Vero/BC-F: an efficient packaging cell line stably expressing F protein to generate single round-infectious human parainfluenza virus type 2 vector.

A stable packaging cell line (Vero/BC-F) constitutively expressing fusion (F) protein of the human parainfluenza virus type 2 (hPIV2) was established for production of the F-defective and single round-infectious hPIV2 vector in a strategy for recombinant vaccine development. The F gene expression has not evoked cytostatic or cytotoxic effects on the Vero/BC-F cells and the F protein was physiologically active to induce syncytial formation with giant polykaryocytes when transfected with a plasmid expressing hPIV2 hemagglutinin-neuraminidase (HN). Transduction of the F-defective replicon RNA into the Vero/BC-F cells led to the release of the infectious particles that packaged the replicon RNA (named as hPIV2ΔF) without detectable mutations, limiting the infectivity to a single round. The maximal titer of the hPIV2ΔF was 6.0 × 10(8) median tissue culture infections dose per ml. The influenza A virus M2 gene was inserted into hPIV2ΔF, and the M2 protein was found to be highly expressed in a human lung cancer cell line after transduction. Furthermore, in vivo airway infection experiments revealed that the hPIV2ΔF was capable of delivering transgenes to hamster tracheal cells. Thus, non-transmissible or single round-infectious hPIV2 vector will be potentially applicable to human gene therapy or recombinant vaccine development.

Existence of leptin receptor protein in chicken tissues: isolation of a monoclonal antibody against chicken leptin receptor.

Leptin receptor belongs to the class I cytokine receptor superfamily, which mediates multiple physiological roles in mammals. However, the leptin system is poorly understood in birds, as the evidence for the existence of a natural ligand of the receptor in birds is controversial. As part of a strategy to reveal the physiological significance of leptin in birds, we isolated a monoclonal antibody (mAb) against a chicken leptin receptor (chLEPR). Based on the cDNA sequence for chLEPR, a peptide coding for the cytoplasmic domain of chLEPR was expressed in Escherichia coli and this was used to immunize mice to obtain the mAb. The anti-chLEPR mAb recognized proteins migrated at approximately 180 kDa by Western blot analysis using cellular extracts prepared from COS-7 cells transfected with chLEPR expression vector. By Western blot analysis using the same mAb, an immunoreactive band migrated at 180 kDa was detected in the chicken brain and Leghorn male hepatoma (LMH) cells, and which was similar to the size observed in the in vitro transfection study. Taken together, the chLEPR mAb obtained in the present study cross-reacted, at least, with long isoform chLEPR, suggesting that LEPR mRNA expressed in chicken tissues is likely to be translated. The chLEPR mAb, which has not been described elsewhere, enables us to explore the expression and localization of the receptor in the chicken tissues at the protein level. Therefore, this antibody would be a powerful tool in studying and understanding the regulation and function of leptin and its receptors in birds.

Hemagglutinin-neuraminidase-independent fusion activity of simian virus 5 fusion (F) protein: difference in conformation between fusogenic and nonfusogenic F proteins on the cell surface.

The fusion (F) protein of simian virus 5 (SV5) strain W3A is known to induce cell fusion in the absence of hemagglutinin-neuraminidase (HN) protein. In contrast, the F protein of SV5 strain WR induces cell fusion only when coexpressed with the HN protein, the same as do other paramyxovirus F proteins. When Leu-22 in the subunit F2 of the WR F protein is replaced with the counterpart (Pro) in the W3A F protein, the resulting mutant L22P induces extensive cell fusion by itself. In the present study, we obtained anti-L22P monoclonal antibodies (MAbs) 21-1 and 6-7, whose epitopes were located in the middle (amino acids [aa] 227 to 320) of subunit F1. The amino-terminal region (aa 20 to 47) of subunit F2 was also involved in the formation of MAb 21-1 epitope. Flow cytometric analysis revealed that both the MAbs reacted very faintly with native WR F protein that was expressed on the cell surface whereas they reacted efficiently with native L22P irrespective of whether it is cleaved into F1 and F2. However, by heating the cells at 47 degrees C after mild formaldehyde fixation, the epitopes for MAb 6-7 and mAb 21-1 in the WR F protein were exposed and the reactivity of the MAbs with the WR F protein became comparable to their reactivity with L22P. Thus, the two MAbs seem to distinguish the difference in native conformation between fusogenic mutant L22P and its parental nonfusogenic WR F protein. The native conformation of L22P may represent an intermediate between native and postfusion conformations of a typical paramyxovirus F protein.

High resistance of human parainfluenza type 2 virus protein-expressing cells to the antiviral and anti-cell proliferative activities of alpha/beta interferons: cysteine-rich V-specific domain is required for high resistance to the interferons.

Human parainfluenza type 2 virus (hPIV-2)-infected HeLa (HeLa-CA) cells and hPIV-2 V-expressing HeLa (HeLa-V) cells show high resistance to alpha/beta interferons (IFN-alpha/beta) irrespective of whether vesicular stomatitis virus or Sindbis virus is used as a challenge virus. When Sindbis virus is used, these cells show high susceptibility to human IFN-gamma. Furthermore, the multiplication of HeLa-V cells is not inhibited by IFN-alpha/beta. HeLa cells expressing the N-terminally truncated V protein show resistance to IFN-alpha/beta, showing that the IFN resistance determinant maps to the cysteine-rich V-specific domain. A complete defect of Stat2 is found in HeLa-CA and HeLa-V cells, whereas the levels of Stat1 expression are not significantly different among HeLa, HeLa-CA, HeLa-P, and HeLa-V cells, indicating that IFN-alpha/beta resistance of HeLa-CA and HeLa-V cells is due to a defect of Stat2. HeLa-SV41V cells show high resistance to all IFNs, and no expression of Stat1 can be detected. Stat2 mRNA is fully detected in HeLa-V cells. Stat2 was scarcely pulse-labeled in the HeLa-V cells, indicating that synthesis of Stat2 is suppressed or Stat2 is very rapidly degraded in HeLa-V cells. The V protein suppresses the in vitro translation of Stat2 mRNA more extensively than that of Stat1 mRNA. An extremely small amount of Stat2 can be detected in HeLa-V cells treated with proteasome inhibitors. The half-life of Stat2 is approximately 3.5 and 2 h in uninfected and hPIV-2-infected HeLa cells, respectively. This study shows that synthesis of Stat2 may be suppressed and Stat2 degradation is also enhanced in hPIV-2-infected HeLa and HeLa-V cells.

Recovery of infectious human parainfluenza type 2 virus from cDNA clones and properties of the defective virus without V-specific cysteine-rich domain.

A full-length cDNA clone was constructed from the genome of the human parainfluenza type 2 virus (hPIV2). First, Vero cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase, and then the plasmid encoding the antigenome sequence was transfected into Vero cells together with polymerase unit plasmids, NP, P, and L, which were under control of the T7 polymerase promoter. Subsequently, the transfected cells were cocultured with fresh Vero cells. Rescue of recombinant hPIV2 (rPIV2) from cDNA clone was demonstrated by finding the introduced genetic tag. As an application of reverse genetics, we introduced one nucleotide change (UCU to ACU) to immediate downstream of the RNA-editing site of the V gene in the full-length hPIV2 cDNA and were able to obtain infectious viruses [rPIV2V(-)] from the cDNA. The rPIV2V(-) possessed a defective V protein that did not have the unique cysteine-rich domain in its carboxyl terminus (the V-protein-specific domain). The rPIV2V(-) showed no growth in CV-1 and FL cells. Replication of the rPIV2V(-) in these cells, however, was partially recovered by adding anti-interferon (IFN)-beta antibody into the culture medium, showing that the rPIV2V(-) is highly sensitive against IFN and that no growth of rPIV2V(-) in CV-1 and FL cells is mainly due to its hypersensitivity to endogenously produced IFN. These findings indicate that the V-protein-specific domain of hPIV2 is related to IFN resistance. On the other hand, the rPIV2V(-) efficiently replicated in Vero cells, which are known as a IFN-non-producers. However, the virus yields of rPIV2V(-) in Vero cells were 10- to100-fold lower than those of control rPIV2, although syntheses of the viral-specific proteins and their mRNAs in rPIV2V(-)-infected Vero cells were augmented up to 48 p.i. in comparison with those of rPIV2. Furthermore, the rPIV2V(-) virions showed anomalous in size as compared with rPIV2 virions. These results suggest that the V protein plays an important role in the hPIV2 assembly, maturation, and morphogenesis.

Cross-talk between RANKL and FRP-1/CD98 Systems: RANKL-mediated osteoclastogenesis is suppressed by an inhibitory anti-CD98 heavy chain mAb and CD98-mediated osteoclastogenesis is suppressed by osteoclastogenesis inhibitory factor.

The two pathways to osteoclastogenesis, RANKL-mediated and CD98-mediated osteoclastogenesis, have recently been reported. RANKL, OCIF, and TIMP-3 mRNAs are not found in monocytes freshly isolated or incubated with anti-FRP-1/CD98hc antibody. RANK, TACE, and M-CSF mRNAs can be detected in these cells. Interestingly, the expressed amount of RANK mRNA increases by cultivation of monocytes with anti-CD98hc antibody and maximal expression is observed in osteoclast-like cells. CD98-mediated cell aggregation and multinucleated giant cell formation are blocked by OCIF. OCIF also suppressed the CD98-mediated induction of Sp1 and c-src mRNAs in monocytes. Soluble RANK shows no effect on CD98-mediated cell aggregation and multinucleated giant cell formation. When blood monocytes were incubated with RANKL and M-CSF, c-src and Sp1 mRNAs were first found in blood monocytes incubated with these cytokines for 7 days. On the contrary, c-src mRNA could be detected 3 h after treatment of blood monocytes with anti-CD98hc mAb. LAT-1 mRNA was not found, and the expression levels of Y(+)LAT-1 and Y(+)LAT-2 mRNAs were not changed in monocytes stimulated without or with anti-CD98hc mAb or RANKL and M-CSF. An inhibitory mAb directed against CD98hc, HBJ 127, shows a suppressive effect on RANKL-mediated cell aggregation and cell fusion. Thus, there is cross-talk between these two pathways.

Involvement of ADAM9 in multinucleated giant cell formation of blood monocytes.

Monocytes-macrophages are converted to multinucleated giant cells by stimulation with various cytokines, and osteoclasts are the multinucleated giant cells derived from a monocyte-macrophage lineage. However, at present, the fusion peptides have not been clearly identified in monocytes-macrophages. The ADAM are a family of transmembrane glycoproteins that have a role in various biological functions. Interestingly, fertilin-alpha, ADAM9, and ADAM11 have potential fusion peptides. In this study, which ADAM was specifically expressed in monocytes stimulated with anti-CD98 antibody or RANKL and which factor(s) was functioning in monocytes as a fusion protein were investigated. ADAM1, 8, 10, 12, 15, 17, 20, and 21 mRNAs are expressed in blood monocytes incubated with control antibody, anti-FRP-1/CD98 antibody, or RANKL + M-CSF, while ADAM2, 7, 11, 13, 19, 23, 29, and 30 mRNAs could not be detected in these blood monocytes. Expression of ADAM9 and ADAM10 mRNAs are enhanced by either RANKL + M-CSF or anti-CD98 antibody. The expression of ADAM9 and ADAM10 is also induced in blood monocytes by anti-CD98 mAb. An anti-ADAM9 antibody enhances CD98-mediated cell aggregation, while it blocks CD98-mediated and RANKL-mediated multinucleated giant cell formation. A hydroxamate-based metalloprotease inhibitor, SI-27, which is found to suppress ADAM9 activity, suppresses multinucleated giant cell formation. New protein synthesis is necessary for the expression of ADAM9 mRNA and genistein suppresses induction of ADAM9 mRNA. This is the first report that ADAM9 is involved in monocyte fusion, such as CD98-mediated and RANKL-mediated cell fusion of blood monocytes. Furthermore, AMAM9 is one candidate for a fusion peptide in blood monocytes.

Induction of c-Src in human blood monocytes by anti-CD98/FRP-1 mAb in an Sp1-dependent fashion.

Freshly isolated human blood monocytes expressed neither c-src mRNA nor c-Src. However, when monocytes were incubated with anti-CD98 heavy chain (HC) mAb, expression of c-src mRNA, c-Src, and activated c-Src was induced. Many binding sites for the ubiquitous transcription factor Sp1 were identified in the promoter region of the c-src gene. Surprisingly, Sp1 and Sp1 mRNA were not found in monocytes that were freshly isolated or incubated with control antibody. Stimulation with anti-CD98HC mAb also resulted in the expression of Sp1 and its translocation to the nucleus. Herbimycin A, genistein, manumycin A, PD-98059, SB203580, and HBJ127 suppressed CD98HC-mediated c-src and Sp1 mRNA induction. On the contrary, H-7, Wortmannin, HA1077, and Y-27632 showed no effect on c-Src and Sp1 induction. Furthermore, anti-CD98HC mAb induced activation of tyrosine kinases and ERK kinases. These findings suggest that the tyrosine kinase(s)-Ras-MAPK-Sp1 pathway(s) is involved in CD98HC-mediated induction of c-Src in human blood monocytes.

Spindle-shaped cells derived from giant-cell tumor of bone support differentiation of blood monocytes to osteoclast-like cells.

Spindle-shaped cells were established from four giant-cell tumors of bone. When human blood monocytes were co-cultured with these cells, multinucleated giant-cell formation of monocytes was induced. Intriguingly, even when a filter (pore size: 0.45 microm) was interposed between monocytes and the spindle-shaped cells, polykaryocytes also appeared. These multinucleated giant cells were positive for tartrate-resistant acid phosphatase, expressed calcitonin receptor, and showed bone-resorption activity, characteristics of osteoclast-like cells. These findings indicate that soluble factors secreted from these cells play an important role in osteoclast-like cell formation from blood monocytes. These data additionally suggest that these cells support osteoclast-like cell formation in giant-cell tumors of bone. The cells also expressed mannose receptor, fibronectin, receptor activator of nuclear factorkappaB, and several cytokine mRNAs, including interleukin-6, receptor activator of nuclear factorkappaB ligand/osteoclast differentiation factor/osteoprotegerin ligand, and macrophage colony-stimulating factor. However, all of these molecules except receptor activator of nuclear factorkappaB ligand mRNA could also be detected in control HeLa and CV-1 cells. Although the soluble receptor activator of nuclear factorkappaB ligand has not been found under physiological conditions, it is possible that it is cleaved by cellular proteases and the truncated receptor activator of nuclear factorkappaB is released from cells. Identification of the soluble factors capable of inducing osteoclast formation from blood monocytes is a pressing problem to be solved.

N-glycosylation contributes to the limited cross-reactivity between hemagglutinin neuraminidase proteins of human parainfluenza virus type 4A and 4B.

cDNAs encoding human parainfluenza virus type 4B (hPIV-4B) hemagglutinin neuraminidase (HN) protein were cloned and the nucleotide sequences were determined. A high degree of identity (81.4%) was observed between the nucleotide sequences of hPIV-4A and -4B HN proteins, and an 87.3% identity was found between the deduced amino acid sequences. This degree of identity is considered to be greater than immunological similarity between hPIV-4A and -4B HN proteins determined using monoclonal antibodies. To elucidate the causes of the antigenic difference between HN proteins of hPIV-4A and -4B, we constructed three cDNAs of hPIV-4B HN whose potential N-glycosylation sites were partially or completely the same as in hPIV-4A HN cDNA. We compared the antigenicity of the expressed wild-type and mutant proteins, and found that the antigenicities of the mutant hPIV-4B HN proteins were more similar to the hPIV-4A HN protein than to the non-mutant hPIV-4B HN protein. This study indicated that the antigenic diversity between hPIV-4A and -4B was partly caused by deletion or creation of glycosylation sites, showing that the point mutations resulting in deletion or creation of glycosylation sites is one of the initial steps leading to the division of virus into subtypes.

Function of fusion regulatory proteins (FRPs) in immune cells and virus-infected cells.

Two molecules that regulate cell fusion have been identified and designated fusion regulatory protein-1 (FRP-1) and FRP-2. FRP-1 is a complex composed of a glycosylated heavy chain and a nonglycosylated light chain that are disulfide linked. FRP-1 heavy chain is identical to 4F2/CD98 heavy chain, whereas FRP-2 is identical to integrin alpha3 subunit. The FRP-1 heavy chain is a multifunctional molecule: that is, fusion regulator, amino acid transporter, integrin regulator, comitogenic factor, Na+-Ca2+ exchanger, oncogenic protein, and so on. Several aspects of the structure and function of the FRP-1 system are reviewed: fusion regulatory molecular mechanisms, cross-talk between the FRP-1 and integrin, the FRP-1 system as amino acid transporter, and FRP-1-mediated T-cell activation. The FRP-1 system is involved in virus-mediated cell fusion and multinucleated giant cell formation of blood monocytes. Monoclonal antibodies against human FRP-1 heavy chain induce polykaryocytes that have properties as osteoclasts. Multiple steps participate in molecular mechanisms regulating cell fusion. The FRP-1 heavy chain supports amino acid transport activity and the FRP-1 light chains have recently been cloned as amino acid transporters that require association with the heavy chain to exhibit their activity. Novel pathways for monocyte-dependent regulation of T-cell activation have recently been found that are mediated by the FRP-1 system. In conclusion, the FRP-1 molecules are essential factors for basic cellular functions.

Incomplete replication of human parainfluenza virus type 4 in LLC-MK2 cells and in L929 cells.

Human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B) were tested for their ability to replicate in the monkey kidney LLC-MK2 cell line (MK2 cells) and the murine L929 cell line (L929 cells). These cells are normally non-permissive for replication of hPIV-4; however, treatment with acetylated trypsin led to virus replication in MK2 cells, but was less effective for L929 cells. Endogenously produced interferon (IFN) played no role in virus replication in L929 cells. Synthesis of virus-specific polypeptides was suppressed in L929 cells. Whereas NP-mRNA and HN-mRNA were detected in MK2 cells, no HN-mRNA was detected in L929 cells. These results indicate that hPIV-4 can infect both MK2 cells and L929 cells. In MK2 cells, when protease exists in the extracellular medium, hPIV-4 exhibits multistep growth. In L929 cells, however, the cause of incomplete replication might be lack of other unknown factors.

Mapping of domains on the human parainfluenza type 2 virus P and NP proteins that are involved in the interaction with the L protein.

Eleven monoclonal antibodies (MAbs) directed against the large (L) protein of human parainfluenza type 2 virus (hPIV-2) were prepared to examine the interactions of the L protein with other viral proteins. Coimmunoprecipitation assays using these MAbs revealed that the L protein directly interacted with the phospho- (P) and nucleocapsid (NP) proteins in vivo and in vitro. Mutational analysis of the P or NP protein was performed to identify the region(s) on these proteins interacting with L protein, indicating that amino acids 278-353 on the P protein and amino acids 403-494 on the NP protein are essential for the binding to the L protein.

Ability of osteoclast formation from peripheral monocytes using anti-fusion regulatory protein-1/CD98/4F2 monoclonal antibodies in patients with osteoporosis.

We investigated the difference in osteoclast formation between patients with osteoporosis and two healthy control groups by inducing it from peripheral blood monocytes with use of anti-fusion regulatory protein- monoclonal antibody. The group of patients with osteoporosis consisted of 35 women and excluded secondary osteoporosis, and the control groups consisted of 12 young healthy volunteers (control I) or 10 individuals age-matched to the patients with osteoporosis (control II). Osteoclast formation declined with age between the two control groups, but this decline was not significant. Fusion rate and the mean number of nuclei in osteoclasts were significantly less in the patients with osteoporosis than in the young or age-matched controls. It was clearly demonstrated that the ability of monocytes to fuse declines significantly in patients with osteoporosis.

An amino acid in the heptad repeat 1 domain is important for the haemagglutinin-neuraminidase-independent fusing activity of simian virus 5 fusion protein.

A canine isolate (strain T1) of simian virus 5 (SV-5) performed multiple replication in BHK cells but did not induce cell fusion for up to 3 days. In contrast, a prototype strain (WR) provoked extensive cell fusion within 2 days during the course of its replication. Accordingly, the fusion (F) protein of the T1 strain did not cause cell fusion even when co-expressed with the SV-5 haemagglutinin-neuraminidase (HN) protein, whereas the WR F protein induced cell fusion in the presence of the HN protein. Differences in the predicted amino acid sequences of the T1 and WR F proteins were found at 12 positions and it was proved that the T1 F protein had a longer cytoplasmic tail than the WR F protein. By reducing the length of the cytoplasmic tail or by replacing the tail with the WR F counterpart, the T1 F protein partly restored its HN-dependent fusing activity. Chimeric and mutational analyses between the T1 F protein and the mutant F protein (L22P) suggested that Glu-132 in the heptad repeat 1 domain was involved in the HN-independent fusing activity in addition to the previously identified Pro-22 at the F(2) N terminus. It was also shown that Ala-290 in the heptad repeat 3 domain contributed to the HN-independent fusing activity to some extent.

Gene expression during osteoclast-like cell formation induced by antifusion regulatory protein-1/CD98/4F2 monoclonal antibodies (MAbs): c-src is selectively induced by anti-FRP-1 MAb.

Human blood monocytes can differentiate into osteoclast-like cells when they are cultured in the presence of anti-FRP-1. Messenger (mRNA) expression of markers related to osteoclasts was analyzed during differentiation of osteoclasts from monocytes. As markers related to osteoclasts, we selected cathepsin-K, carbonic anhydrase (CA) II, vacuolar H(+)-ATPase (v-ATPase), vitronectin receptor (VNR), tartrate-resistant acid phosphatase (TRAP), osteopontin (OPN), galectin-3, c-src, c-fos, and c-fms. The mRNAs other than c-src mRNA were expressed in freshly isolated monocytes or monocytes incubated with control antibody or anti-FRP-1 monoclonal antibody (MAb) for 14 days. Of these mRNAs, cathepsin-K, CA II, v-ATPase, VNR, TRAP, OPN, and c-fms mRNAs were expressed at higher levels in the osteoclast-like cells than those in monocytes cultured with control antibody. On the other hand, galectin-3 mRNA was expressed at lower levels in the osteoclast-like cells, and there was no significant difference in c-fos mRNA expression between the monocytes cultured with control antibody and anti-FRP-1 MAb. c-src mRNA could not be detected in monocytes freshly isolated or incubated with control antibody. Surprisingly, expression of c-src mRNA was induced in monocytes by anti-FRP-1 MAb and was detectable as early as 3 h after anti-FRP-1 MAb treatment, indicating that c-src is selectively induced by anti-FRP-1 MAb treatment. Furthermore, the osteoclast-like cells expressed calcitonin receptor. Receptor activator of NF-kappaB (RANK) mRNA was detectable in freshly isolated monocytes or monocytes cultured with control antibody or anti-FRP-1 MAbs. Maximal expression of RANK was observed in osteoclast-like cells. On the other hand, no receptor activator of NF-KB ligand (RANKL) mRNA was detectable in any of the samples, suggesting that anti-FRP-1 mAb can induce osteoclast-like cells from blood monocytes without RANKL.

Suppression of FRP-1/CD98-mediated multinucleated giant cell and osteoclast formation by an anti-FRP-1/CD98 mAb, HBJ 127, that inhibits c-src expression.

When anti-CD98 mAb 6-1-13, 4-5-1, or 38-2-2 was added to the culture fluids of monocytes, extensive cell aggregation and polykaryocyte formation were induced. These multinucleated giant cells were tartrate-resistant acid phosphatase (TRAP) positive. On the other hand, when monocytes were incubated with another anti-CD98 mAb, HBJ 127, polykaryocyte formation was not detected, although extensive cell aggregation was induced. When HBJ 127 and 6-1-13 were simultaneously added to the culture fluids, anti-CD98 mAb-induced cell fusion was inhibited almost completely. HBJ 127 suppressed formation of 6-1-13-induced cell fusion in a dose-dependent manner. If, however, HBJ 127 was added after incubation of monocytes with mAb 6-1-13 for 6 h, an appreciable degree of TRAP-positive polykaryocyte formation was found. The bindings of 6-1-13 and HBJ 127 were not mutually competed. When monocytes were incubated with 6-1-13 or HBJ 127, 6-1-13 induced c-src mRNA, while HBJ 127 did not. Furthermore, when monocytes were incubated with both 6-1-13 and HBJ 127, c-src mRNA could not be detected, showing that HBJ 127 suppresses c-src expression. Therefore, CD98-mediated osteoclast formation can be regulated by modification of CD98 system.

Isolation and characterization of monoclonal antibodies directed against murine FRP-1/CD98/4F2 heavy chain: murine FRP-1 is an alloantigen and amino acid change at 129 (P<-->R) is related to the alloantigenicity.

Nineteen mAb directed against murine fusion regulatory protein-1 (mFRP-1)/4F2/CD98 were isolated and their biological properties were analysed. Intriguingly, mFRP-1 was found to be an alloantigen, namely, FRP-1.1 (DBA/2 and CBA mice type) and FRP-1.2 (BALB/c, C57BL/6 and C3H/He mice type). The nucleotide sequences of FRP-1.1 and FRP-1.2 were determined, demonstrating that amino acid change at 129 (P<-->R) is related to the alloantigenicity. mFRP-1 is expressed on thymocytes, on spleen cells, on peripheral lymphocytes and on blood monocytes, suggesting that the physiological role in vivo of murine FRP-1 is different from that of human FRP-1. The biological activities of antimFRP-1 mAbs showed by the present study are: (i) enhancement of Newcastle disease virus-induced cell fusion; (ii) suppression of HIVgp160-mediated cell fusion; and (iii) induction of aggregation and multinucleated giant cells of monocytes/macrophages.

Cutting edge: primary structure of the light chain of fusion regulatory protein-1/CD98/4F2 predicts a protein with multiple transmembrane domains that is almost identical to the amino acid transporter E16.

The CD98 light chain (CD98LC) was copurified from HeLa S3 cells by an affinity chromatography using a mAb specific for the fusion regulatory protein-1 (FRP-1) which is identical to the CD98 heavy chain. On the basis of the N-terminal sequence (63 amino acids) of purified CD98LC polypeptide, we have cloned a PCR fragment (155 bp) from a HeLa S3 cDNA library and finally obtained a full cDNA clone encoding the CD98LC. Fluorescence in situ hybridization analysis using the cDNA assigned the CD98LC gene to the long arm of human chromosome 16 (16q24). The predicted amino acid sequence suggested that CD98LC is a protein with multiple transmembrane domains and is almost identical to the amino acid transporter E16. Resting monocytes and lymphocytes expressed CD98LC as analyzed by a newly isolated anti-CD98LC mAb, which showed cross-reactivity with insect Sf9 cells as well as with various mammalian cell lines.