当前位置: 首页 > 医学版 > 期刊论文 > 基础医学 > 病菌学杂志 > 2005年 > 第16期 > 正文
编号:11202975
Envelope Targeting: Hemagglutinin Attachment Speci
     Molecular Medicine Program and Virology and Gene Therapy Track, Mayo College of Medicine, Rochester, Minnesota 55905

    Hygiene-Institut der Universitt Heidelberg, Abteilung Virologie, 69120 Heidelberg, Germany

    ABSTRACT

    To engineer a targeting envelope for gene and oncolytic vector delivery, we characterized and modified the envelope proteins of Tupaia paramyxovirus (TPMV), a relative of the morbilli- and henipaviruses that neither infects humans nor has cross-reactive relatives that infect humans. We completed the TPMV genomic sequence and noted that the predicted fusion (F) protein cleavage-activation site is not preceded by a canonical furin cleavage sequence. Coexpression of the TPMV F and hemagglutinin (H) proteins induced fusion of Tupaia baby fibroblasts but not of human cells, a finding consistent with the restricted TPMV host range. To identify the factors restricting fusion of non-Tupaia cells, we initially analyzed F protein cleavage. Even without an oligo- or monobasic protease cleavage sequence, TPMV F was cleaved in F1 and F2 subunits in human cells. Edman degradation of the F1 subunit yielded the sequence IFWGAIIA, placing the conserved phenylalanine in position 2, a novelty for paramyxoviruses but not the cause of fusion restriction. We then verified whether the lack of a TPMV H receptor limits fusion. Toward this end, we displayed a single-chain antibody (scFv) specific for the designated receptor human carcinoembryonic antigen on the TPMV H ectodomain. The H-scFv hybrid protein coexpressed with TPMV F mediated fusion of cells expressing the designated receptor, proving that the lack of a receptor limits fusion and that TPMV H can be retargeted. Targeting competence and the absence of antibodies in humans define the TPMV envelope as a module to be adapted for ferrying ribonucleocapsids of oncolytic viruses and gene delivery vectors.

    INTRODUCTION

    Several viruses, including the paramyxoviruses Measles virus (MV) (47), Mumps virus (MuV) (1), and Newcastle disease virus (NDV) (32), are being developed as novel agents for the treatment of human cancer (3, 39). Neutralizing antibodies, even if they do not abolish efficacy of human viruses in some xenograft models of oncolysis (14) or viral replication after challenge of hosts with preexisting immunity (36), may reduce their efficacy in currently open clinical trials (9, 16, 55, 58). To circumvent neutralizing antibodies prevalent in the general population through vaccination or natural disease, we envisage enclosing the replicative units of human viruses in envelopes of animal viruses that do not have cross-reactive relatives in humans.

    We have begun exploring the use of the Tupaia paramyxovirus (TPMV) envelope glycoproteins as a vector delivery module. Tupaias or tree shrews are small mammals widely present in southeast Asia. After having been classified as either primates or insectivores, they were finally assigned to a separate order, Scandentia. Tupaias have yielded a rich crop of viruses, including a herpesvirus (2), adenovirus (43), and rhabdovirus (46) that have recently been characterized at the genomic level. TPMV was isolated from spontaneously degenerating primary kidney cells of an apparently healthy tree shrew (Tupaia belangeri) (53, 54). The virus is growing in a trypsin-independent manner in Tupaia cells but not in any other cell line tested thus far. The cytopathic effect includes multinucleated syncytia followed by cell lysis. The nucleotide sequence of the N and P genes revealed that the virus is most closely related to the morbilli- and henipaviruses (54), but the glycoproteins have not been characterized.

    The glycoproteins are important determinants of paramyxovirus tropism: the attachment protein because it targets one or more receptors and the fusion (F) protein because it can be activated by tissue-specific proteases. The attachment protein is a type II transmembrane protein named hemagglutinin (H), hemagglutinin-neuraminidase (HN) or, when the protein does not have these functions, glycoprotein (G). Viruses relying on oligosaccharides as receptors have neuraminidase activity to eliminate these from the cell surface at late infections stages and allow efficient particle release. Viruses with peptidylic epitopes of specific proteins as receptors do not need neuraminidase activity.

    The F protein of paramyxoviruses is a trimeric type I transmembrane protein that executes membrane fusion. All F proteins characterized thus far are cleaved in an N-terminal F2 and a C-terminal F1 fragment; a hydrophobic fusion peptide is amino terminal to the F1 fragment. The cleavage site is preceded by either one or several arginines and lysines, i.e., it is oligo- or monobasic (18). Proteins with oligobasic cleavage sites are cleaved in the trans-Golgi network by the ubiquitous protease furin or related proteases, whereas proteins with monobasic cleavage sites are cleaved by trypsin-like proteases outside the cell. The nature of the protease cleavage site and the localization of the F protein activating protease can determine viral tropism and pathogenicity. The proteases activating Sendai virus (SeV), tryptase Clara and mini-plasmin, are only present in the respiratory tract of the murine host, thereby limiting the infection to this organ (27, 51). F protein cleavage is a tropism determinant also in the avian paramyxovirus NDV: the cleavage sites of pathogenic strains are oligobasic, whereas those of nonpathogenic strains are monobasic (28, 33, 45). Paramyxoviruses with monobasic cleavage sites often require trypsin for efficient propagation in cultured cells. As an exception, the recently characterized Hendra virus (HeV) and Nipah virus (NiV) have only one lysine or arginine preceding the fusion peptide, respectively, but nevertheless propagate efficiently in a variety of cultured cells without trypsin (64) and may be cleaved inside the cells by as-yet-unidentified proteases (31).

    We present here a functional analysis of the TPMV glycoproteins and discuss their sequences that we deposited in the GenBank (AF079780). We show that F-protein cleavage activation is not a limiting factor in human cells, even if the structure of the F1 subunit amino terminus is unprecedented. We also show that a specificity domain added to the TPMV H ectodomain is sufficient to mediate fusion of target cells, thereby defining receptor recognition as tropism determinant.

    MATERIALS AND METHODS

    Cells and viruses. TPMV was propagated in Tupaia baby fibroblasts (TBF) (11) as described previously (54). 293T, Vero, and HT1080 were from the American Type Culture Collection. MC38cea cells (40) were kindly provided by Jeffrey Schlom, National Institutes of Health. All cells were grown in Dulbecco modified Eagle medium containing 10% fetal calf serum with penicillin-streptomycin except for the Vero cells that were supplemented with 5% fetal calf serum.

    Nucleotide sequencing and computer-assisted sequence analysis. To complete the TPMV genomic sequence, degenerated oligonucleotide primers corresponding to conserved regions in the M gene of the morbilliviruses and henipaviruses were constructed, taking into account the TPMV codon usage in the N and P genes. These primers were combined with primers in the P gene in PCR to amplify and sequence the beginning of the M gene. The M gene sequence was then completed by 3'-rapid amplification of cDNA ends (RACE). The same strategy was used for the remaining F, H, and L genes. The TPMV trailer sequence was also determined by 3'-RACE. DNA sequencing and RACE procedures were carried out as described previously (54). Protein similarities were calculated with the CLUSTAL W program (52) on the website of the Ple Bioinformatique Lyonnais (http://npsa-pbil.ibcp.fr/).

    Molecular cloning of the TPMV glycoproteins. Total RNA was isolated from infected TBF cells with the RNeasy kit (QIAGEN, Hilden, Germany). The F and H coding sequences were PCR amplified with the oligonucleotide primers TPMV-F-S (5'-TTTGGGGGATCCCCAAGGATGGCATCACTGCTA-3'), TPMV-F-AS (5'-TTTGGGTCTAGATTATCCACTTATATCTGTACTG-3'), TPMV-H-S (5'-TTTGGGGGATCCATTATGGATTATCATTCACACACG-3'), and TPMV-H-AS (5'-TTTGGGTCTAGATTACTTAGTATTAGGACATGTAC-3') (restriction sites used for cloning are underlined) and cloned into the eukaryotic expression vector pCG, resulting in the plasmids pCG-TPMV-F and pCG-TPMV-H. The F protein was additionally cloned with a Flag tag (DYKDDDDK, one-letter amino acid code) fused to its cytoplasmic tail (pCG-TPMV-FFlag). The nucleotide sequence of the cloned genes differed slightly from the deposited sequence, resulting in two different amino acids in the F (T485A and I502T) and H (V257A and Y313S) proteins. Since the proteins were functional and the sequences found in different clones, the plasmids were not corrected.

    Retargeted TPMV H. The expression plasmid pCG-TPMV-HXCEA that encodes a fusion protein of TPMV H and a single-chain antibody (scFv) to human carcinoembryonic antigen (CEA) was generated by amplification of the H open reading frame (ORF) with the primers TPMV-H-S and T-HXL-SphI (5'-TTTGGGGCATGCGCGCGCCCCTTCCCTCGATCTTAGTATTAGGACATGTAC-3'), adding the sequence encoding a factor Xa cleavage site and a BssHII site to the 3' end of the H ORF. The modified ORF was then cloned by using BamHI and BssHII into the plasmid pCG-H5-XL (15) that contains the coding sequence for the scFv MFE-23 (8) with a 16-amino-acid linker between the VH and VL domains (as described in reference 15).

    Expression of the TPMV glycoproteins. Various cell lines, including TBF, 293T, HT1080, Vero, and MC38cea, were transfected in 12-well plates with 2 μg of plasmid DNA by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and observed for syncytium formation or lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 1.0% Triton X-100, 50 mM Tris-HCl [pH 8.0]) with complete protease inhibitor (Roche Diagnostics, Mannheim, Germany) for Western blot analysis.

    Generation of antisera and Western blot analysis. The peptides (C)PSHHRSNGHQNHSFSTDISG, corresponding to the TPMV-F amino acids 534 to 553 with an N-terminal cysteine and MDYHSHTTQTGSNET(C) corresponding to the first 15 TPMV H amino acids with a C-terminal cysteine were synthesized by the Mayo Clinic protein core facility, coupled to keyhole limpet hemocyanin, and used to generate peptide antisera in rabbits (Cocalico Biologicals, Reamston, PA). For Western blotting, protein lysates were cleared by centrifugation for 15 min at 5,000 x g at 4°C and mixed with an equal volume of urea buffer (200 mM Tris [pH 6.8], 8 M urea, 5% sodium dodecyl sulfate [SDS], 0.1 mM EDTA, 0.03% bromophenol blue) containing 1.5% dithiothreitol. Some protein samples were digested with PNGase F or Endo H (New England Biolabs, Beverly, MA) according to the manufacturer's instructions. After denaturation at 65°C for 10 min, the proteins were fractionated on SDS-polyacrylamide gels of various concentrations (Bio-Rad, Hercules, CA) and blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The antipeptide sera were used in a concentration of 1:10,000. The membranes were subjected to enhanced chemiluminescence detection with a horseradish-peroxidase conjugated secondary antibody and the ECL system (Amersham Biosciences, Piscataway, NJ).

    Edman degradation. 293T cells were transfected (Lipofectamine 2000; Invitrogen) with the plasmid pCG-TPMV-FFlag, lysed in radioimmunoprecipitation assay buffer after 36 h, and immunoprecipitated with ANTI-FLAG M2 affinity gel (Sigma). The immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), blotted to a polyvinylidene difluoride membrane (Sequi-Blot; Bio-Rad), and visualized by Coomassie blue staining. Another membrane segment was probed with anti-Flag antibodies to identify the F protein bands. The F0 and F1 protein fragments were excised and sequenced by Edman degradation (Mayo Clinic protein core facility) with an Applied Biosystems 492 Procise cLC sequencer (Applied Biosystems Inc, Foster City, CA).

    RESULTS

    The TPMV genome and the amino acid sequences of the glycoproteins. The completed TPMV genomic sequence was submitted to GenBank by C. A. Tidona and G. Darai (AF079780). We will restrict our comments to the general characteristics of the genome and the predicted glycoprotein sequences. The 17,904-nucleotide genome is rule-of-six compliant (20, 37) and larger than those of all known paramyxovirus except the henipaviruses. TPMV shows the typical paramyxovirus gene order N-P/C/V-M-F-H-L (Fig. 1C), and all genes are separated by the nontranscribed trinucleotide CUU (antigenome). The trailer region is 590 nucleotides long, much larger than all other trailer regions in the family.

    The F gene codes for a protein of 553 amino acids (aa), including the signal peptide. As in other paramyxovirus fusion proteins, three hydrophobic regions exist that were named the signal peptide (SP), the fusion peptide (FP), and the transmembrane domain (TM) (Fig. 1D). The TPMV F protein sequence shows the highest similarity to those of the recently identified Mossman virus (25) (34% amino acid identity), the morbilliviruses (33.4% identity to MV) and the henipaviruses (31.8% identity to NiV). The fusion peptide is predicted to begin with a nearly invariable phenylalanine (aa 109) and is highly conserved, including the three glycines that have recently been shown to regulate F protein activation (41). However, in contrast to all other known paramyxovirus F proteins, there is neither a mono- nor an oligobasic cleavage site preceding F109 (Fig. 1E, top line). This raises the possibility that the TPMV F protein is cleaved by an unknown protease that may or may not exist in cells of mammals other than tree shrews. Four possible N glycosylation sites (consensus sequence NXS/T) are present in the ectodomain of the TPMV F protein: three on the F2 fragment (N63, N71, and N90) and one on the F1 fragment (N434) (Fig. 1D, triangles).

    The attachment protein gene codes for a 665-aa protein. Since TPMV virions hemagglutinate Tupaia erythrocytes but have no neuraminidase activity (21), this protein is named hemagglutinin (H). H has a predicted molecular mass of 74 kDa without the three predicted oligosaccharides (N glycosylation sites at N199, N349, and N509; Fig. 1B, triangles). Sequence identity to the attachment proteins of other paramyxoviruses is low (<20%), but the spacing of cysteines and other structurally important residues allows accurate alignment (data not shown). It is noteworthy that the 94 residue H cytoplasmic tail has twice as many residues as any other paramyxovirus attachment protein cytoplasmic tail (Fig. 1A, bottom line).

    Characterization of the TPMV glycoproteins. The ORFs of the TPMV F and H proteins were amplified by reverse transcription-PCR from infected TBF cells and cloned into the eukaryotic expression vector pCG (7). The proteins were transiently expressed in different cell lines and analyzed by Western blotting with rabbit antisera. To raise these antisera, peptides corresponding to aa 534 to 553 of the F protein and aa 1 to 15 of the H protein were synthesized, coupled to a carrier protein, and used for rabbit immunization.

    Two F protein bands with an estimated molecular masses of 65 and 48 kDa were detected after expression in Tupaia cells (Fig. 2A, lane F). These estimated sizes correspond to the predicted molecular mass of the uncleaved F0 protein and the F1 fragment, respectively. Since the antibody is directed against the carboxy-terminal cytoplasmic tail, the amino-terminal F2 fragment cannot be detected. This result suggests that the TPMV F protein is cleaved, despite the absence of a mono- or oligobasic sequence upstream of the putative fusion peptide. Digestion with PNGase F resulted in faster migration of both fragments, confirming that they are both glycosylated (Fig. 2A). The shifts in apparent molecular mass of the F0 and F1 bands were consistent with the addition of one oligosaccharide on F1 and several on F0. To determine how many of the four potential N glycosylation sites in F0 are used, we performed a partial PNGase F digestion. The concentration of the enzyme was varied so that the number of partially digested bands could be counted. Figure 2B shows the result of this analysis, detecting four bands in addition to the completely deglycosylated protein. Thus, all potential glycosylation sites are used.

    To determine whether F0 or F1 are transported to the cell surface, we digested total cellular protein extracts with endoglycosidase H (Endo H). Endo H-resistant glycans are acquired in the Golgi compartment, and proteins containing these glycans are generally transported rapidly to the cell surface (35). F1 was almost completely resistant to Endo H, whereas most F0 was Endo H sensitive (Fig. 2A, right lane). This suggests that the bulk of F1 is on the cell surface, whereas F0 is mainly intracellular.

    We then sought to determine whether TPMV F2/F1 cleavage may be restricted to Tupaia cells and explain tropism. Cleavage occurred in all cell lines tested, including human (Fig. 2C, 293T and HT1080), simian (Fig. 2C, Vero), and murine cells (Fig. 2C, MC38cea). Cleavage efficiency varied in the different cells lines and was highest in HT1080 cells that are known to secrete matrix-metalloproteinases (MMP) (Fig. 2C, compare the intensity of the F0 and F1 bands). However, the broad-spectrum MMP inhibitor GM 6001 (13) did not inhibit fusion of TBF cells by the TPMV glycoproteins (see below), indicating that proteases other than MMPs activate TPMV F in these cells (data not shown). Since cleavage is ubiquitous, it cannot explain tropism.

    When protein lysate from transfected or infected TBF cells was separated on a more concentrated gel, an additional small band of ca. 10 kDa was detected (Fig. 2D). We have recently identified similar F1b fragments generated by a partial membrane-proximal cleavage of the F proteins of the paramyxoviruses MV, Canine distemper virus (CDV), and MuV, and have shown that partial membrane-proximal cleavage is important for protein function (60).

    We also sought to determine whether the TPMV H protein has the expected molecular mass and is glycosylated. A Western blot with H-protein specific antibodies revealed a band of ca. 80 kDa (Fig. 2E) corresponding to the predicted molecular mass. Digestion with PNGase F reduced the apparent molecular mass of the protein, demonstrating that it is glycosylated (Fig. 2E, right lane).

    Analysis of the F protein amino terminus by Edman degradation reveals an amino acid upstream of the conserved phenylalanine. Since the TPMV F protein does not have a detectable mono- or oligobasic cleavage site upstream of the fusion peptide, we attempted to determine the exact F2/F1 cleavage site. The F0 and F1 proteins were Flag tagged at the common carboxy terminus and purified by using an anti-Flag antibody from human 293T cells transfected with an expression plasmid. After separation on SDS-PAGE and Western blotting, Coomassie blue-stained bands were excised and analyzed by Edman degradation. F0 analysis yielded the amino acid sequence EPTPKSQL (one-letter code), corresponding to aa 20 to 27 of the precursor protein. This indicates that the signal peptidase cleaves the protein between aa 19 and 20 (full sequence of the signal peptide MASLLKTICYIYLITYAKL). The signal peptide was correctly predicted by the computer program SignalP (4). The result for the F1 protein was IFWGAIIA (aa 108 to 116), demonstrating that the protein was cleaved between aa 107 and 108, leaving an isoleucine upstream of the phenylalanine that is conserved as the first amino acid of a large majority of the paramyxovirus F proteins (Fig. 1E).

    The TPMV glycoproteins selectively fuse Tupaia cells. TPMV replicates selectively in Tupaia cells and not in cells of other mammals (54). To verify whether impaired membrane fusion of non-Tupaia cells by the two TPMV glycoproteins may account for tropism restriction, TPMV glycoprotein expression plasmids were cotransfected into Tupaia (TBF) and non-Tupaia (293T, HT1080, Vero, and MC38cea) cell lines. In TBF cells, cotransfection elicited large, multinucleated syncytia (Fig. 3, bottom panel), a finding comparable to the typical TPMV cytopathic effect in these cells (not shown). In contrast, there was no syncytium formation when the proteins were expressed separately (Fig. 3, top panel, F protein alone) or in any other cell type tested. Control transfections of primate cells (293T, HT1080, and Vero) with plasmids encoding MV F and H resulted in extensive fusion (data not shown). Cotransfection of TPMV H with MV F and vice versa in TBF and Vero cells did also not lead to syncytium formation, indicating that these proteins cannot functionally interact. Altogether, the experiments described above suggest the lack of a receptor on non-Tupaia cells as a possible reason for the lack of function of the TPMV glycoproteins.

    Receptor-binding conferred by a single-chain antibody displayed on the TPMV H protein restores its fusion support function in non-Tupaia cells. To test whether the lack of a specific receptor accounts for lack of fusion of non-Tupaia cells by the TPMV glycoproteins, we took advantage of the observation that another paramyxoviral attachment protein (MV H) can be targeted to bind designated receptors through added specificity domains, including single-chain antibodies (scFv) (15, 42). Analogous to what was done with MV H, we constructed a plasmid coding for a TPMV H protein with a human CEA scFv added to its carboxy terminus (Fig. 4A, shaded box). H was connected to the scFv by the four-residue linker IEGR (Fig. 4A, bottom right) that can be cleaved by factor Xa protease for removal of the displayed specificity domain. Upon transfection of 293T cells and Western blot analysis a protein of the expected size was detected (Fig. 4B, lane HXCEA), and factor Xa digestion confirmed that this protease can be used to remove the scFv (Fig. 4B, lane HXCEA/FXa).

    We then sought to determine whether, in combination with the TPMV F protein, TPMV HXCEA can induce fusion of murine MC38cea cells stably expressing human CEA (40). These cells cannot be fused when the TPMV glycoproteins are coexpressed (Fig. 4C, upper panel), but when standard H is substituted with the H protein displaying the anti-CEA scFv, the cells form syncytia (Fig. 4C, center panel) at least as efficiently as with the retargeted MV envelope protein pair (Fig. 4C, lower panel). The modified H protein coexpressed with the F protein was still able to induce syncytium formation in TBF cells, albeit with reduced efficiency (data not shown). Thus, we conclude that receptor attachment is the factor limiting efficiency of fusion of non-Tupaia cells by the TPMV glycoproteins.

    DISCUSSION

    Paramyxovirus glycoproteins mediate attachment to host cells and fusion of viral and cellular membranes (reviewed in reference 22). We analyzed the glycoproteins of a paramyxovirus that infects mammals of the order Scandentia and has no cross-reactive relatives that infect humans. We disclosed a unique F-protein cleavage sequence but also verified that F-protein cleavage is not a factor limiting fusion of human cells. On the other hand, we showed that the TPMV H protein has a narrow receptor specificity and that fusion of non-Tupaia cells expressing a designated receptor occurs only after the display of a cognate specificity domain on TPMV H.

    A ubiquitous protease cleaves TPMV F, producing a unique F1 terminus. The F1 N termini of several paramyxoviruses, including SeV, Simian virus 5 and NDV (38), MV (56), CDV (57), MuV (44), Respiratory syncytial virus (12), HeV (24), and NiV (26) have been determined by amino acid sequencing, and this information has been used to align the cleavage sites and define the preceding mono- and oligobasic signature sequences. Sequencing of the TPMV F1 amino terminus by Edman degradation indicated that an isoleucine is found upstream of phenylalanine 109, in spite of the fact that all paramyxoviral F1 protein sequences examined to date begin with a phenylalanine or with a leucine in the equivalent position. This observation has important implications for F protein engineering (see below). The sequence preceding the first isoleucine is RDGGT, not a signature protease cleavage site to our knowledge.

    The henipaviruses are closely related to TPMV and are also unique within the paramyxoviruses since they have a monobasic cleavage site that is nevertheless cleaved intracellularly (31). Moreover, the first amino acid of their fusion peptide is a leucine instead of a phenylalanine (24, 26) (Fig. 1E). Interestingly, it was recently shown that all of the amino acids, including a basic one preceding the cleavage site in the NiV F protein, can be individually mutated with only minor effects on protein function (26). It was also shown that the protease cleaving the nearly identical HeV F protein requires calcium as a cofactor (31). Since the NiV and HeV carboxyl-terminal sequences are not homologous to the corresponding TPMV sequences, we suspect that the protease cleaving TPMV F may be different from that cleaving HeV and NiV F and from furin. Since TPMV F cleavage occurs efficiently in cells originating from different tissues of four different species, the protease cleaving TPMV F must be ubiquitous.

    TPMV envelope as a vector delivery module. These observations not only indicate that F protein cleavage is not a factor limiting TPMV tropism but also suggest that the fusion peptide of paramyxoviruses can be extended at its amino terminus without impairing F protein function. Tropism of another paramyxovirus, MV, was restricted without changing the F1 amino terminus by introducing two point mutations in the furin cleavage sequence, making it specific for trypsin or trypsin-like proteases, such as tryptase Clara in lungs (23). The opportunity of leaving one or more additional residues at the F-protein amino terminus facilitates engineering of paramyxoviruses activated by certain proteases excreted by tumors (MMPs) that cleave in the middle of a 6-aa recognition sequence. Indeed, recent experiments with a third paramyxovirus suggest that up to three additional residues can be left upstream of the conserved phenylalanine without compromising fusion efficiency: a recombinant MMP-activated SeV reduces human MMP-secreting xenografts in mice (17). Similarly, TPMV F may be modified for tumor-specific activation.

    The fact that a scFv can be functionally displayed on TPMV H without affecting fusion-support function suggests another level at which the TPMV envelope can be engineered for the targeting of oncolytic vectors. It was previously shown that the H protein of MV, a human paramyxovirus, can be targeted by displaying either small ligands such as EGF and IGF (42) or larger specificity domains such as scFvs (5, 15, 30, 34), and TPMV H may be similarly tolerant to ligand display. The TPMV envelope has two distinct advantages over the MV envelope as a vector delivery module. First, it is derived from an animal virus, and since there are no indications that TPMV or TPMV-like viruses infect humans, neutralizing antibodies may not be an issue. Second, TPMV does not recognize a receptor on human cells, and therefore "de-targeting" from known and unknown human receptors is not necessary (29, 61, 63).

    How straightforward is it to enclose a genetic cargo in a TPMV envelope We have shown that envelope exchange within the morbilliviruses yields high-titer viruses (62), and other groups have enclosed paramyxoviral ribonucleocapsids in envelopes from different paramyxovirus subgroups and confirmed efficacy of the chimeric viruses as vaccines (6, 48-50, 59). Therefore, we anticipate that a paramyxoviral chimera with a TPMV envelope may efficiently spread in vivo. In this perspective, the ribonucleocapsids of MV and MuV, human paramyxoviruses used in clinical trials of oncolysis, are premier candidates for enclosure in a TPMV envelope.

    Finally, it might be possible to use the TPMV glycoproteins to pseudotype retroviral and lentiviral vectors. These vectors are already used to deliver genes in clinical protocols (10, 65), but in many cases the target cells have to be transduced ex vivo because no efficient targeting is available. Since successful pseudotyping of retroviruses with the SeV glycoproteins has been reported (19), pseudotyping with the TPMV glycoprotein may also be possible. The availability of targeting at the receptor attachment level and the perspective of tropism restriction at the F protein cleavage-activation level make the TPMV envelope a promising module for a wealth of clinical vector applications.

    ACKNOWLEDGMENTS

    We thank Sompong Vongpunsawad for excellent technical support, Markus Moll for helpful advice on Edman degradation, Ben Madden from the Mayo Clinic Proteomics Research Center for excellent service, and Jeffrey Schlom for the MC38cea cells.

    This study was supported by research grants of the Mayo and Siebens Foundation, the NIH grant R01 CA90636, and a research scholarship (SP 694/1-1) from the German research foundation (DFG) to C.S.

    REFERENCES

    Asada, T. 1974. Treatment of human cancer with mumps virus. Cancer 34:1907-1928.

    Bahr, U., and G. Darai. 2001. Analysis and characterization of the complete genome of tupaia (tree shrew) herpesvirus. J. Virol. 75:4854-4870.

    Bell, J. C., B. Lichty, and D. Stojdl. 2003. Getting oncolytic virus therapies off the ground. Cancer Cell 4:7-11.

    Bendtsen, J. D., H. Nielsen, G. von Heijne, and S. Brunak. 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340:783-795.

    Bucheit, A. D., S. Kumar, D. M. Grote, Y. Lin, V. von Messling, R. B. Cattaneo, and A. K. Fielding. 2003. An oncolytic measles virus engineered to enter cells through the CD20 antigen. Mol. Ther. 7:62-72.

    Buchholz, U. J., H. Granzow, K. Schuldt, S. S. Whitehead, B. R. Murphy, and P. L. Collins. 2000. Chimeric bovine respiratory syncytial virus with glycoprotein gene substitutions from human respiratory syncytial virus (HRSV): effects on host range and evaluation as a live-attenuated HRSV vaccine. J. Virol. 74:1187-1199.

    Cathomen, T., C. J. Buchholz, P. Spielhofer, and R. Cattaneo. 1995. Preferential initiation at the second AUG of the measles virus F mRNA: a role for the long untranslated region. Virology 214:628-632.

    Chester, K. A., R. H. Begent, L. Robson, P. Keep, R. B. Pedley, J. A. Boden, G. Boxer, A. Green, G. Winter, O. Cochet, and R. E. Hawkins. 1994. Phage libraries for generation of clinically useful antibodies. Lancet 343:455-456.

    Chu, R. L., D. E. Post, F. R. Khuri, and E. G. Van Meir. 2004. Use of replicating oncolytic adenoviruses in combination therapy for cancer. Clin. Cancer Res. 10:5299-5312.

    Curiel, D. T., and J. T. Douglas (ed.). 2002. Vector targeting for therapeutic gene delivery. Wiley-Liss, Hoboken, N.J.

    Darai, G., B. Matz, R. M. Flügel, A. Grfe, H. Gelderblom, and H. Delius. 1980. An adenovirus from Tupaia (tree shrew): growth of the virus, characterization of viral DNA, and transforming ability. Virology 104:122-138.

    Elango, N., M. Satake, J. E. Coligan, E. Norrby, E. Camargo, and S. Venkatesan. 1985. Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N terminus of F1 subunit. Nucleic Acids Res. 13:1559-1574.

    Grobelny, D., L. Poncz, and R. E. Galardy. 1992. Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids. Biochemistry 31:7152-7154.

    Grote, D., S. J. Russell, T. I. Cornu, R. Cattaneo, R. Vile, G. A. Poland, and A. K. Fielding. 2001. Live attenuated measles virus induces regression of human lymphoma xenografts in immunodeficient mice. Blood 97:3746-3754.

    Hammond, A. L., R. K. Plemper, J. Zhang, U. Schneider, S. J. Russell, and R. Cattaneo. 2001. Single-chain antibody displayed on a recombinant measles virus confers entry through the tumor-associated carcinoembryonic antigen. J. Virol. 75:2087-2096.

    Hirasawa, K., S. G. Nishikawa, K. L. Norman, M. C. Coffey, B. G. Thompson, C. S. Yoon, D. M. Waisman, and P. W. Lee. 2003. Systemic reovirus therapy of metastatic cancer in immune-competent mice. Cancer Res. 63:348-353.

    Kinoh, H., M. Inoue, K. Washizawa, T. Yamamoto, S. Fujikawa, Y. Tokusumi, A. Iida, Y. Nagai, and M. Hasegawa. 2004. Generation of a recombinant Sendai virus that is selectively activated and lyses human tumor cells expressing matrix metalloproteinases. Gene Ther. 11:1137-1145.

    Klenk, H. D., and W. Garten. 1994. Host cell proteases controlling virus pathogenicity. Trends Microbiol. 2:39-43.

    Kobayashi, M., A. Iida, Y. Ueda, and M. Hasegawa. 2003. Pseudotyped lentivirus vectors derived from simian immunodeficiency virus SIVagm with envelope glycoproteins from paramyxovirus. J. Virol. 77:2607-2614.

    Kolakofsky, D., T. Pelet, D. Garcin, S. Hausmann, J. Curran, and L. Roux. 1998. Paramyxovirus RNA synthesis and the requirement for hexamer genome length: the rule of six revisited. J. Virol. 72:891-899.

    Kurz, H. W. 1984. Isolierung und Charakterisierung von Tupaia Paramyxovirus und Rhabdovirus. Medical doctoral thesis. Heidelberg University, Heidelberg, Germany.

    Lamb, R. A., and D. Kolakofsky. 2001. Paramyxoviridae: the viruses and their replication, p. 1305-1340. In D. M. Knipe and P. M. Howley (ed.), Fields virology, vol. 1. Lippincott/The Williams & Wilkins Co., Philadelphia, Pa.

    Maisner, A., B. Mrkic, G. Herrler, M. Moll, M. A. Billeter, R. Cattaneo, and H. D. Klenk. 2000. Recombinant measles virus requiring an exogenous protease for activation of infectivity. J. Gen. Virol. 81:441-449.

    Michalski, W. P., G. Crameri, L. Wang, B. J. Shiell, and B. Eaton. 2000. The cleavage activation and sites of glycosylation in the fusion protein of Hendra virus. Virus Res. 69:83-93.

    Miller, P. J., D. B. Boyle, B. T. Eaton, and L. F. Wang. 2003. Full-length genome sequence of Mossman virus, a novel paramyxovirus isolated from rodents in Australia. Virology 317:330-344.

    Moll, M., S. Diederich, H. D. Klenk, M. Czub, and A. Maisner. 2004. Ubiquitous activation of the Nipah virus fusion protein does not require a basic amino acid at the cleavage site. J. Virol. 78:9705-9712.

    Murakami, M., T. Towatari, M. Ohuchi, M. Shiota, M. Akao, Y. Okumura, M. A. Parry, and H. Kido. 2001. Mini-plasmin found in the epithelial cells of bronchioles triggers infection by broad-spectrum influenza A viruses and Sendai virus. Eur. J. Biochem. 268:2847-2855.

    Nagai, Y., H. D. Klenk, and R. Rott. 1976. Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology 72:494-508.

    Nakamura, T., K. W. Peng, M. Harvey, S. Greiner, I. A. Lorimer, C. D. James, and S. J. Russell. 2005. Rescue and propagation of fully retargeted oncolytic measles viruses. Nat. Biotechnol. 23:209-214.

    Nakamura, T., K. W. Peng, S. Vongpunsawad, M. Harvey, H. Mizuguchi, T. Hayakawa, R. Cattaneo, and S. J. Russell. 2004. Antibody-targeted cell fusion. Nat. Biotechnol. 22:331-336.

    Pager, C. T., M. A. Wurth, and R. E. Dutch. 2004. Subcellular localization and calcium and pH requirements for proteolytic processing of the Hendra virus fusion protein. J. Virol. 78:9154-9163.

    Pecora, A. L., N. Rizvi, G. I. Cohen, N. J. Meropol, D. Sterman, J. L. Marshall, S. Goldberg, P. Gross, J. D. O'Neil, W. S. Groene, M. S. Roberts, H. Rabin, M. K. Bamat, and R. M. Lorence. 2002. Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers. J. Clin. Oncol. 20:2251-2266.

    Peeters, B. P., O. S. de Leeuw, G. Koch, and A. L. Gielkens. 1999. Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence. J. Virol. 73:5001-5009.

    Peng, K. W., K. A. Donovan, U. Schneider, R. Cattaneo, J. A. Lust, and S. J. Russell. 2003. Oncolytic measles viruses displaying a single-chain antibody against CD38, a myeloma cell marker. Blood 101:2557-2562.

    Pfeffer, S. R., and J. E. Rothman. 1987. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu. Rev. Biochem. 56:829-852.

    Polack, F. P., S. H. Lee, S. Permar, E. Manyara, H. G. Nousari, Y. Jeng, F. Mustafa, A. Valsamakis, R. J. Adams, H. L. Robinson, and D. E. Griffin. 2000. Successful DNA immunization against measles: neutralizing antibody against either the hemagglutinin or fusion glycoprotein protects rhesus macaques without evidence of atypical measles. Nat. Med. 6:776-781.

    Rager, M., S. Vongpunsawad, W. P. Duprex, and R. Cattaneo. 2002. Polyploid measles virus with hexameric genome length. EMBO J. 21:2364-2372.

    Richardson, C. D., A. Scheid, and P. W. Choppin. 1980. Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N termini of the F1 or HA2 viral polypeptides. Virology 105:205-222.

    Ring, C. J. 2002. Cytolytic viruses as potential anti-cancer agents. J. Gen. Virol. 83:491-502.

    Robbins, P. F., J. A. Kantor, M. Salgaller, P. H. Hand, P. D. Fernsten, and J. Schlom. 1991. Transduction and expression of the human carcinoembryonic antigen gene in a murine colon carcinoma cell line. Cancer Res. 51:3657-3662.

    Russell, C. J., T. S. Jardetzky, and R. A. Lamb. 2004. Conserved glycine residues in the fusion peptide of the paramyxovirus fusion protein regulate activation of the native state. J. Virol. 78:13727-13742.

    Schneider, U., F. Bullough, S. Vongpunsawad, S. J. Russell, and R. Cattaneo. 2000. Recombinant measles viruses efficiently entering cells through targeted receptors. J. Virol. 74:9928-9936.

    Schndorf, E., U. Bahr, M. Handermann, and G. Darai. 2003. Characterization of the complete genome of the Tupaia (tree shrew) adenovirus. J. Virol. 77:4345-4356.

    Server, A. C., J. A. Smith, M. N. Waxham, J. S. Wolinsky, and H. M. Goodman. 1985. Purification and amino-terminal protein sequence analysis of the mumps virus fusion protein. Virology 144:373-383.

    Shengqing, Y., N. Kishida, H. Ito, H. Kida, K. Otsuki, Y. Kawaoka, and T. Ito. 2002. Generation of velogenic Newcastle disease viruses from a nonpathogenic waterfowl isolate by passaging in chickens. Virology 301:206-211.

    Springfeld, C., G. Darai, and R. Cattaneo. 2005. Characterization of the Tupaia rhabdovirus genome reveals a long open reading frame overlapping with P and a novel gene encoding a small hydrophobic protein. J. Virol. 79:6781-6790.

    Springfeld, C., A. K. Fielding, K. W. Peng, E. Galanis, C. J. Russell, and R. Cattaneo. 2004. Measles virus: improving natural oncolytic properties by genetic engineering, p. 459-480. In J. C. Horvarth and J. G. Sinkovics (ed.), Virus therapy of human cancer. Marcel Dekker, New York, N.Y.

    Stope, M. B., A. Karger, U. Schmidt, and U. J. Buchholz. 2001. Chimeric bovine respiratory syncytial virus with attachment and fusion glycoproteins replaced by bovine parainfluenza virus type 3 hemagglutinin-neuraminidase and fusion proteins. J. Virol. 75:9367-9377.

    Tao, T., A. P. Durbin, S. S. Whitehead, F. Davoodi, P. L. Collins, and B. R. Murphy. 1998. Recovery of a fully viable chimeric human parainfluenza virus (PIV) type 3 in which the hemagglutinin-neuraminidase and fusion glycoproteins have been replaced by those of PIV type 1. J. Virol. 72:2955-2961.

    Tao, T., M. H. Skiadopoulos, F. Davoodi, J. M. Riggs, P. L. Collins, and B. R. Murphy. 2000. Replacement of the ectodomains of the hemagglutinin-neuraminidase and fusion glycoproteins of recombinant parainfluenza virus type 3 (PIV3) with their counterparts from PIV2 yields attenuated PIV2 vaccine candidates. J. Virol. 74:6448-6458.

    Tashiro, M., Y. Yokogoshi, K. Tobita, J. T. Seto, R. Rott, and H. Kido. 1992. Tryptase Clara, an activating protease for Sendai virus in rat lungs, is involved in pneumopathogenicity. J. Virol. 66:7211-7216.

    Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.

    Tidona, C. A., and G. Darai. 2002. Tupaia paramyxovirus, p. 635-673. In C. A. Tidona and G. Darai (ed.), The Springer index of viruses. Springer-Verlag, Heidelberg, Germany.

    Tidona, C. A., H. W. Kurz, H. R. Gelderblom, and G. Darai. 1999. Isolation and molecular characterization of a novel cytopathogenic paramyxovirus from tree shrews. Virology 258:425-434.

    Varghese, S., and S. D. Rabkin. 2002. Oncolytic herpes simplex virus vectors for cancer virotherapy. Cancer Gene Ther. 9:967-978.

    Varsanyi, T. M., H. Jornvall, and E. Norrby. 1985. Isolation and characterization of the measles virus F1 polypeptide: comparison with other paramyxovirus fusion proteins. Virology 147:110-117.

    Varsanyi, T. M., H. Jornvall, C. Orvell, and E. Norrby. 1987. F1 polypeptides of two canine distemper virus strains: variation in the conserved N-terminal hydrophobic region. Virology 157:241-244.

    Vile, R., D. Ando, and D. Kirn. 2002. The oncolytic virotherapy treatment platform for cancer: unique biological and biosafety points to consider. Cancer Gene Ther. 9:1062-1067.

    von Messling, V., and R. Cattaneo. 2004. Toward novel vaccines and therapies based on negative-strand RNA viruses. Curr. Top. Microbiol. Immunol. 283:281-312.

    von Messling, V., D. Milosevic, P. Devaux, and R. Cattaneo. 2004. Canine distemper virus and measles virus fusion glycoprotein trimers: partial membrane-proximal ectodomain cleavage enhances function. J. Virol. 78:7894-7903.

    von Messling, V., N. Oezgun, Q. Zheng, S. Vongpunsawad, W. Braun, and R. Cattaneo. 2005. Nearby clusters of hemagglutinin residues sustain SLAM-dependent canine distemper virus entry in peripheral blood mononuclear cells. J. Virol. 79:5857-5862.

    von Messling, V., G. Zimmer, G. Herrler, L. Haas, and R. Cattaneo. 2001. The hemagglutinin of canine distemper virus determines tropism and cytopathogenicity. J. Virol. 75:6418-6427.

    Vongpunsawad, S., N. Oezgun, W. Braun, and R. Cattaneo. 2004. Selectively receptor-blind measles viruses: identification of residues necessary for SLAM- or CD46-induced fusion and their localization on a new hemagglutinin structural model. J. Virol. 78:302-313.

    Wang, L., B. H. Harcourt, M. Yu, A. Tamin, P. A. Rota, W. J. Bellini, and B. T. Eaton. 2001. Molecular biology of Hendra and Nipah viruses. Microbes Infect. 3:279-287.

    Wickham, T. J. 2003. Ligand-directed targeting of genes to the site of disease. Nat. Med. 9:135-139.(Christoph Springfeld, Ver)