Specific Infectionof CD4+ Target Cells by Recombinant Rabies Virus Pseudotypes Carrying the HIV-1 Envelope Spike Protein

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11366-11370, October 1996 Colloquium Paper

This paper was presented at a colloquium entitled "Genetic Engineering of Viruses and Virus Vectors," organized by Bernard Roizman and Peter Pakse (Co-chairs), held June 9-11, 1996, at the National Academy ofSciences in Irvine, CA.

Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein TESHOME MEBATSION AND KARL-KLAUS CONZELMANN* Department of Clinical Virology, Federal Research Centre for Virus Diseases of Animals, Paul-Ehrlich-Strasse 28, D-72076 Tiubingen, Germany

ABSTRACT A recombinant rabies virus (RV) mutant infectious pseudotype viruses are usually obtained, represent- deficient for the surface spike glycoprotein (G) gene was used ing the major drawback in clinical application. to study the incorporation of envelope proteins from HIV-1 Another group of viruses that are able to bud in the absence expressed from transfected plasmids. A hybrid HIV-1 protein of their viral spike glycoprotein are the rhabdoviruses (7) in which the cytoplasmic domain was replaced with that of RV which include VSV and rabies virus (RV). Rhabdoviruses have G was incorporated into the virus envelope and rescued the a single negative strand RNA genome of 11,000-12,000 nt and infectivity of the RV mutant. The RV(HIV-1) pseudotype replicate in the cytoplasm (8). Virus assembly and budding viruses could infect only CD4+ cells, and their infectivity was takes place at the cell membrane where the viral ribonucleo- neutralized specifically by anti-HIV-1 sera. In contrast to the protein complex is enwrapped into an envelope containing an chimeric protein, wild-type HIV-1 envelope protein or mu- internal matrix protein and the single transmembrane spike tants with truncated cytoplasmic domains failed to produce glycoprotein (G) (9). By applying a system that allows genetic pseudotyped particles. This indicates the presence ofa specific engineering of RV (10, 11), we could recently recover a signal in the RV G cytoplasmic domain, allowing correct recombinant RV mutant deficient for the G gene and dem- incorporation of a spike protein into the envelope of rhab- onstrate that, in the absence of G protein, bullet-shaped dovirus particles. The possibility of directing the cell tropism spikeless rhabdovirus particles are released from the infected of RV by replacement of the RV G with proteins of defined cells (7). In the same study, we could also confirm that, similar receptor specificity should prove useful for future develop- to VSV G (12) and in contrast to retroviruses (13), the ment of targetable gene delivery vectors. cytoplasmic domain of the RV G protein contributes considerably to sorting and incorporation of the protein into the viral The host range of viruses is determined primarily by the envelope. Moreover, previous studies confined to a tempera- interaction of viral envelope glycoproteins with specific pro- ture-sensitive G protein mutant of VSV (tsO45) have indicated teins on the host cell surface that act as receptors. This that the cytoplasmic domain of VSV G protein is sufficient for interaction is very often species-specific and, in some cases, directing a foreign protein, the HIV-lEnv, into VSV (14). even tissue-specific. As a result, some viruses, like retroviruses, In the present study, we exploited the G deficiency of a have a quite limited host range, whereas others, such as recombinant RV mutant to confirm that the RV G cytoplasmic rhabdoviruses, can infect a variety of cell types including in tail provides a signal allowing specific incorporation of HIV-1 many cases cells of human, animal, and insect origin. Devel- Env, expressed from transfected plasmids, into the envelope of oping means for redefining the receptor specificity of viral virus particles and to verify that no residual G protein is particles would have numerous applications in basic research required to initiate rhabdovirus pseudotype formation. The and clinical application. On the one hand, this might allow generated RV(HIV) pseudotype particles possess the tropism better infection of cells of therapeutic interest that are poorly of HIV-1 in that they can infect HeLa cells expressing the amenable to retrovirus infection, such as hepatocytes or early major HIV receptor CD4, but not CD4- HeLa cells. The hematopoietic progenitors. On the other hand, this would also transient G-deficient RV mutant system provides a versatile allow targeting of specific cell types in complex cell popula- and safe tool to rapidly analyze incorporation and function of tions. In the in vtivo situation, the availability of targeting a wide range of surface protein constructs. Moreover, since methods should, for example, permit the development of new foreign genes in RV and VSV genomes are highly stable (15, gene therapy models. 16), recombinant rhabdoviruses represent promising candi- Alteration of the tropism of viruses by incorporation of a dates for future development of targetable, nonintegrative foreign envelope protein, however, is hampered in most virus viral vectors for delivery of therapeutic or protective genes. systems by the fact that the presence of the viral spike protein(s) is required also to drive the viral assembly and budding process (for review, see ref. 1). Retroviruses represent MATERIALS AND METHODS one example where this does not apply (2, 3). In the past, Cells, Viruses, cDNA, and Antibodies. The following re- retroviral vectors deficient for the spike glycoprotein gene agents were obtained through the AIDS Research and Ref- (env) have been used to generate pseudotype viruses carrying erence Reagent Program (National Institute of Allergy and foreign proteins in their envelopes, predominantly the vesic- Infectious Diseases, National Institutes of Health, Bethesda). ular stomatitis rhabdovirus (VSV) G protein. As expected, the HeLa CD4+ and HeLa CD8+ cells were from Richard Axel pseudotype viruses exhibit the expanded host range of the (17), and human anti-HIV-1 immune globulin was from Alfred foreign glycoprotein (4-6). Since incorporation of the heter- Prince (18, 19). The recombinant vaccinia virus vTF73, ex- ologous spike proteins is apparently nonspecific, low titers of Abbreviations: RV, rabies virus; SAD, Street-Alabama-Dufferin The publication costs of this article were defrayed in part by page charge strain of RV; VSV, vesicular stomatitis virus; G, glycoprotein. paymenit. This article must therefore be hereby marked "advertisemnent" in *To whom reprint requests should be addressed. e-mail: klaus. accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected].

11366 Colloquium Paper: Mebatsion and Conzelmann Proc. Natl. Acad. Sci. USA 93 (1996) 11367 pressing T7 RNA polymerase (20), was kindly provided by T. buffer (10 mM Tris, pH 7.4/50 mM NaCl/1 mM EDTA), Fuerst and B. Moss, and the HIV-1 (NL-43) gpl60 cDNA was layered on continuous 10-50% sucrose gradients, and centri- from W. Garten and H.-D. Klenk (Institute of Virology, fuged at 27,000 rpm in an SW 41 rotor for 1 hr. Virus proteins University of Marburg, Germany). The G-deficient Street- from 12 equal gradient fractions were resolved by SDS/PAGE Alabama-Dufferin RV mutant, SAD AG, was recovered and and transferred to nitrocellulose membranes using a semidry propagated after deletion of the entire G protein coding region transfer apparatus (Hoefer). After incubation with a blocking from a full-length RV cDNA in cells expressing RV G from a solution (2.5% dry milk/0.05% Tween 20 in PBS), the blot was transfected plasmid in the vaccinia/T7 polymerase system (7). incubated overnight with a mixture of human HIV-1 IgG Construction of Expression Plasmids. An expression plas- (1:10,000) and rabbit sera raised against purified RV G protein mid encoding wild-type HIV-1 Env (pEnv) was generated by (S72, 1:20,000), RV ribonucleoprotein complex (S50, 1:20,000) insertion of the BbsI-XhoI-Klenow fragment spanning the or a peptide deduced from the the RV matrix protein sequence entire gpl60 coding region from the HIV NL-43 cDNA in the (M1-B4, 1:10,000) in PBST. After three successive washes in EcoRV site of pT7T (21). To replace the HIV cytoplasmic PBST, the blot was incubated for 2 hr with a mixture of domain, an HpaI site (underlined) was introduced by PCR peroxidase-conjugated goat anti-human and goat anti-rabbit using a primer (5'- GCTCTAGACTAGTTAACTATA- IgG (Dianova) diluted 1:10,000 in PBST. The blot was washed GAAAGTACAGC-3') overlapping the transmembrane/ as above, stained with an Enhanced Chemiluminescence West- cytoplasmic domain-encoding region and a second primer ern blot detection kit (Amersham) for 1 min, and exposed to (5'-AACAATTACACAAGCTTAAT-3') spanning an up- x-ray films (Biomax MR, Kodak). stream unique Hindlll site (underlined) of the Env cDNA. An HpaI site was also introduced by PCR (5'-AAGTCGACCGT- TAACAGAAGAGTCAATCGATCA-3') upstream of the RESULTS pT7T-G sequence encoding the RV SAD B19 G cytoplasmic The HIV-1 env protein is synthesized as a precursor (gpl60) tail. After ligation of the HIV-derived HindIII-HpaI fragment that is cleaved during transport to the plasma membrane into and the G-derived HpaI-PstI fragment, the construct was used two noncovalently associated subunits, gpl20, which is in- to replace the sequence downstream of Hindlll in pEnv to volved in binding to the HIV receptor, and gp4l, which yield pEnv-RVG. The removal of an HpaI fragment of pEnv contains the membrane anchor and a carboxyl-terminal cyto- resulted in pEnv-A107; the encoded protein possessed a de- plasmic domain of 150 amino acids (22, 23). To determine the letion of 107 amino acids adjacent to the transmembrane influence of both sequence and length of the cytoplasmic domain (see Fig. 1). The Env construct possessing a carboxyl- domain on formation of RV(HIV) pseudotypes, various terminal truncation (pEnv-44) was generated by introduction cDNA constructs resulting in altered cytoplasmic domains of a translational stop codon (complementary sequence un- were prepared in the expression plasmid pT7T (21), which is derlined) into the cytoplasmic tail-encoding sequence of under the control of the T7 RNA polymerase promoter. pHIV-Env by PCR mutagenesis using a primer with the Starting from a plasmid encoding the authentic HIV-1 NL-43 sequence 5'-ACGAATTCATTAGTTCACTAATCGAATG- Env protein (pEnv), we prepared a construct that encodes a 3 . protein in which the entire Env cytoplasmic domain is replaced Expression of Envelope Proteins. BSR cells were first in- by the 44 amino acid cytoplasmic domain of RV G (Env- fected with vTF7-3 at a multiplicity of infection of 5. After 1 RVG). To investigate whether the considerable length of the hr of infection, cells were transfected with CsCl-purified HIV Env cytoplasmic domain may affect incorporation into plasmids by using the Stratagene mammalian transfection kit RV particles, two other plasmids encoding proteins with short as described (21). To adjust the level of cell surface expression, Env cytoplasmic domains, similar in length to that of RV G, various concentrations of plasmid DNA were used for trans- were constructed. Env-44 represented a protein with a car- fection (see below). After incubation for 16 hr, cells were fixed boxyl-terminally truncated cytoplasmic domain, whereas in with 4% paraformaldehyde and incubated at 4°C for 30 min pEnv-A107 an internal deletion removed the membrane- with human anti-HIV-1 IgG (1:500 dilution) or a monoclonal proximal 107 amino acids and fused the carboxyl-terminal 43 antibody directed against RV G protein (diluted 1:100). Cells residues to the membrane anchor domain (Fig. 1). were stained with fluorescein isothiocyanate-conjugated goat Transient Expression of Glycoproteins. To analyze expres- anti-human IgG or goat anti-mouse IgG (Dianova, Hamburg, sion of the recombinant glycoproteins on the cell surface, Germany). For double staining, rhodamine-conjugated goat which represents a prerequisite for incorporation into RV anti-mouse IgG (Dianova) was used in combination with particles, plasmids were transfected into BSR cells that had fluorescein isothiocyanate-conjugated goat anti-human IgG. been infected with the recombinant vaccinia virus vTF7-3 Complementation of SAD AG with Envelope Proteins. BSR providing T7 RNA polymerase (20). Surface expression was cells were infected at a multiplicity of infection of 1 with SAD compared by indirect immunofluorescence with human anti- AG phenotypically complemented with RV G protein and HIV IgG (18) after transfection of equal amounts of protein- were then superinfected with vTF7-3. Plasmids encoding the encoding plasmids. Interestingly, compared with the authentic spike proteins, pT7T-G, pEnv, pEnv-A107, pEnv-44, and HIV-Env, the level of cell surface expression was higher for all pEnv-RVG (1, 7.5, 5, 3, and 3 ,tg, respectively) were then Env constructs with modified cytoplasmic domains (data not transfected as described above. After incubation at 37°C for 24 shown). To obtain similar levels of expression, the amounts of hr, cell culture media were harvested and cleared of cell debris. the plasmids in transfection experiments were therefore ad- To determine the infectious titers of pseudotype viruses, justed. After transfection of 7.5 ,ug pT7T-Env and of 5, 3, and supernatants were serially diluted and used for inoculation of 3 ,tg of pEnv-A107, pEnv-44, and pEnv-RVG, respectively, confluent monolayers of CD4+ or CD8+ HeLa cells. After 24 surface fluorescence intensity and the number of expressing hr of infection cells were fixed with 80% acetone and expres- cells were similar. Representative micrographs are shown in sion of RV nucleoprotein was examined by direct immuno- Fig. 2. By double labeling cells expressing both RV G and each fluorescence after staining with an fluorescein isothiocyanate- of the plasmids encoding an Env protein, no differences in the conjugate directed against RV nucleoprotein (Centocor). distribution of proteins on the cell surface were observed (data Sucrose Gradients and Western Blotting. Supernatants not shown). from complementation experiments (approximately 6 x 106 We next determined whether the introduced mutations cells) were harvested 24 hr after transfection, and virions were affected the function of the HIV envelope proteins in attach- pelleted through a 10% sucrose cushion at 19,000 rpm in a ment to the HIV receptor and induction of membrane fusion. Beckman SW 41 rotor. Pellets were resuspended in TEN The adjusted amounts of plasmids were transfected into HeLa 11368 Colloquium Paper: Mebatsion and Conzelmann Proc. Natl. Acad. Sci. USA 93 (1996)

transmembrane cytoplasmic RV G: ... MLIIFLMTCCRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGETRL*

Env-RVG: ... IVFAVLSIVNRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGETRL* Env-44: ... IVFAVLSIVNRVRQGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVN* HIV-Env: ... IVFAVLS IVNRVRQGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDL/ /RSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVN/ /LLNATAIAVAEGTDRVIEVLQAAYRAIRHIPRRIRQGLERILL* Env-A107: ... IVFAVLSIVNLLNATAIAVAEGTDRVIEVLQAAYRAIRHIPRRIRQGLERILL* FIG. 1. Organization of wild-type and hybrid envelope proteins. The amino acid sequence of part of the transmembrane domains (underlined) and of the entire cytoplasmic domains of mutants and wild-type proteins are shown. cells constitutively expressing the major HIV receptor, CD4 proteins or, as a control, were transfected with RV G. After (CD4+ HeLa, ref. 17), and that had been infected with vTF7-3. incubation for 24 hr, the supernatants were collected and the At 14 hr after transfection, each mutant caused formation of infectivity of virus particles was determined on HeLa CD4+ syncytia equivalent to that produced by the HIV-Env protein and HeLa CD8+ cells by direct immunofluorescence. RV G (Fig. 3). In addition, analysis of 35S-labeled proteins from cell protein rescued the infectivity of SAD AG for both cell lines, extracts and media had revealed correct proteolytic cleavage of regardless of the presence of CD4 at the cell surface (Fig. 4A mutant precursors to generate gpl20 (data not shown). Taken and B). In addition, rescue was observed in one of the four Env together, these results indicated correct transport to the cell protein constructs, Env-RVG, which is the Env protein pos- surface, proteolytic processing, and fusogenicity of the mu- sessing the cytoplasmic tail of RV G. In contrast to RV G, tants. however, particles complemented with Env-RVG could only Rescue of SAD AG by Engineered Env Protein Mutants. The infect CD4+ HeLa cells (Fig. 4 E and F) and their infectivity construction and recovery of a G-deficient RV mutant lacking could be specifically neutralized by anti-HIV-1 serum (data the entire G gene (SAD AG) were described previously (7). not shown). Thig indicates that infection is mediated by the Stocks of phenotypically complemented SAD AG were pro- hybrid Env protein. Compared with RV G, the infectious titers duced in BSR cells expressing RV G from transfected pT7T-G. obtained with Env-RVG were in average 25 times lower (Table Upon inoculation of noncomplementing BSR cell cultures, 1). No fluorescent cells were observed after complementation infection was restricted to the cells initially infected by the of SAD AG with the wild-type Env or the constructs possessing G-complemented particles and was not able to spread through the short gp4l cytoplasmic tails, indicating that the presence of the monolayers. The supernatants obtained from such cultures defined sequences of the RV G tail is required for specific contained spikeless rhabdovirus particles that could not infect incorporation into the viral envelope. fresh BSR cells (7). As for BSR cells, infection of HeLa cells Protein Composition of RV(HIV) Pseudotype Viruses. To was found to require the presence of the viral spike protein. verify the incorporation of the Env-RVG protein into the To determine whether any of the mutant Env proteins could envelope of SAD AG particles, virions were purified by velocity rescue the infectivity of SAD AG, BSR cells were infected with centrifugation in continuous 10-50% sucrose gradients. The both G-complemented SAD AG and vTF7-3 and were then protein composition of gradient fractions was analyzed by transfected with plasmids encoding wild-type or hybrid Env Western blotting with a combination of sera directed against

-; ,R 4g i 5 i; g

0 C'~~~~~~~~~~0

FIG. 2. Surface expression of envelope protein constructs. Cells FIG. 3. Fusion activity of envelope protein constructs. CD4+ HeLa were infectedwith vTF7-3 and transfedted with plasmids encoding Env cells were infected with vTF7-3 and transfected with pEnv (A), (A), Env-A107 (B), Env-44 (C), Env-RVG (D), or RV G (E) (7.5, 5, pEnv-A107 (B), pEnv-44 (C), pEnv-RVG (D), and pT7T-G (E) by 3, 3, and 1 utg, respectively). (F) Mock-transfected cells. Sixteen hours using the amounts of plasmids determined to yield similar surface after transfection, cells were processed for indirect immunofluores- expression of proteins (see Fig. 2). (F) Mock-transfected cells. Syn- cence as described. cytium formation is shown after an incubation of 14 hr at 37°C. Colloquium Paper: Mebatsion and Conzelmann Proc. Natl. Acad. Sci. USA 93 (1996) 11369 CD4+ HeLa cells CD8' HeLa cells 1 6 12 6 12 .gpl2O -100- -*- - G- -."*,4-I 68 -

N -_"NMN,06. -N - 43 -

_'gp41"_p, - 29 -

kd top top RV G Env-RVG FIG. 5. Protein composition of pseudotype particles. SAD AG particles phenotypically complemented with RV G protein or Env- RVG were analyzed by velocity centrifugation in 10-50% sucrose gradients and Western blotting. Infectivity and the majority of virus proteins are located in gradient fractions 6 and 7. In addition to the RV nucleoprotein (N), phosphoprotein (P), and matrix protein (M), RV(HIV) pseudotype viruses contain a gp4l-derived chimeric transmembrane subunit protein of -37 kDa ("gp4l") and the gpl20 subunit. Env proteins (data not shown). Taken together, these results confirmed true incorporation of the hybrid Env-RVG protein FIG. 4. Rescue of the infectivity of SAD AG by engineered Env and revealed the requirement of the RV G cytoplasmic tail to proteins. BSR cells were infected with G-complemented SAD AG and promote this process. vTF7-3 and transfected with Env protein-coding plasmids. At 24 hr after transfection, supernatants were used to inoculate monolayers of DISCUSSION CD4+ and CD8+ HeLa cells. Cells were then fixed after 24 hr of incubation and examined by direct immunofluorescence with a con- The possibility of generating recombinant RV pseudotype jugate directed against RV nucleoprotein. (A and B) RV G. (C and D) viruses with altered cell specificity was suggested by several HIV-Env. (E and F) Env-RVG. ForA and B, 100 ,ul and for C-F 1 ml previous observations. The recent availability of a defined, of the respective supernatant from 106 transfected BSR cells was used for inoculation. recombinant RV mutant lacking the entire G gene revealed that the only viral surface protein, or part of it, is not required RV proteins and anti-HIV IgG. Both the peak of infectivity to drive budding of particles from the cell surface membrane. and the majority ofvirus proteins were found in fractions 6 and In addition, the cytoplasmic tail of RV G was found to 7 for particles pseudotyped with RV G or Env-RVG (Fig. 5). considerably contribute to sorting of G spikes into the virions In addition to RV nucleoprotein, phosphoprotein, and matrix (7). Finally, a temperature-sensitive VSV mutant containing at protein, RV(Env-RVG) pseudotype particles contained a nonpermissive temperature only carboxyl-terminal VSV G protein reacting with the anti-HIV serum and migrating at protein fragments (25) was successfully rescued by a chimeric -37 kDa. This corresponds well to the size predicted for the HIV Env protein possessing the VSV G cytoplasmic tail. This hybrid subunit composed of the HIV gp4l extracellular and indicated that the G cytoplasmic domain sequence might be transmembrane domains fused to the RV G cytoplasmic tail. even sufficient to direct foreign proteins into virions (14). In addition, a faint band of high molecular weight, which The data presented here demonstrate that the cytoplasmic should correspond to the gpl20 subunit, was identified. Since tail of the RV G contains a signal sufficient and necessary to shedding of the gpl20 subunit from virus particles during direct a chimeric HIV Env spike protein, Env-RVG, into the sucrose gradient centrifugation is apparently notorious (24), envelope of "rabies" virus particles in the absence of G or of this finding was not unexpected. In contrast to Env-RVG, parts of it. In contrast to RV, which is able to infect all culture which was the only construct able to rescue infectivity of SAD cells analyzed so far, the tropism of the pseudotype particles is AG, no HIV-related proteins could be detected in virions restricted and is obviously determined by the specificity of the generated in the presence of the wild-type or the other mutant incorporated spike protein and its interaction with the HIV-1 CD4 receptor complex. This implies that, in contrast to RV, Table 1. Rescue of SAD AG by recombinant spike proteins which enters cells after receptor-mediated endocytosis and Titers of infectious pseudotype particles fusion of membranes in acidic lysosomes, RV(HIV) per ml* pseudotype membranes should fuse with the cell surface membrane at neutral pH. Subsequent successful viral replica- Spike protein CD4+ HeLa cells CD8+ HeLa cells tion and protein expression could be monitored directly. As a Env 0 0 consequence of the genetic deficiency for a spike protein, Env-A107 0 0 infection was restricted to the primarily infected cells, allowing Env-44 0 0 direct determination of the yield of infectious pseudotype Env-RVG 1 x 103-4 x 103 0 viruses. RV G 2 x 104-1 x 105 3 x 104-2 x 105 In contrast to Env-RVG, wild-type HIV-1 Env or the two Mock 0 0 constructs with Env-derived cytoplasmic tails identical or *Titers were determined by counting fluorescing cells, assuming that similar in length to that of G were not capable to rescue the nucleoprotein-expressing cells result from infection with one SAD infectivity of SAD AG particles, although they were proteo- AG particle. Titer ranges result from four independent experiments. lytically processed and expressed at the cell surface and also 11370 Colloquium Paper: Mebatsion and Conzelmann Proc. Natl. Acad. Sci. USA 93 (1996) induced syncytium formation in CD4+ HeLa cells in a level 0311171 from the Bundesministerium fur Bildung, Wissenschaft, comparable to that of Env-RVG. Thus, rather than the length Forschung und Technologie. of -44 amino acids, the specific sequence of the RV cytoplasmic tail is required to promote or to allow incorporation of 1. Simons, K. & Garoff, H. (1980) J. Gen. Virol. 50, 1-21. 2. Delchambre, M., Gheysen, D., Thines, D., Thiriart, C., Jacobs, E., spikes into the viral envelope. This is strongly supported by Verdin, E., Horth, M., Burny, A. & Bex, F. (1989) EMBO J. 8, preliminary data on an additional Env mutant capable of 2653-2660. rescuing the infectivity of SAD AG. The mutant has a cyto- 3. Gheysen, D., Jacobs, E., deForesta, F., Thiriart, C., Francotte, plasmic tail of 65 amino acids, the carboxyl terminus of which M., Thines, D. & DeWilde, M. (1989) Cell 59, 103-112. corresponds to the sequence of the RV G tail. As suggested by 4. Dong, J., Roth, M. G. & Hunter, E. (1992) J. Virol. 66, 7374- the ability of the G protein from another lyssavirus serotype to 7382. replace RV G (26) and by comparison of available lyssavirus 5. Burns, J. C., Friedmann, T., Driever, W., Burrascano, M. & Yee, G the critical motif be located in the car- J.-K. (1993) Proc. Natl. Acad. Sci. USA 90, 8033-8037. sequences, might 6. Yee, J. K., Miyanohara, A., LaPorte, P., Bouic, K., Burns, J. & boxyl-terminal region of the tail in which 7 of 14 residues are Friedman, T. (1994) Proc. Natl. Acad. Sci. USA 91, 9564-9568. conserved with regard to other lyssaviruses (unpublished 7. Mebatsion, T, Konig, M. & Conzelmann, K.-K. (1996) Cell 84, data). 941-951. As demonstrated recently, similar to VSV G (27), RV G 8. Rose, J. K. & Schubert, M. (1987) in The Rhabdoviruses, ed. protein possesses an independent exocytosis activity (7) and, in Wagner, R. R. (Plenum, New York), pp. 129-166. the presence of G, the yield of rhabdovirus particles is aug- 9. Wagner, R. R. & Rose, J. K. (1996) in Fields Virology, eds. Fields, mented. As estimated from the an B. N., Knipe, D. M., Howley, P. M., Chomock, R. M., Melnick, gradient analyses, approx- J. L., Monath, T. P., Roizman, B. & Straus, S. E. (Lippincott- imately 5-fold higher amount of particles complemented with Raven, Philadelphia), pp. 1121-1135. G compared with RV(HIV) pseudotype particles was ob- 10. Schnell, M. J., Mebatsion, T. & Conzelmann, K.-K. (1994) EMBO served. Although substantial amounts of Env-RVG spikes J. 13, 4195-4203. were incorporated into the particles, as shown directly by the 11. Conzelmann, K.-K. (1996) J. Gen. Virol. 77, 381-389. presence of the anchored, gp4l-derived chimeric 37 kDa 12. Whitt, M. A., Chong, L. & Rose, J. K. (1989) J. Virol. 63, subunit (Fig. 5), the infectious titers differed by a factor of 3569-3578. 20-25. Since the infectivity of viruses is largely governed by the 13. Hunter, E. (1994) Semin. Virol. 5, 71-83. 14. Owens, R. J. & Rose, J. K. (1993) J. Virol. 67, 360-365. features of their spike proteins and the interaction with 15. Schnell, M. J., Buonocore, L., Whitt, M. A. & Rose, J. K. (1996) receptors, the discrepancy in infectious titers may not directly J. Virol. 70, 2318-2323. reflect the efficiency by which the hybrid protein can substitute 16. Mebatsion, T., Schnell, M. J., Cox, J. H., Finke, S. & Conzel- for RV G in formation of infectious virions. mann, K.-K. (1996) Proc. Natl. Acad. Sci. USA 93, 7310-7314. It is not known so far whether oligomerization has an 17. Maddon, P. J., Dalgleish, A. G., McDougal, J. S., Clapham, P. R., influence on the putative interaction of the cytoplasmic tail Weiss, R. A. & Axel, R. (1986) Cell 47, 333-348. with internal virus proteins. Similar to the RV spike (28), 18. Prince, A. M., Reesink, H., Pascual, D., Horowitz, B., Hewlett, I., Murthy, K. K., Cobb, K. E. & Eichberg, J. W. (1991) AIDS Res. HIV-Env may probably form trimers (29). The successful Hum. Retroviruses 7, 971-973. incorporation of Env-RVG suggests that this protein may 19. Prince, A. M., Horowitz, B., Baker, L., Shulman, R. W., Ralph, present the tail(s) in an appropriate configuration. Incorpo- H., et al. (1988) Proc. Natl. Acad. Sci. USA 85, 6944-6948. ration of the monomeric cellular glycoprotein CD4 into VSV 20. Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. (1986) Proc. tsO45 has been reported previously (30). However, particles Natl. Acad. Sci. USA 83, 8122-8126. containing CD4, or a chimeric CD4 with the VSV cytoplasmic 21. Conzelmann, K.-K. & Schnell, M. J. (1994) J. Virol. 68, 713-719. tail, were observed only at permissive temperature, suggesting 22. Wain-Hobson, S., Sonigo, P., Danos, O., Cole, S. & Alizon, M. (1985) Cell 40, 9-17. that VSV G protein is required for incorporation of CD4 into 23. Willey, R. L., Bonifacino, J. S., Potts, B. J., Martin, M. A. & VSV. Klausner, R. D. (1988) Proc. Natl. Acad. Sci. USA 85, 9580-9584. Our results verified that a recombinant rhabdovirus can be 24. Schneider, J., Kaaden, O., Copeland, T. D., Orozlan, S. & generated whose tropism is exclusively determined by a foreign Hunsmann, G. (1986) J. Gen. Virol. 67, 2533-2538. surface glycoprotein incorporated specifically into the viral 25. Metsikko, K. & Simons, K. (1986) EMBO J. 5, 1913-1920. envelope. Due to the transient nature of the assay, the 26. Mebatsion, T., Schnell, M. J. & Conzelmann, K.-K. (1995) J. Vi- described system should provide a versatile and, most impor- rol. 69, 1444-1451. 27. Rolls, M. M., Webster, P., Balba, N. H. & Rose, J. K. (1994) Cell tantly, a safe tool in determining whether other glycoproteins 79, 497-506. might be able to direct rhabdovirus vectors to specific target 28. Gaudin, Y., Tuffereau, C., Segretain, D., Knossow, M. & Fla- cells. mand, A. (1992) Virology 187, 627-632. 29. Gelderblom, H. R. (1991) AIDS 5, 617-638. We thank Veronika Schlatt and Karin Kegreiss for perfect technical 30. Schubert, M., Joshi, B., Blondel, D. & Harmison, G. G. (1992) assistance. This work was supported by Grants BEO 21/0310118A and J. Virol. 66, 1579-1589.