The seventh transmembrane domain of cc chemokine receptor 5 is critical for MIP-1beta binding and receptor activation: role of MET 287.

CC chemokine receptor 5 (CCR5) is a high-affinity receptor for macrophage inflammatory protein (MIP)-1beta and functions as the major coreceptor for entry of macrophage-tropic (M-tropic) human immunodeficiency virus type 1 (HIV-1). To evaluate the role of transmembrane domains (TM) in the receptor function of CCR5, the seventh transmembrane domain (TM7) was examined in a series of chimeric receptor constructs including CCR5TM (CCR5 backbone/CCR5 TM7 replaced with CCR1 TM7) and mutants of CCR5TM. The CCR5TM chimera exhibited a dramatic reduction in receptor activation, as well as little or no MIP-1beta binding. Further mutational analysis revealed that Met 287 in TM7 of CCR5 is a critical molecular determinant for both MIP-1beta binding and receptor activation. Interestingly, all of the chimeric/mutated receptors were biologically active in an HIV-1 coreceptor fusion assay, demonstrating that chemokine binding is independent of HIV-1 coreceptor activity.

CC chemokine receptors 1 and 3 are differentially regulated by IL-5 during maturation of eosinophilic HL-60 cells.

CC chemokine receptors 1 and 3 (CCR1 and CCR3) are expressed by eosinophils; however, factors regulating their expression and function have not previously been defined. Here we analyze chemokine receptor expression and function during eosinophil differentiation, using the eosinophilic cell line HL-60 clone 15 as a model system. RNA for CCR1, -3, -4, and -5 was not detectable in the parental cells, and the cells did not specifically bind CC chemokines. Cells treated with butyric acid acquired eosinophil characteristics; expressed mRNA for CCR1 and CCR3, but not for CCR4 or CCR5; acquired specific binding sites for macrophage-inflammatory protein-1alpha and eotaxin (the selective ligands for CCR1 and CCR3, respectively); and exhibited specific calcium flux and chemotaxis responses to macrophage-inflammatory protein-1alpha, eotaxin, and other known CCR1 and CCR3 agonists. CCR3 was expressed later and at lower levels than CCR1 and could be further induced by IL-5, whereas IL-5 had little or no effect on CCR1 expression. Consistent with the HIV-1 coreceptor activity of CCR3, HL-60 clone 15 cells induced with butyric acid and IL-5 fused with HeLa cells expressing CCR3-tropic HIV-1 envelope glycoproteins, and fusion was blocked specifically by eotaxin or an anti-CCR3 mAb. These data suggest that CCR1 and CCR3 are markers of late eosinophil differentiation that are differentially regulated by IL-5 in this model.

Patterns of CCR5, CXCR4, and CCR3 usage by envelope glycoproteins from human immunodeficiency virus type 1 primary isolates.

Coreceptor usage by Envs from diverse primary human immunodeficiency virus type 1 isolates was analyzed by a vaccinia virus-based expression and assay system. Usage of recombinant CCR5 and CXCR4 correlated closely with fusogenicity toward macrophages and T-cell lines expressing endogenous coreceptors. Surprisingly, recombinant CCR3 was utilized by most primary and T-cell-line-adapted Envs. Endogenous CXCR4 in macrophages was functional as a coreceptor.

HIV-1 coreceptor activity of CCR5 and its inhibition by chemokines: independence from G protein signaling and importance of coreceptor downmodulation.

HIV-1 infection requires the presence of specific chemokine receptors on CD4+ target cells to enable the fusion reactions involved in virus entry. CCR5 is a major fusion coreceptor for macrophage-tropic HIV-1 isolates. HIV-1 entry and fusion are mediated by the viral envelope glycoprotein (Env) and are inhibited by CCR5 ligands, but the mechanisms are unknown. Here, we test the role of G protein signaling and CCR5 surface downmodulation by two separate approaches: direct inactivation of CCR5 signaling by mutagenesis and inactivation of G(i)-type G proteins with pertussis toxin. A CCR5 mutant lacking the last 45 amino acids of the cytoplasmic C-terminus (CCR5306) was created that was expressed on transfected cells at levels comparable to cells expressing CCR5 and displayed normal chemokine binding affinity. CCR5 ligands induced calcium flux and receptor downmodulation in cells expressing CCR5, but not in cells expressing CCR5306. Nevertheless, CCR5 or CCR5306, when coexpressed with CD4, supported comparable HIV-1 Env-mediated cell fusion. Consistent with this, treatment of CCR5-expressing cells with pertussis toxin completely blocked ligand-induced transient calcium flux, but did not affect Env-mediated cell fusion or HIV-1 infection. Also, pertussis toxin did not block chemokine inhibition of Env-mediated cell fusion or HIV-1 infection. However, chemokines inhibited Env-mediated cell fusion less efficiently for CCR5306 than for CCR5. We conclude that the C-terminal domain of CCR5 is critical for G protein signaling and receptor downmodulation from the surface, but that neither function is required for CCR5 fusion coreceptor activity. The contrasting phenotypes of CCR5 and CCR5306 suggest that coreceptor downmodulation and direct blockage of Env interaction sites both contribute to chemokine inhibition of HIV-1 infection.

CC chemokine receptor 5-mediated signaling and HIV-1 Co-receptor activity share common structural determinants. Critical residues in the third extracellular loop support HIV-1 fusion.

There is a close correspondence between the ability of RANTES and macrophage inflammatory proteins 1alpha and 1beta to activate CC chemokine receptor 5 (CCR5) and the ability to inhibit CCR5-dependent membrane fusion mediated by the envelope glycoprotein of human immunodeficiency virus (HIV), type 1. This finding suggests that some of the structural determinants for CC chemokine/CCR5 interactions and CCR5 HIV-1 fusion co-receptor activity may be shared. Recent studies using human CCR5/CCR2B chimeras have suggested that the determinants of CCR5 co-receptor activity are complex and may involve multiple extracellular receptor domains and that viral co-receptor activity is dissociable from ligand-dependent signaling responses. However, conclusive evidence demonstrating an important role for the second and third extracellular regions of human CCR5 is lacking. Furthermore, to determine whether the determinants for CCR5 co-receptor activity overlap with those required for agonist activity, studies that compare the chemokine specificity for inhibition of envelope-mediated cell fusion and the agonist profile of chimeric receptors are necessary. In the present report, using a series of CCR5/CCR2B chimeras we ascribe an important role for the second and third extracellular loop of CCR5 in supporting the co-receptor activity of CCR5. We also provide evidence that the intracytoplasmic tail of CCR5 does not play an important role in supporting HIV-1 entry. The hypothesis that the structural determinants for CC chemokine/CCR5 interactions and CCR5 HIV-1 fusion co-receptor activity may be shared was confirmed by two novel observations: first, the fusion activity supported by two hybrid receptors could be inhibited by both RANTES and monocyte chemoattractant protein-1, chemokines specific to CCR5 and CCR2B, respectively; and second, the chemokine specificity for inhibition of envelope-mediated cell fusion matched the agonist profile of these hybrid receptors. These data shed new light on the structural determinants involved in these distinct activities of CCR5 and may have important implications for the development of CCR5-targeted anti-viral compounds.

Determinants of HIV-1 coreceptor function on CC chemokine receptor 3. Importance of both extracellular and transmembrane/cytoplasmic regions.

The chemokine receptors CXCR4, CCR2b, CCR3, and CCR5 are cell entry coreceptors for HIV-1. Using an HIV-1 envelope (Env)-dependent cell-cell fusion model of entry, we show that CCR3 can interact with Envs from certain macrophage (M)-tropic strains (which also use CCR5), T cell line (TCL)-tropic laboratory-adapted strains (which also use CXCR4), and a dual-tropic primary isolate (which also uses CCR2b, CCR5, and CXCR4). Paradoxically, CCR1 is the closest homologue to CCR3 (63% amino acid identity), but lacked HIV-1 coreceptor activity. These results confirm and extend previous reports. Replacing the N-terminal segment of CCR3 with that of CCR1 abolished activity of the resulting chimera for M-tropic and TCL-tropic Envs, but not for the dual-tropic Env. Replacing extracellular loop 2 of CCR3 with that of CCR1 abolished activity for TCL-tropic Envs, but not for M- and dual-tropic Envs. A chimera containing all four extracellular regions of CCR3 on a backbone of CCR1 lacked any activity. Env-CCR3 interactions were strongly inhibited by the major CCR3 ligand eotaxin, but weakly or not at all by other CCR3 ligands. With primary macrophages, eotaxin induced transient calcium flux and partially inhibited fusion with cells expressing M-tropic Envs. We conclude that specificity determinants for different Envs are located in shared and distinct extracellular regions of CCR3, the transmembrane/cytoplasmic domains make major contributions to coreceptor function, and CCR3 may be used by certain HIV-1 strains as a cell fusion factor on macrophages.

STRL33, A novel chemokine receptor-like protein, functions as a fusion cofactor for both macrophage-tropic and T cell line-tropic HIV-1.

The chemokine receptors CXCR4, CCR2B, CCR3, and CCR5 have recently been shown to serve along with CD4 as coreceptors for HIV-1. The tropisms of HIV-1 strains for subgroups of CD4(+) cells can be explained, at least partly, by the selective use of G protein-coupled receptors (GPCRs). We have identified a novel human gene, STRL33, located on chromosome 3 that encodes a GPCR with sequence similarity to chemokine receptors and to chemokine receptor-like orphan receptors. STRL33 is expressed in lymphoid tissues and activated T cells, and is induced in activated peripheral blood lymphocytes. When transfected into nonhuman NIH 3T3 cells expressing human CD4, the STRL33 cDNA rendered these cells competent to fuse with cells expressing HIV-1 envelope glycoproteins (Envs). Of greatest interest, STRL33, in contrast with CXCR4 or CCR5, was able to function as a cofactor for fusion mediated by Envs from both T cell line-tropic and macrophage-tropic HIV-1 strains. STRL33-transfected Jurkat cell lines also supported enhanced productive infection with HIV-1 compared with control Jurkat cells. Despite the sequence similarities between STRL33 and chemokine receptors, STRL33-transfected cell lines did not respond to any in a panel of chemokines. Based on the pattern of tissue expression of the STRL33 mRNA, and given the ability of STRL33 to function with Envs of differing tropisms, STRL33 may play a role in the establishment and/or progression of HIV-1 infection.

Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk.

BACKGROUND: CC chemokine receptor 5 (CCR5) is a cell entry cofactor for macrophage-tropic isolates of human immunodeficiency virus-1 (HIV-1). Recently, an inactive CCR5 allele (designated here as CCR5-2) was identified that confers resistance to HIV-1 infection in homozygotes and slows the rate of progression to AIDS in heterozygotes. The reports conflict on the effect of heterozygous CCR5-2 on HIV-1 susceptibility, and race and risk levels have not yet been fully analyzed. Here we report our independent identification of CCR5-2 and test its effects on HIV-1 pathogenesis in individuals with contrasting clinical outcomes, defined race, and quantified risk. MATERIALS AND METHODS: Mutant CCR5 alleles were sought by directed heteroduplex analysis of genomic DNA from random blood donors. Genotypic frequencies were then determined in (1) random blood donors from North America, Asia, and Africa; (2) HIV-1+ individuals; and (3) highly exposed-seronegative homosexuals with quantified risk. RESULTS: CCR5-2 was the only mutant allele found. It was common in Caucasians, less common in other North American racial groups, and not detected in West Africans or Tamil Indians. Homozygous CCR5-2 frequencies differed reciprocally in highly exposed-seronegative (4.5%, n = 111) and HIV-1-seropositive (0%, n = 614) Caucasians relative to Caucasian random blood donors (0.8%, n = 387). This difference was highly significant (p < 0.0001). By contrast, heterozygous CCR5-2 frequencies did not differ significantly in the same three groups (21.6, 22.6, and 21.7%, respectively). A 55% increase in the frequency of heterozygous CCR5-2 was observed in both of two cohorts of Caucasian homosexual male, long-term nonprogressors compared with other HIV-1+ Caucasian homosexuals (p = 0.006) and compared with Caucasian random blood donors. Moreover, Kaplan-Meier estimates indicated that CCR5-2 heterozygous seroconvertors had a 52.6% lower risk of developing AIDS than homozygous wild-type seroconvertors. CONCLUSIONS: The data suggest that homozygous CCR5-2 is an HIV-1 resistance factor in Caucasians with complete penetrance, and that heterozygous CCR5-2 slows the rate of disease progression in infected Caucasian homosexuals. Since the majority (approximately 96%) of highly exposed-seronegative individuals tested are not homozygous for CCR5-2, other resistance factors must exist. Since CCR5-2 homozygotes have no obvious clinical problems, CCR5 may be a good target for the development of novel antiretroviral therapy.

Cellular distribution of a natural killer cell tumour recognition-related surface antigen in purified human lymphocytes.

Natural killer (NK) cells are large granular lymphocytes capable of human leucocyte antigen (HLA) unrestricted killing of tumour cells. A putative NK cell tumour-recognition molecule (NK-TR) was previously isolated and cloned. The predicted primary structure of the NK-TR revealed that the amino terminus of the protein shared high homology with cyclophilin proteins. In this study, we used rabbit antibodies directed against synthetic peptides corresponding to amino acids 476-497 of the NK-TR protein, to examine the expression of the NK-TR antigen in freshly purified human lymphocytes. Cell-surface staining experiments using these peptide antibodies indicated the presence of the NK-TR protein on the surface of human CD3+ T-cell populations purified from peripheral blood. There were individual donor differences in the levels of cell-surface expression of this antigen ranging from 35 to 90% in T lymphocytes and, NK cells purified from different healthy volunteers. The immunoreactivity of our peptide antibodies in immunoprecipitation showed that the NK-TR-related protein expressed in purified T cells is similar to that expressed in NK cells in terms of its electrophoretic mobility. Cell-surface staining experiments using the peptide antibodies revealed that the NK-TR-related protein is more abundantly expressed on the surface of purified T cells compared with NK cells. Northern blot analysis of the mRNA species transcribed in human lymphocytes revealed abundant expression of NK-TR-specific mRNA species in purified T cells. Furthermore, another mRNA species smaller than 7 kb was detected in both NK and T-cell populations of lymphocytes freshly isolated from peripheral blood. Expression at the cell surface of a cyclophilin-homologous protein in purified human T lymphocytes may indicate another function for the reported NK-TR protein, that is, distinct from tumour-cell recognition and cytosis.

Cell type-specific fusion cofactors determine human immunodeficiency virus type 1 tropism for T-cell lines versus primary macrophages.

Work in this laboratory previously demonstrated that the tropism of different human immunodeficiency type 1 isolates for infection of human CD4+ continuous cell lines (e.g., T-cell lines and HeLa-CD4 transformants) versus primary macrophages is associated with parallel intrinsic fusogenic specificities of the corresponding envelope glycoproteins (Envs). For T-cell line-tropic isolates, it is well established that the target cell must also contain a human-specific fusion cofactor(s) whose identity is unknown. In this study, we tested the hypothesis that the Env fusion specificities underlying T-cell line versus macrophage tropism are determined by distinct cell type-specific fusion cofactors. We applied a recombinant vaccinia virus-based reporter gene assay for Env-CD4-mediated cell fusion; the LAV and Ba-L Envs served as prototypes for T-cell line-tropic and macrophage-tropic isolates, respectively. We examined CD4+ promyeloctic and monocytic cell lines that are infectible by T-cell line-tropic isolates and become susceptible to macrophage-tropic strains only after treatment with differentiating agents. We observed parallel changes in fusion specificity: untreated cells supported fusion by the LAV but not the Ba-L Env, whereas cells treated with differentiating agents acquired fusion competence for Ba-L. These results suggest that in untreated cells, the block to infection by macrophage-tropic isolates is at the level of membrane fusion; furthermore, the differential regulation of fusion permissiveness for the two classes of Envs is consistent with the existence of distinct fusion cofactors. To test this notion directly, we conducted experiments with transient cell hybrids formed between CD4-expressing nonhuman cells (murine NIH 3T3) and different human cell types. Hybrids formed with HeLa cells supported fusion by the LAV Env but not by the Ba-L Env, whereas hybrids formed with primary macrophages showed the opposite specificity; hybrids formed between HeLa cells and macrophages supported fusion by both Envs. These results suggest the existence of cell type-specific fusion cofactors selective for each type of Env, rather than fusion inhibitors for discordant Env-cell combinations. Finally, analyses based on recombinant protein expression and antibody blocking did not support the proposals by others that the CD44 or CD26 antigens are involved directly in the entry of macrophage-tropic isolates.

CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1.

Human immunodeficiency virus-type 1 (HIV-1) entry requires fusion cofactors on the CD4+ target cell. Fusin, a heterotrimeric GTP-binding protein (G protein)-coupled receptor, serves as a cofactor for T cell line-tropic isolates. The chemokines RANTES, MIP-1alpha, and MIP-1beta, which suppress infection by macrophage-tropic isolates, selectively inhibited cell fusion mediated by the corresponding envelope glycoproteins (Envs). Recombinant CC CKR5, a G protein-coupled receptor for these chemokines, rendered CD4-expressing nonhuman cells fusion-competent preferentially with macrophage-tropic Envs. CC CKR5 messenger RNA was detected selectively in cell types susceptible to macrophage-tropic isolates. CC CKR5 is thus a fusion cofactor for macrophage-tropic HIV-1 strains.

Characterization of a cleavage mutant of the measles virus fusion protein defective in syncytium formation.

Membrane fusion caused by measles virus (MV) is a function of the fusion (F) protein. This process is essential for penetration into the host cell and subsequent initiation of the virus replicative cycle. The biological activity of the MV F protein is generated by endoproteolytic cleavage of a precursor protein (F0) into a large F1 subunit and a smaller F2 subunit held together by disulfide bonds. The cleavage site consists of a cluster of five basic amino acids (amino acids 108 to 112) within the predicted primary structure of the F protein. To investigate the role of the arginine residue at the carboxy terminus of the F2 subunit (arginine 112), site-directed mutagenesis was used to construct a cleavage mutant of the MV F protein in which this arginine residue was changed to a leucine residue. The mutated F gene, encoding four out of the five basic amino acids at the cleavage site, was inserted into the genome of vaccinia virus. The resulting recombinant virus was used to study expression of the mutant F protein in infected cells. Analysis of the Leu-112 mutant protein made in infected cells demonstrated that this single-amino-acid substitution resulted in a reduced rate of transport of the mutant protein to the cell surface, despite its efficient cleavage to yield F1 and F2 subunits. However, the electrophoretic mobilities of the Leu-112 polypeptides suggested that the protein was cleaved incorrectly. This aberrant cleavage appears to have abolished the ability of the F protein to cause syncytium formation. The data indicate that the arginine 112 residue is critical for the correct proteolytic cleavage that is required for the membrane fusion activity of the MV F protein.

Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes.

Nonreplicating viral vectors as potential vaccines: recombinant canarypox virus expressing measles virus fusion (F) and hemagglutinin (HA) glycoproteins.

The development of canarypox virus (CPV) recombinants expressing the hemagglutinin (HA) and fusion (F) glycoproteins of measles virus (MV) is described. Inoculation of the CPV-MV recombinants into avian or nonavian tissue culture substrates led to the expression of authentic MVF and MVHA as determined by radioimmunoprecipitation and surface immunofluorescence. In contrast to avian-derived tissue culture, no productive replication of the CPV recombinant was evident in tissue culture cells derived from nonavian origin. On inoculation of dogs, a species restricted for avipoxvirus replication, the recombinants elicited a protective immune response against a lethal canine distemper virus (CDV) challenge. The level of MV neutralizing antibodies and the level of protection induced against CDV challenge achieved by the host-restricted CPV vector were equivalent to that obtained by vaccinia virus vectors expressing the same MV antigens.

Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge.

Intracellular processing, glycosylation, and cell-surface expression of the measles virus fusion protein (F) encoded by a recombinant adenovirus.

The membrane fusion protein of measles virus (MVF) is a surface glycoprotein which is essential for initiation of viral infection. The F protein mediates penetration of the host cell through a process of membrane fusion between the viral envelope and the host cell plasma membrane. To study the structure-function relationship of the MVF protein, a recombinant adenovirus, Ad5MVF, was constructed which expressed the F protein in mammalian cells. The MVF gene was inserted into the Ad5 genome by homologous recombination, which resulted in replacement of most of the E1 region. This recombinant virus was stable and replicated efficiently in the 293 cell line which complemented the deleted E1 functions. Human 293 cells infected with Ad5MVF synthesized an authentic MVF protein precursor (F0) which appeared to be cleaved efficiently to the F1 and F2 polypeptides. This recombinant F protein was glycosylated, transported to the cell surface, and found to be capable of inducing syncytia formation and hemolysis of monkey erythrocytes. The hemagglutinin protein (HA), provided by a coinfecting adenovirus, was not able to increase the biological activity of the F protein. Treatment of MV or Ad5MVF-infected cells with tunicamycin, an inhibitor of N-linked glycosylation, abolished processing of the F protein. This observation suggests that glycosylation might play an important role in cleavage-dependent activation of the precursor F0 protein or in its transport to the subcellular region where proteolytic cleavage occurs.

Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene.

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.

Expression of bicistronic measles virus P/C mRNA by using hybrid adenoviruses: levels of C protein synthesized in vivo are unaffected by the presence or absence of the upstream P initiator codon.

The measles virus (MV) P/C mRNA is functionally bicistronic. Translation is presumed to initiate at both the first and second 5'-proximal AUG codons, leading, respectively, to synthesis of the P and C polypeptides from different overlapping reading frames. To study the function and differential expression of these polypeptides, we have constructed hybrid human adenoviruses capable of expressing high levels of P and C together or of C alone. Cloned cDNA corresponding to the MV P/C gene was coupled to the adenovirus type 2 (Ad2) major late promoter, most of the Ad2 tripartite leader sequence, and the simian virus 40 3'-end processing signal and then used to replace most of the E1a-E1b region of the Ad5 genome in two hybrid adenoviruses: one (Ad5MV/PC13) which contained both 5'-proximal AUG codons of the P/C mRNA and another (Ad5MV/C3) which retained only the second. The sequence context for the P protein initiator AUG codon in Ad5MV/PC13 was made more favorable (GAGAUGG) than the relatively unfavorable context (CCGAUGG) seen in the native MV P/C mRNA. After infection of 293 cells (which provide complementary E1a-E1b functions), both viruses directed equal amounts of P/C-specific mRNA transcription. Ad5MV/PC13 directed the synthesis of both P and C proteins, while Ad5MV/C3 directed the synthesis of C protein alone. Ad5-expressed P protein was phosphorylated, while C was not. C protein had a similar diffuse cytoplasmic localization in both MV and Ad5-infected cells. Ad5MV/C3 and Ad5MV/PC13 directed equal amounts of C protein expression in 293 cells at a level approximately 15 times greater than that seen in MV-infected cells. Thus the level of C protein expression was unaffected by the presence or absence of an out-of-frame upstream AUG codon in a favorable sequence context. This observation cannot be explained by the scanning model for ribosomal initiation and suggests that ribosomes may be binding directly at an internal mRNA site at or near the initiator AUG codon for the C protein.

High-level eucaryotic in vivo expression of biologically active measles virus hemagglutinin by using an adenovirus type 5 helper-free vector system.

The entire measles virus (MV) hemagglutinin (HA)-coding region was reconstructed from cloned cDNAs and used as part of a hybrid transcription unit to replace a region of the adenovirus type 5 genome corresponding to the entire E1a transcription unit and most of the E1b transcription unit. The resulting recombinant virus was stable and able to replicate to high titers in 293 cells (which constitutively express the complementary E1a-E1b functions) in the absence of helper virus. During infection of 293 cells, the hybrid virus expressed MV HA protein which was indistinguishable from that expressed in MV-infected cells in terms of immunoreactivity, gel mobility, glycosylation, subcellular localization, and biologic activity. Infection of 293 cells with the hybrid virus led to high-level synthesis of the MV HA protein (equivalent to 65 to 130% of the level seen in MV-infected cells). At late times after high-multiplicity hybrid virus infection of HeLa and Vero cells (which do not express E1 functions), the level of HA protein synthesis was at least 35% of that seen in 293 cells. This MV-adenovirus recombinant will be useful in the study of the biologic properties of the MV HA protein and in assessment of the potential usefulness of hybrid adenoviruses as live-virus vaccine vectors.