermore, the presence of additional genes in other rhabdoviruses, paramyxoviruses, and filoviruses (14), which share vm~v0M pq o0 0 the basic strategy of expression, supported the idea that RV has the capacity to encode and express an additional gene. The virus dilution (log10) -3 -4 -5 -6 -7 -8 -3 -4 -5 -6 -7 -8 successful recovery of RVs with genomes consisting of a succes- virus titer ffu/100 1 5x105 2.2xle0 sion of six instead of five functional protein-encoding genes passage number 3 25 confirmed this assumption. Moreover, the observation of biological properties highly similar to those of standard RV demon- FIG. 3. Expression of CAT activity in cells infected with SAD strated that the presence ofan additional gene did not impede the XCAT or SAD VCAT during serial cell culture passages. The auto- biological fitness of the novel recombinant viruses. radiograph shows CAT activity of cells infected with 100 ,l of 10-fold Multiple passages revealed an unprecedented genetic stability dilutions of supernatants harvested after the 3rd or 25th passage. Virus titers of the supernatants, as determined by immunofluorescence of the foreign sequences in the recombinant RV RNA genomes, microscopy, are indicated. illustrating the suitability of rhabdoviruses for vector use. An indication for the outstanding potential of members of the order served for SAD B19, SAD L16 is pathogenic for adult mice only Mononegavirales to retain nonessential sequences has previously after intracerebral inoculation (unpublished experiments). Three been obtained by the successful recovery of fully viable recom- groups of five NMRI mice were, therefore, infected by intrace- binant RV (6) and also of measles paramyxovirus (15), the rebral injection of a dosis of3000 ffu per mouse with SAD VCAT, genomes of which possessed deletions in noncoding regions. In SAD XCAT, and SAD L16, respectively. All animals showed the case of RV, the 0.4-kb pseudogene present in all members of identical, typical rabies symptoms and died within 7-8 days after the lyssavirus genus analyzed so far was found to be dispensable infection. Brain samples from deceased mice were directly pro- for normal replication in cell culture. The mutant virus grew to cessed for CAT assays and for virus reisolation, as detailed in wild-type titers and also caused the typical rabies symptoms in Materials and Methods. In brain samples of all animals infected mice after intracerebral injection (unpublished data). A deletion with SAD XCAT or SAD VCAT, efficient CAT expression of504 nucleotides in the 5' noncoding region of the measles virus could be demonstrated (Fig. 4). In addition, cell cultures fusion glycoprotein (F) gene, which is typical for members of the inoculated with aliquots of the brain suspensions displayed subfamily Paramyxovirinae was also found not to interfere with CAT activity (data not shown). replication in cell culture. These findings were amazing, since in RNA virus genomes, the amount of nonessential sequences is usually limited to a minimum. Continuous streamlining of ge- DISCUSSION nomes and elimination of sequences not contributing to a selec- A variety of DNA viruses and several positive strand RNA tive advantage is achieved in most positive strand RNA viruses virus-based systems have been exploited for the expression of through inter- and intramolecular recombination by copy-choice. heterologous genes and have brought forth important tools for The mechanism involves detachment of the polymerase together both basic and clinical research (1). The development of with the nascent RNA chain from the template, reattachment of techniques allowing the recovery of negative strand RNA the complex to another template in a region fully or partially viruses from cDNA enabled us to address the potential of these complementary to the nascent RNA, and resumption of RNA viruses in this important technology. Two unique features of synthesis (for a recent discussion, see ref. 16). As evidenced by the nonsegmented negative strand RNA viruses were anticipated appearance of defective interfering particles, processes similar to to facilitate successful vector use, namely the simple modular copy-choice recombination also exist in negative stranded RNA organization of their genomes, allowing easy engineering of a viruses, but obviously occur less frequently than in most positive complete cistron into the genome, and the RNP nature of the strand RNA viruses. Most probably, this depends on the fact that viral genetic information, assumed to preserve the integrity of genome RNAs are exclusively present in the form of RNP nonessential sequences. complexes. Members of the rhabdovirus and paramyxovirus Several previous observations had suggested the feasibility of families possess tight RNP structures that protect the enwrapped introducing a complete additional gene into the RV genome. On RNA against RNase attack at any salt concentration and that Downloaded by guest on October 2, 2021 7314 Microbiology: Mebatsion et al. Proc. Natl. Acad. Sci. USA 93 (1996) remain highly active even after banding in CsCl density gradients RV and VSV (unpublished data). As demonstrated by the (17, 18). Since both template and product RNAs are tightly successful recovery of chimeric VSV, the G protein of another encapsidated by nucleoprotein, hybridization of complementary serotype or strain, respectively, can replace the homologous G sequences in an intermolecular copy-choice process appears protein (28, 29). Most promising is our recent observation that in unlikely. Moreover, the overwhelming amount of negative strand the absence of the RV G, spikeless rhabdovirions are assembled virus defective interfering particles is of the copyback type, the and budded (30). Contribution of a recombinant surface protein formation of which does not require a switch to another RNP to virus formation is, therefore, not necessary, indicating that template (19). Therefore, it appears that intermolecular homol- rhabdoviruses with a wide spectrum of glycoproteins and speci- ogous recombination is a very rare event in rhabdoviruses and ficity can be generated in the future. paramyxoviruses. RNPs of the segmented influenza virus are less rigidly structured (20). Interestingly, this is not only reflected by Note Added in Proof. Schnell et al. (31) have recently described stable the observation that the RNA component of influenza RNPs is expression of CAT from VSV. susceptible to RNase, but also that influenza RNPs can be reconstituted in vitro (4, 5). Although homologous recombination We thank Uli Wulle, Veronika Schlatt, and Karin Kegreiss for their has not been observed, sporadic "illegitimate" recombination excellent technical assistance and G. Meyers for critical comments on between viral genome RNAs and cellular mRNAs, as detected in the manuscript. This work was supported by Grant BEO21/0310118A positive strand RNA viruses (21, 22), has been described for from the Bundesministerium fur Bildung, Wissenschaft, Forschung influenza virus (23). und Technologie. Due to these considerations, the observed high genetic stability 1. Bredenbeek, P. J. & Rice, C. M. (1992) Semin. Virol. 3, 297-310. of the CAT gene in the nonsegmented RV genome is not totally 2. Emerson, S. U. & Wagner, R. R. (1972) J. Virol. 10, 297-309. unexpected and may be attributed to the lack of efficient recom- 3. Conzelmann, K.-K. (1996) J. Gen. Virol. 77, 381-389. bination in rhabdoviruses. Thus, in comparison to vaccine vectors 4. Garcia-Sastre, A. & Palese, P. (1995) Biologicals 23, 171-178. based on various positive strand RNA viruses, as recently pro- 5. Li, S., Polonis, V., Isobe, H., Zaghouani, H., Guinea, R., Moran, posed for polio virus (24), rhabdoviruses appear to be more T., Bona, C. & Palese, P. (1993) J. Virol. 67, 6659-6666. suitable. 6. Schnell, M. J., Mebatsion, T. & Conzelmann, K.-K. (1994) EMBO The helical nature of the rhabdovirus nucleocapsid is antici- J. 13, 4195-4203. pated to be another clue for future successful application of 7. Mebatsion, T, Schnell, M. & Conzelmann, K.-K. (1995) J. Virol. rhabdovirus vectors. While in icosahedral RNA virus cores 69, 1444-1451. packaging constraints impose limits on the length ofthe insert (1), 8. Conzelmann, K.-K., Cox, J. H., Schneider, L. G. & Thiel H.-J. not (1990) Virology 175, 485-499. this is expected to apply to rhabdoviruses. Truncated genomes 9. Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. (1986) Proc. of defective interfering particles or of artificial model genomes of Natl. Acad. Sci. USA 83, 8122-8126. VSV and RV are assembled into nucleocapsids and enwrapped 10. Conzelmann, K.-K. & Schnell, M. (1994) J. Virol. 68, 713-719. into short virus particles (ref. 25; unpublished data). As shown 11. Gorman, C. M., Moffat, L. F. & Howard, H. (1982) Mol. Cell. here, the enlargement of the RV genome by 635 and 1035 Biol. 2, 1044-1051. nucleotides to values of 12,567 and 12,967 nucleotides in SAD 12. Conzelmann, K.-K., Cox, J. H. & Thiel H.-J. (1991) Virology 184, VCAT and SAD XCAT, respectively, did not interfere with 655-663. amplification and incorporation of genomes into virion particles. 13. Schnell, M. & Conzelmann, K.-K. (1995) Virology 214, 522-530. Another recombinant RV with a genome of 13,600 nucleotides 14. Tordo, N., De Haan, P., Goldbach, R. & Poch, 0. (1992) Semin. and possessing the open reading frame of the firefly luciferase Virol. 3, 341-357. gene instead of the CAT sequences was recently recovered 15. Radecke, F., Spielhofer, P., Schneider, H., Kaelin, K., Huber, M., Dotsch, C., Christiansen, G. & Billeter, M. A. (1995) EMBOJ. 14, (unpublished data). This virus (SAD flash) transcribes an addi- 5773-5784. tional mRNA of 1862 templated nucleotides encoding a protein 16. Mindich, L. (1995) Semin. Virol. 6, 75-83. of 550 amino acids and grows to titers similar to that of standard 17. Lynch, S. & Kolakofsy, D. (1977) J. Virol. 28, 584-589. RV. Since the expression machinery of nonsegmented negative 18. Heggeness, M. H., Scheid, A. & Choppin, P. W. (1981) Virology strand RNA viruses can cope with genomes approaching 20 kb, 114, 555-562. as illustrated by filoviruses, we suspect that packaging of even 19. Lazzarini, R. A., Keene, J. D. & Schubert, M. (1981) Cell 26, longer rhabdovirus genomes into elongated, bullet-shaped virions 145-154. is possible. Constraints similar to those displayed by particular 20. Baudin, F., Bach, C., Cusack, S. & Ruigrok, R. W. (1994) EMBO paramyxoviruses, namely the requirement of genomic RNAs to J. 13, 3158-3165. consist of a multiple of six nucleotides to be replicated efficiently 21. Meyers, G., Riimenapf, T. & Thiel, H.-J. (1989). Nature (London) (15, 26), and thus embarrassing manipulations, apparently also do 341, 491. 22. Meyers, G., Rumenapf, T., Tautz, N., Dubovi, E. J. & Thiel, H.-J. not apply to rhabdoviruses. Also taking into account that rhab- (1991) Arch. Virol. Suppl. 3, 133-142. dovirus vectors may provide the options to adjust the level of 23. Katchikian, D., Orlich, M. & Rott, R. (1989) Nature (London) expression of a heterologous gene by selecting the insertion site 340, 156-157. and to manipulate the ratio between replication and transcrip- 24. Andino, R., Silvera, D., Suggett, S. D., Achacoso, P. L., Miller, C. J., tion, as demonstrated for VSV (25) and Sendai virus (27), these Baltimore, D. & Feinberg, M. B. 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