Mokola Virus Glycoprotein and Chimeric Proteins ... - Journal of Virology

JOURNAL OF VIROLOGY, Mar. 1995, p. 1444–1451 0022-538X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 69, No. 3

Mokola Virus Glycoprotein and Chimeric Proteins Can Replace Rabies Virus Glycoprotein in the Rescue of Infectious Defective Rabies Virus Particles TESHOME MEBATSION, MATTHIAS J. SCHNELL,

AND

KARL-KLAUS CONZELMANN*

Federal Research Centre for Virus Diseases of Animals, D-72076 Tu ¨bingen, Germany Received 27 July 1994/Accepted 16 November 1994

A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713–719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells.

The mature RV G consists of an amino-terminal N-glycosylated ectodomain of 439 amino acids, a transmembrane anchor sequence of 22 amino acids, and a cytoplasmic domain of 44 amino acids (1, 36). In the extracellular domain of the G protein of the challenge virus standard (CVS), two major antigenic sites (sites II and III) and one minor site (minor site a) that bind virus-neutralizing monoclonal antibodies were defined (2). Two additional epitopes have been delineated on the Evelyn-Rokitnicky-Abelseth (ERA) RV strain G protein (22). The structure and locations of antigenic regions on the G proteins of rabies-related viruses, however, have not yet been defined. The RV G is transported from the endoplasmic reticulum to the Golgi apparatus and then to the plasma membrane, where it is incorporated into the membrane of budding virus particles. Interaction between the cytoplasmic tail of the viral glycoprotein and internal viral components is thought to be required for virus assembly and budding. Direct interaction between the cytoplasmic tail of alphavirus spike glycoproteins and the nucleocapsid protein has been demonstrated recently (25). At least, incorporation of a glycoprotein into virions was also shown to be directed by the cytoplasmic domain of the G protein of vesicular stomatitis virus (VSV), the prototype rhabdovirus (27, 32). Functional analysis of mutated RV G proteins in order to investigate the interactions with viral and cellular proteins has been hampered considerably by the technical difficulty in manipulating the genomes of nonsegmented negative-stranded RNA viruses. In addition, temperature-sensitive mutants that would allow complementation by DNA-encoded G protein, as used successfully for VSV (27, 32), were not available. Recently, however, we described a reverse genetics approach which allowed reconstitution of the entire viral life cycle en-

The genus Lyssavirus within the family Rhabdoviridae contains classical rabies viruses (RVs) (serotype 1) and rabiesrelated viruses which are divided into serotypes 2 to 6 (3). RV, the prototype of the genus Lyssavirus, is an enveloped negative-strand RNA virus containing five structural proteins. Three of the proteins, the nucleoprotein (N), the phosphoprotein (P), and the polymerase protein (L), together with the RV genome form a helical ribonucleoprotein complex (RNP) or nucleocapsid. The RNP is surrounded by an envelope which contains the matrix protein (M), located at the inner side of the lipid bilayer, and the transmembrane glycoprotein (G). The glycoproteins of lyssaviruses appear to exhibit similar overall structure; however, considerable antigenic differences were noted between RV and rabies-related viruses by using antiglycoprotein monoclonal antibodies (10). Serologic analyses, cross-protection experiments, and molecular characterization have demonstrated that classical RV and Mokola virus (lyssavirus serotype 3) are the most divergent lyssaviruses (3, 10, 23, 24). The RV G is present as a trimer at the viral surface (16, 31). It is responsible for induction of virus-neutralizing antibodies (8, 33) and mediates binding of the virus to specific receptors on the cell membrane (35), which have not yet been characterized. After attachment to the host cell and internalization of the virion by endocytosis, the G protein mediates fusion of the viral envelope with the endosomal membrane (15, 17, 31) to allow the release of the RNP into the cytoplasm.

* Corresponding author. Mailing address: Institute for Clinical Virology, Federal Research Centre for Virus Diseases of Animals, PaulEhrlich-Strasse 28, D-72076 Tu ¨bingen, Germany. Phone: 49 7071 967 205. Fax: 49 7071 967 303. 1444

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FUNCTIONAL REPLACEMENT OF RABIES VIRUS GLYCOPROTEIN

tirely from cDNA-encoded proteins and RNAs (6, 29). Here, this system was used to determine whether heterologous and chimeric lyssavirus G proteins could assemble and function in RV-like particles which possess a model genome encoding the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. We therefore cloned and expressed the G gene of a Mokola virus isolate (Eth-16 [23, 24]) as well as chimeric Mokola virus-RV G constructs. In spite of highly heterogeneous cytoplasmic and transmembrane domain sequences, the Eth-16 virus G could replace RV G in all steps of the virus life cycle, as demonstrated by successful rescue of particles which were able to infect cells. In addition, infectious RV-like particles were rescued by chimeric proteins in which either the entire ectodomain or a short surface epitope was replaced. MATERIALS AND METHODS Molecular cloning and sequence determination. Eth-16 virus was isolated from the brain of a rabid cat in Ethiopia and adapted to cell culture by five successive passages in BHK-21 cells. It was identified as a Mokola virus (lyssavirus serotype 3) by using monoclonal antibodies (24). Genomic cDNA was synthesized and cloned in lambda Zap II phages (23). The nucleotide sequence of the Eth-16 virus G gene was determined from exonuclease III deletion clones (19) of two independent cDNA clones, p16-A15 and p16-A3, using the Pharmacia T7 DNA polymerase sequencing kit. Construction of expression plasmids. The construction of plasmids pSDI-CAT and pT7T-G, used to generate the RV genome analog SDI-CAT and for expression of the RV G, respectively, has been described previously (6). A plasmid encoding the Eth-16 virus G (pE16) was constructed by insertion of a 2.1-kb BspHI restriction fragment (beginning at position 21 and ending 54 nucleotides downstream of the Eth-16 glycoprotein gene sequence; Fig. 1A) from p16-A15 after Klenow fill-in of recessive ends into the EcoRV site of the pSK-T7T transcription vector (6). To prepare pTCD, the 59-terminal 1.3-kb fragment of the pE16 coding region was removed by restriction with HincII (position 1368; Fig. 1A) and an upstream multiple cloning site enzyme (HindIII) and replaced by a corresponding 1.3-kb HincII fragment (position 4663 of SAD B19 sequence [4]) of pT7T-G. To construct pE16/SAD, a pE16 HindIII (at the multiple cloning site of pSKT7T vector)-MaeII 0.8-kb fragment (position 818; Fig. 1A) and a pT7T-G BanI-PstI 1-kb fragment (positions 4126 to 5138 of the SAD B19 sequence) were fused by a synthetic oligonucleotide linker (59-CGAAACCAAATG-39, coding strand; 59-GCACCATTTGGTTT-39, noncoding strand). Site-directed mutagenesis by the method of Kunkel et al. (21) was performed with a 63-mer oligonucleotide to exchange 10 nucleotides of pT7T-G. The resulting plasmid (pSIII) encoded a modified RV G protein in which five amino acids of antigenic site III were replaced by the corresponding amino acids of Eth-16 virus G (Fig. 1B). Virus infections and DNA transfections. Transfection of BSR cells was carried out as described previously (6). First, cells were infected with the recombinant vaccinia virus vTF7-3, expressing T7 RNA polymerase (14) (kindly provided by T. Fuerst and B. Moss) at a multiplicity of infection of 5. One hour postinfection, plasmids were transfected by using the Stratagene mammalian transfection kit (CaPO4 transfection protocol). For transient expression and analyses of glycoprotein constructs (Fig. 2), cells were transfected with 2 mg of the respective plasmids and processed for fluorescence or immunoprecipitation (see below). For rescue experiments, a plasmid mix containing pSDI-CAT (2 mg) and the protein-encoding plasmids pT7T-N, -P, -L, and -M (5, 2.5, 2.5, and 2 mg, respectively) was transfected together with 2 mg of a plasmid coding for a wild-type or engineered G protein. Cells and supernatants were harvested 48 h posttransfection for further analysis and passaging experiments. Infection of BSR cells with RV SAD B19 was performed as described previously (5). Indirect immunofluorescence. At 6 h posttransfection, cells were incubated at 48C for 30 min with a 1:300 dilution of a rabbit serum (S72) raised against purified RV G, washed, and further incubated with a fluorescein isothiocyanateconjugated anti-rabbit immunoglobulin G antibody (Sigma). Cells were fixed in 4% paraformaldehyde for 5 min at 48C and examined by fluorescence microscopy. Metabolic labeling and immunoprecipitation. Approximately 106 BSR cells were infected and transfected in a 3.2-cm-diameter culture dish as described above and labeled with 125 mCi each of [35S]methionine and [35S]cysteine (.1,000 Ci/mmol each; Amersham) at 16 h posttransfection for 18 h. For pulsechase experiments, cells were labeled for 10 min with 100 mCi of [35S]methionine at 16 h posttransfection and incubated with chase medium for increasing times. Cells were lysed in lysis buffer (1% Triton X-100, 0.1% deoxycholic acid, 0.1% sodium dodecyl sulfate [SDS], 20 mM Tris [pH 7.6], 100 mM NaCl, 1 mM EDTA, 0.2% bovine serum albumin [BSA]), and the extracts were centrifuged at 45,000 rpm (TLA-45 rotor) for 1 h and incubated with specific antisera. Precipitates were formed with cross-linked Staphylococcus aureus (20), analyzed by

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SDS–10% polyacrylamide gel electrophoresis (PAGE), and processed for fluorography with En3Hance (New England Nuclear). CAT assays. CAT assays were done by standard procedures adapted from Gorman et al. (18). Cell extracts were prepared by three cycles of freezing and thawing. Equal amounts of proteins were adjusted as described before (6) and incubated with 0.25 mCi of [14C]chloramphenicol (53 mCi/mmol; Amersham) and 5 ml of 4 mM acetylcoenzyme A (Boehringer) at 378C for 1 h. Nucleotide sequence accession number. The nucleotide sequence of the glycoprotein gene of the Eth-16 virus (Mokola virus) was deposited with GenBank under accession number U17064.

RESULTS Sequence analysis of Eth-16 virus glycoprotein gene. Since classical RV and Mokola virus are the most divergent lyssaviruses, the genome of an Ethiopian Mokola virus isolate, Eth-16, was cloned (23), and the G gene was analyzed. It encodes a protein of 522 amino acids, which is 2 amino acids shorter than RV G proteins (1, 4, 30, 36). According to the deduced hydropathic profile, the Eth-16 virus G reveals structural domains similar to those of RV G proteins, namely, an amino-terminal signal peptide, a large ectodomain, and short transmembrane and cytoplasmic domains (Fig. 1B). The overall amino acid identity between the Eth-16 and RV (SAD B19) G proteins was 56.7%. Virtually no sequence homology was displayed by the signal sequences or transmembrane and cytoplasmic domains of Eth-16 and RV G proteins (15.7, 21, and 26% amino acid identity, respectively). The ectodomains of Eth-16 virus and RV G proteins are conserved, with 62% amino acid identity. The regions where RV major antigenic sites are located, however, are poorly conserved (Fig. 1B). Eth-16 virus G possesses two potential N-glycosylation sites at positions 202 and 319, the latter site being present in all lyssavirus G proteins analyzed so far. All 14 cysteine residues in the ectodomain of RV G are conserved in the Eth-16 virus G, suggesting similar folding through disulfide bridges. Construction of expression plasmids. In order to analyze whether Eth-16 virus G could replace RV G in the rescue of infectious RV particles, an expression plasmid (pE16) containing the authentic Eth-16 virus G coding region under the control of a T7 promoter was constructed. With the purpose to investigate whether a glycoprotein composed of domains from different viruses may retain a functional structure, cDNA encoding the ectodomain of RV G (amino acids 219 to 430) was fused to cDNA encoding the entire transmembrane and cytoplasmic domain of Eth-16 virus G (amino acids 431 to 503; pTCD). Two other constructs were assembled which encoded proteins engineered within the ectodomain. One of these, pE16/SAD, encoded a protein with a hybrid ectodomain whose amino-terminal half was derived from Eth-16 virus G (amino acids 219 to 247) and whose carboxy-terminal half, including transmembrane and cytoplasmic domains (amino acids 248 to 505), was derived from RV G (Fig. 1B and 2). It possesses two potential N-glycosylation sites at positions 202 and 319, the first from Eth-16 virus G and the latter from RV G. For construction of another chimeric protein (SIII), the RV Gencoding plasmid pT7T-G (6) was used as a basis for in vitro mutagenesis. By using a synthetic 63-base oligonucleotide, the 9-amino-acid stretch representing continuous antigenic site III of RV G (2) was exchanged with the corresponding sequence of Eth-16 G, which differs by 5 amino acid residues (Fig. 1B and 2). Transient expression of glycoproteins. The recombinant glycoproteins were expressed after transfection of the respective plasmids into cells which had been infected with vaccinia virus vTF7-3, expressing T7 RNA polymerase. First, expression was analyzed by immunoprecipitation and SDS-PAGE (Fig. 3). With a mouse serum (M16) directed against Eth-16 virus (24),

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FIG. 1. (A) Nucleotide sequence of the Eth-16 virus (Mokola virus, lyssavirus serotype 3) G gene, presented as a DNA positive strand. The putative transcriptional start and stop/polyadenylation signals are underlined. The translation start and stop codons of the G open reading frame are marked by asterisks. Restriction sites used in the construction of plasmids encoding E16 or hybrid proteins are indicated. (B) Deduced amino acid sequence and putative domains of Eth-16 virus G (E16) and comparison to the G protein (SAD) of RV strain SAD B19 (4). Only sequence deviations are shown in the SAD sequence. The potential N-glycosylation sites are indicated by asterisks. The two major RV antigenic sites (discontinuous site II and linear site III) are boxed (2). Junction sites and the respective amino- and carboxy-terminal regions of the chimeric G proteins are indicated (E16/SAD, SIII, and TCD).

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FIG. 2. Schematic diagram of RV (SAD), Eth-16 virus (E16), and chimeric (SIII, E16/SAD, and TCD) glycoproteins. RV G, with a signal peptide (sp) of 19 amino acids, ectodomain (ed) of 439 amino acids, transmembrane domain (tm) of 22 amino acids, and cytoplasmic domain (cd) of 44 amino acids, is depicted on the top line. Potential N-glycosylation sites are indicated. The location of RV antigenic site III is marked. Open bars, RV-derived sequences; solid bars, Eth-16 virus-derived sequences.

all proteins could be demonstrated (Fig. 3A). From the apparent molecular weight, the proteins expressed from pT7T-G (SAD) and pE16 (E16) were indistinguishable from the glycoproteins generated in cells infected with the SAD B19 and Eth-16 viruses, respectively. SIII protein, differing only in five amino acid residues from SAD protein, was recognized better than the latter, showing that antibodies directed against the introduced region were present in the anti-Eth-16 serum. In contrast, SIII and SAD proteins were recognized similarly by monoclonal antibody 120-6, directed to a region downstream of antigenic site III (34). Conversely, a monoclonal antibody (P44) specific for antigenic site III (28) failed to precipitate SIII protein (Fig. 3C). The ectodomain hybrid E16/ SAD was recognized by anti-Eth-16 serum as well as by both monoclonal antibodies (Fig. 3). As expected, the reaction of

FIG. 3. Immunoprecipitation analysis of recombinant glycoproteins. BSR cells were infected with Eth-16 virus or SAD B19 strain RV or transfected with one of the plasmids encoding RV (SAD), Eth-16 virus (E16), and chimeric (SIII and E16/SAD) G proteins and labeled at 16 h after infection or transfection with 125 mCi each of [35S]methionine and [35S]cysteine for 18 h. Cell extracts were incubated with mouse serum M16 (24), raised against purified Eth-16 virus (A); anti-RV G antibody 120-6 (33), directed to a region downstream of antigenic site III (B); or anti-RV G antibody P44 (28), directed against antigenic site III of RV (C). Lane vTF7-3, cells infected with vTF7-3 and transfected with vector plasmid. Arrows indicate the positions of glycoproteins (G), nucleoproteins (N), and the phosphoprotein (P). Sizes are indicated in kilodaltons.

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TCD protein, possessing the SAD ectodomain, with the above antibodies was similar to that shown for authentic SAD protein (not shown). Since expression of glycoproteins on the cell membrane is a prerequisite for incorporation into RV-like particles, we examined surface expression of wild-type and engineered G proteins. By indirect immunofluorescence staining of live cells, the presence of all glycoproteins on the cell surface could be demonstrated as early as 4 h posttransfection. After transfection of equal amounts of plasmid DNA, an apparently similar level of cell surface expression was obtained for all G constructs in a range from 4 to 48 h posttransfection (Fig. 4, at 6 h posttransfection). The rate at which G protein is transported from the endoplasmic reticulum to the Golgi apparatus may influence its accumulation on the plasma membrane and incorporation into virions. Thus, we monitored the acquisition of endoglycosidase-H (endo-H)-resistant sugars for wild-type and recombinant proteins, which is indicative of the arrival of G proteins in the medial cisternae of the Golgi complex (12). BSR cells infected with vTF7-3 and transfected with G protein-encoding plasmids were pulse labeled for 10 min with [35S]methionine and then chased for increasing times. Aliquots of immunoprecipitated proteins were treated with endo-H and analyzed by SDS-PAGE. After a 45-min chase, approximately half of E16 protein acquired endo-H-resistant sugars. The same result was obtained for SAD, SIII, TCD, and E16/SAD G proteins (not shown). Thus, the expressed G proteins are transported from the endoplasmic reticulum to the medial Golgi apparatus at a similar rate. For comparison, the G protein of another strain of RV (CVS) acquires endo-H-resistant sugars with a half-time of 50 min (31). Rescue of SDI-CAT particles by engineered G proteins. A previous study showed that an intracellularly transcribed RV genome analog containing terminal RV sequences and a CAT reporter gene cistron was rescued by simultaneously expressed RV proteins (6). The formation of infectious particles (SDICAT) required expression of both RV envelope proteins, M and G. The infectivity of rescued particles could easily be determined by monitoring CAT activity after transfer of supernatants from transfected cell cultures to fresh cells expressing RV helper proteins needed for expression of CAT from the recombinant genome. To determine whether the heterologous Eth-16 virus G or one of the chimeric proteins may functionally replace standard RV G in the formation of infectious SDICAT particles, rescue experiments were performed with these plasmids instead of pT7T-G for transfection. BSR cells were first infected with vTF7-3 and transfected 1 h later with a plasmid mix containing pSDI-CAT, plasmids encoding RV N, P, M, and L proteins, and 2 mg of the G-encoding plasmids. Cell extracts and supernatants from transfection experiments were collected 48 h after transfection. Equal aliquots of supernatants were then used to inoculate monolayers of BSR cells. After 1 h of incubation, the cells were washed, superinfected with SAD B19 helper virus, and harvested at 48 h postinfection. As shown in Fig. 5A, CAT activity was transmitted not only by the supernatant from cells expressing standard RV G, but also by that from cells transfected with pSIII, pE16, and pTCD, indicating that the respective G proteins were able to rescue infectious particles. In contrast, supernatant from cells expressing the chimeric E16/SAD protein failed to transmit CAT activity, although nucleocapsids expressing CAT were present after transfection of cells (Fig. 5A). Generation of infectious particles containing the particular glycoproteins was observed only in the presence of M protein (Fig. 5B), indicating the

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FIG. 4. Cell surface expression of glycoproteins. BSR cells were infected with recombinant vaccinia virus (vTF7-3) expressing T7 RNA polymerase. After 1 h of infection, cells were transfected with plasmids encoding wild-type or chimeric G proteins. Live cells were processed for indirect immunofluorescence at 6 h posttransfection as described in Materials and Methods. vTF7-3, mock-transfected cells; SAD and E16, authentic RV and Eth-16 virus G proteins, respectively; SIII, TCD, and E16/SAD, chimeric G proteins.

formation of viral envelopes corresponding to that of standard RV. To quantitate the amount of infectious SDI-CAT particles rescued by the G proteins, serial dilutions of supernatants from transfected cultures were used to infect fresh cells. At 2 days postinfection, CAT activity was measured, and the highest dilution resulting in detectable CAT transmission was determined from three independent experiments. Identical CAT titers of 7.5 3 104/ml were obtained for SDI-CAT particles rescued by the SAD and SIII proteins. Expression of the heterologous E16 protein and the chimeric TCD containing the transmembrane and cytoplasmic domains of E16 protein, however, gave rise to lower CAT titers of 2.2 3 103 and 1.2 3 104/ml, respectively. In order to confirm that CAT transfer was mediated by the glycoproteins incorporated into the envelope of particles, neutralization assays were carried out. Prior to infection of cells, supernatants from transfection experiments were incubated with specific antisera or monoclonal antibodies. The infectivity of particles obtained after expression of the SAD, SIII, and TCD proteins, whose ectodomains possess at least large parts of the SAD B19 G protein, could be neutralized by a rabbit serum (S72) raised against purified RV G protein. In contrast, this serum failed to inhibit CAT transmission by particles obtained after expression of the heterologous E16 protein (Fig.

6A). Monoclonal antibody P44, which is specific for RV antigenic site III, neutralized SAD and TCD protein-containing particles, but not those rescued by SIII or E16 proteins (Fig. 6B). The infectivity of particles with E16 protein could only be neutralized by the anti-Eth-16 virion serum (Fig. 6C). Thus, the recombinant glycoproteins were incorporated into the viral envelope in a way similar to that of RV G and determined the infectivity of the SDI-CAT particles. In the above-described neutralization assay, reduction of infectivity of particles possessing SIII protein was not observed after incubation with anti-Eth-16 antiserum, although specific antibodies against the introduced Eth-16 epitope appeared to be present (see Fig. 3). To investigate whether the antiserum contained small amounts of neutralizing antibodies that react with the introduced region of Eth-16 G sequence, 10-fold serial dilutions of transfection supernatants were incubated with anti-Eth-16 serum diluted only 1:20 instead of 1:50. Under these conditions, anti-Eth-16 serum reduced the CAT titer of SIII particles approximately 100-fold compared with particles containing SAD protein. For the latter, only a slight decrease in CAT activity was observed (Fig. 7). This indicated that the Eth-16 virus-specific epitope was present on the surface of SIII protein incorporated into virions. In addition, it suggested that the corresponding region of the Eth-16 virus G represents an antigenic site against which neutralizing antibodies are raised.

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FIG. 7. Neutralization of infectivity of SDI-CAT particles containing SAD and SIII G proteins. Transfection supernatants were serially diluted (10-fold) and incubated with anti-Eth-16 serum M16 (diluted 1:20). Infection and CAT assays were performed as described for Fig. 6.

FIG. 5. Rescue of SDI-CAT particles by engineered glycoproteins. (A) Expression of CAT activity was analyzed in cells after infection with vTF7-3 and transfection with a plasmid mix containing pSDI-CAT, plasmids encoding RV N, P, M, and L proteins, and G protein-encoding plasmids (transfection) and after transfer of the supernatants to fresh monolayer BSR cells expressing helper virus proteins 48 h after infection or transfection (passage). 2G, plasmid encoding G protein was omitted from the plasmid mix used for transfection. (B) The plasmid encoding RV M protein was omitted during transfection to demonstrate the specific requirement for M protein expression for the generation of infectious SDI-CAT particles.

DISCUSSION The single glycoprotein of rhabdoviruses plays a crucial role in the formation of virions as well as in recognition and infection of target cells. Although the G protein of RV is one of the best-known viral antigens (2, 11, 13, 33), investigation of structure-function relations has been hindered considerably so far by the lack of systems with which to introduce exclusively mutated G proteins into viral progeny or to manipulate the negative-stranded RNA genome of RV. In this study, a recombinant DNA technology assay which allows us to address directly the requirements for mutant or heterologous G proteins to functionally replace homologous G protein and which in

FIG. 6. Neutralization of the infectivity of SDI-CAT particles. Supernatants from rescue experiments were incubated with antisera or a monoclonal antibody for 30 min and transferred to fresh cells. Cells were superinfected with helper virus SAD B19 at 1 h postinfection, and CAT activity was determined at 48 h postinfection. Neutralization with anti-RV glycoprotein serum S72 (A), anti-RV site III-specific antibody P44 (B), and anti-Eth-16 serum M16 (C) is shown. The dilutions of the antibodies are indicated.

addition avoids handling of infectious RV was used. RV-like particles encoding a CAT reporter enzyme were generated in cells expressing individual viral proteins and a viral genome analog from transfected plasmids. Successful assembly and budding of infectious virions were monitored by CAT assays. Assembly of viral glycoproteins into the membrane of enveloped viruses is most likely a necessary step in the formation of virus particles. The finding of a C-terminal fragment of the VSV G protein in spikeless particles of a temperature-sensitive VSV mutant (tsO45) suggests that this portion, namely, the cytoplasmic and transmembrane domains, of VSV G protein is required to initiate budding (26). This mutant has been also used to demonstrate incorporation of hybrid or foreign glycoproteins, including that of RV strain CVS (27, 31). In these experiments, although the titer of infectious particles increased, there was no significant increase in particle numbers (31, 32), suggesting that the rescued virions resulted from incorporation of G proteins into particles already containing the VSV C-terminal G protein fragment. Thus, it was not possible to determine whether the foreign or hybrid G proteins could actually replace VSV G. The complete absence of RV G in the described rescue system enabled us to demonstrate that the heterologous Eth-16 virus G as well as hybrid proteins containing portions of Eth-16 virus and RV G proteins can functionally replace authentic RV G in all steps of the viral life cycle required to produce infectious virions. The infectivity of rescued ‘‘pseudotype’’ particles was mediated by the incorporated G proteins, as demonstrated by specific neutralizations. The cytoplasmic domain of the Eth-16 virus G, despite extensive sequence heterogeneity, may thus interact heterotypically with the internal RV proteins. A stringent sequence conservation of the entire cytoplasmic tail sequence therefore appears not to be critical for the formation of infectious rhabdovirus particles. As suggested by the CAT titers obtained in rescue experiments, approximately 30-fold fewer infectious particles were generated after expression of the heterologous E16 protein compared with the homologous SAD protein. Probably, most of this difference was caused by a reduced ability of the cytoplasmic domain of E16 protein to interact with the RV internal proteins. Attempts to increase E16 virion titers by transfection of larger amounts of pE16 failed, indicating that the level of E16 protein surface expression was not the limiting factor in the rescue experiments. In addition, longer incubation with supernatants for infection of cells in order to allow more time for attachment and fusion did not reduce the titer differences observed for the E16 and SAD virions.

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However, the titer difference between SAD and E16 virions may not be attributed entirely to features of the cytoplasmic domains of the two G proteins. Expression of the chimeric TCD protein, composed of E16 cytoplasmic and transmembrane domain sequences and the SAD ectodomain, resulted in higher titers than expression of the authentic E16 protein. Previous results obtained with the VSV tsO45 mutant indicated that the internal and external portions of the rhabdovirus G protein may carry out their functions independently of each other; namely, incorporation into virions on the one hand and trimer formation, attachment to cells, and fusion on the other hand. A chimeric protein possessing the VSV G cytoplasmic tail and the human immunodeficiency virus gp160 ectodomain was incorporated into VSV virions and could also mediate infection of CD4-expressing cells (27). On the other hand, truncated VSV G proteins lacking the cytoplasmic and transmembrane domains, which are therefore secreted, were shown to form trimers (9). The ability of TCD to rescue infectious SDI-CAT particles confirms that entire domains of lyssavirus G proteins may be exchanged without affecting the functions of the complete G protein. Moreover, the finding of an increased titer after replacement of the E16 ectodomain with the corresponding SAD domain suggests that there are differences in the ability of SAD and E16 ectodomain modules to mediate infection of BSR culture cells. These may also contribute to the different virus titers obtained in BSR cells after infection with the fixed virus strain SAD B19 or the recent street virus isolate Eth-16 (about 108 and 106 FFU/ml, respectively). Critical residues of the ectodomains influencing cell infection might be identified in the future by rescue experiments with mutated G proteins. No differences in infectious particle titers were observed after substitution of only a short region of the RV G ectodomain, antigenic site III, with the corresponding sequence of Eth-16 virus G. This indicated that the alteration affected neither the ability of the G protein to support formation of infectious particles nor its ability to mediate infection. Antigenic site III of RV G was selected because it represents a wellcharacterized continuous RV surface epitope recognized by several distinct neutralizing monoclonal antibodies (2). In addition, the only RV virulence marker so far identified (R333) is located here (7, 11). The exchange of five amino acids in antigenic site III of RV G resulted in the failure of a site III-directed monoclonal antibody to neutralize particles. Conversely, specific neutralization by anti-Eth-16 serum was observed, indicating that antigenic site III may have a similar location on the G proteins of RV and Eth-16 virus and be interchangeable. The only chimeric protein that did not give rise to infectious particles was E16/SAD. In contrast to TCD protein, whose entire ectodomain module except for a few amino acids close to the transmembrane domain was derived from one virus, and to SIII, in which only a short surface epitope was exchanged within the ectodomain, E16/SAD possessed a real hybrid ectodomain. The rate of transport and the level of surface expression were similar to those of the other G constructs. The presence of E16/SAD protein in the cellular membrane, mediated correctly by the carboxy-terminal part of the protein derived from SAD, lets us assume that this protein is also incorporated into the viral envelope. However, although all cysteine residues of the E16/SAD ectodomain were conserved and the pattern of potential N-glycosylation sites corresponded to that of E16 protein, folding of the hybrid ectodomain was suspected to result in a configuration that may not allow either correct oligomerization, attachment to a receptor, or the con-

J. VIROL.

formational changes necessary to mediate fusion of membranes (15, 17). The described system provides a way to address the two general functions of RV G. On the one hand, a correct structure of the ectodomain is needed to mediate infection of cells. On the other hand, initiation of budding and formation of virions most likely requires an appropriate cytoplasmic domain interacting with the RV nucleocapsid and/or the M protein. Further use of mutated G and/or M proteins in this system or, where appropriate, incorporation of mutant genes into the genomes of infectious RV should provide basic information on general mechanisms involved in virus assembly, budding, cell recognition, and fusion. ACKNOWLEDGMENTS We thank Veronika Schlatt and Heike Bo ¨hli for expert technical assistance. This work was supported by grant BEO21/0310118A from the Bundesministerium fu ¨r Forschung und Technologie. REFERENCES 1. Anilionis, A., W. H. Wunner, and P. J. Curtis. 1981. Structure of the glycoprotein gene of rabies virus. Nature (London) 294:275–278. 2. Benmansour, A., H. Leblois, P. Coulon, C. Tuffereau, Y. Gaudin, A. Flamand, and F. Lafay. 1991. Antigenicity of rabies virus glycoprotein. J. Virol. 65:4198–4203. 3. Bourhy, H., B. Kissi, and N. Tordo. 1993. Molecular diversity of the Lyssavirus genus. Virology 194:70–81. 4. Conzelmann, K.-K., J. H. Cox, L. G. Schneider, and H.-J. Thiel. 1990. Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B19. Virology 175:485–499. 5. Conzelmann, K.-K., J. H. Cox, and H.-J. Thiel. 1991. An L (polymerase) deficient rabies virus defective interfering particle RNA is replicated and transcribed by heterologous helper virus L proteins. Virology 184:655–663. 6. Conzelmann, K.-K., and M. Schnell. 1994. Rescue of synthetic rabies virus genome analogs by plasmid-encoded proteins. J. Virol. 68:713–719. 7. Coulon, P., P. E. Rollin, and A. Flamand. 1983. Molecular basis of rabies virus virulence. II. Identification of a site on the CVS glycoprotein associated with virulence. J. Gen. Virol. 64:693–696. 8. Cox, J. H., B. Dietzschold, and L. G. Schneider. 1977. Rabies virus glycoprotein. II. Biological and serological characterization. Infect. Immun. 16: 754–759. 9. Crise, B., A. Ruusala, P. Zagouras, A. Shaw, and J. K. Rose. 1989. Oligomerization of glycolipid-anchored and soluble forms of the vesicular stomatitis virus glycoprotein. J. Virol. 63:5328–5333. 10. Dietzschold, B., C. E. Rupprechet, M. Tollis, M. Lafon, J. Mattei, T. J. Wiktor, and H. Koprowski. 1988. Antigenic diversity of the glycoprotein and nucleocapsid proteins of rabies and rabies-related viruses: implications for epidemiology and control of rabies. Rev. Infect. Dis. 10:785–798. 11. Dietzschold, B., W. H. Wunner, T. J. Wiktor, A. D. Lopes, M. Lafon, C. L. Smith, and H. Koprowski. 1983. Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus. Proc. Natl. Acad. Sci. USA 80:70–74. 12. Dunphy, W. G., and J. E. Rothman. 1985. Compartmental organization of the Golgi stack. Cell 42:13–21. 13. Flamand, A., H. Raux, Y. Gaudin, and R. W. H. Ruigrok. 1993. Mechanisms of rabies virus neutralization. Virology 194:302–313. 14. Fuerst, T. R., E. G. Niles, F. W. Studier, and B. Moss. 1986. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 83:8122–8126. 15. Gaudin, Y., R. W. H. Ruigrok, M. Knossow, and A. Flamand. 1993. Low-pH conformational changes of rabies virus glycoprotein and their role in membrane fusion. J. Virol. 67:1365–1372. 16. Gaudin, Y., R. W. H. Ruigrok, C. Tuffereau, M. Knossow, and A. Flamand. 1992. Rabies virus glycoprotein is a trimer. Virology 187:627–632. 17. Gaudin, Y., C. Tuffereau, D. Segretain, M. Knossow, and A. Flamand. 1991. Reversible conformational changes and fusion activity of rabies virus glycoprotein. J. Virol. 65:4853–4859. 18. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044–1051. 19. Henikoff, S. 1984. Unidirectional digestion of exonuclease III in DNA sequence analysis. Methods Enzymol. 155:156–165. 20. Kessler, S. W. 1981. Use of protein A-bearing staphylococci for the immunoprecipitation and isolation of antigens from cells. Methods Enzymol. 73: 442–459.

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21. Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367–382. 22. Lafon, M., J. Ideler, and W. H. Wunner. 1984. Investigation of the antigenic structure of rabies virus glycoprotein by monoclonal antibodies. Dev. Biol. Stand. 57:219–225. 23. Mebatsion, T., J. H. Cox, and K.-K. Conzelmann. 1993. Molecular analysis of rabies-related viruses from Ethiopia. Onderstepoort J. Vet. Res. 60:289– 294. 24. Mebatsion, T., J. H. Cox, and J. W. Frost. 1992. Isolation and characterization of 115 street rabies virus isolates from Ethiopia by using monoclonal antibodies: identification of two isolates as Mokola and Lagos bat viruses. J. Infect. Dis. 166:972–977. 25. Metsikko ¨, K., and H. Garoff. 1990. Oligomers of the cytoplasmic domain of the p62/E2 membrane protein of Semliki Forest virus bind to the nucleocapsid in vitro. J. Virol. 64:4678–4683. 26. Metsikko ¨, K., and K. Simons. 1986. The budding mechanism of spikeless vesicular stomatitis virus particles. EMBO J. 5:1913–1920. 27. Owens, R. J., and J. K. Rose. 1993. Cytoplasmic domain requirement for incorporation of a foreign envelope protein into vesicular stomatitis virus. J. Virol. 67:360–365. 28. Schneider, L., and S. Meyer. 1981. Antigenic determinants of rabies virus as demonstrated by monoclonal antibody, p. 947–953. In D. H. L. Bishop and R. W. Compans (ed.), The replication of negative strand viruses.

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Elsevier/North-Holland, Amsterdam. 29. Schnell, M. J., T. Mebatsion, and K.-K. Conzelmann. 1994. Infectious rabies viruses from cloned cDNA. EMBO J. 13:4195–4203. 30. Tordo, N., O. Poch, A. Ermine, G. Keith, and F. Rougeon. 1986. Walking along the rabies genome: is the large G-L intergenic region a remnant gene? Proc. Natl. Acad. Sci. USA 83:3914–3918. 31. Whitt, M. A., L. Buonocore, C. Prehaud, and J. K. Rose. 1991. Membrane fusion activity, oligomerization and assembly of the rabies virus glycoprotein. Virology 185:681–688. 32. Whitt, M. A., L. Chong, and J. K. Rose. 1989. Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant. J. Virol. 63:3569–3578. 33. Wiktor, T. J., E. Gyo¨rgy, H. D. Schlumberger, F. Sokol, and H. Koprowski. 1973. Antigenic properties of rabies virus components. J. Immunol. 110:269– 276. 34. Wunner, W. H., B. Dietzschold, R. I. Macfarlan, C. L. Smith, E. Golub, and T. J. Wiktor. 1985. Localization of immunogenic domains on the rabies virus glycoprotein. Ann. Virol. Inst. Pasteur Paris 136E:353–362. 35. Wunner, W. H., K. J. Reagan, and H. Koprowski. 1984. Characterization of saturable binding sites for rabies virus. J. Virol. 50:691–697. 36. Yelverton, E., S. Norton, J. F. Obijeski, and D. V. Goeddel. 1983. Rabies virus glycoprotein analogs: biosynthesis in Escherichia coli. Science 219:614– 620.