Attachment of Madin-Darby canine kidney cells to extracellular matrix: role of a laminin binding protein related to the 37/67 kDa laminin receptor in the development of plasma membrane polarization.

Madin-Darby canine kidney (MDCK) cells have been extensively used as a model for the study of epithelial polarization. The contacts between the cell and extra-cellular matrix (ECM) provide a signal for the polarization of apical membrane markers. In order to study the molecular basis of these contacts, MDCK cells extracts in Triton X-100 were affinity-purified on laminin, yielding polypeptides of 100-110 and 36 kDa, but only the second one could be enzymatically iodinated from the cell surface. This protein was also recognized by an antibody against the 37/67-kDa laminin/elastin family of proteins. Different polypeptides were purified by the same method on type I collagen. An antibody developed against the polypeptides purified on laminin recognized also a 67-kDa protein, blocked 125I-laminin binding to a population of high affinity (1.5 nM KD) binding sites and caused a significant decrease in cell attachment and spreading to laminin or endogenous ECM. This antibody did not interfere with MDCK cell attachment to fibronectin or collagen matrices, but still impaired cell spreading. An apical MDCK plasma membrane protein (184 kDa), fully polarized in untreated cells, was partially mispolarized after treatment with anti-36 kDa antibody. These results are consistent with a model of various ECM receptors operating together in these cells, and show an important role of a non-integrin 36-kDa laminin binding protein related to the 67-kDa laminin receptor family in cell attachment, spreading and polarization.     

Identification of two surface proteins from C6/36 cells that bind dengue type 4 virus.

Dengue viruses infect cells by attaching to a surface receptor, probably through the envelope (E) glycoprotein, located on the surface of the viral membrane. However, the identity of the dengue virus receptor in the mosquito and in mammalian host cells remains unknown. To identify and characterize the molecules responsible for binding dengue virus, overlay protein blot and binding assays were performed with labeled virus. Two glycoproteins of 40 and 45 kDa located on the surface of C6/36 cells bound dengue type 4 virus. Virus binding by total and membrane proteins obtained from trypsin-treated cells was inhibited, while neuraminidase treatment did not inhibit binding. Periodate treatment of cell proteins did not reduce virus binding, but it modified the molecular weight of the polypeptide detected by overlay assays. Preincubation of C6/36 cells with electroeluted 40- and 45-kDa proteins or with specific antibodies raised against these proteins inhibited virus binding. These results strongly suggest that the 40- and 45-kDa surface proteins are putative receptors or part of a receptor complex for dengue virus.     

The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor.     

Serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor

Dengue virus, the causative agent of dengue fever, dengue shock syndrome, and dengue hemorrhagic fever, infects susceptible cells by initially binding to a receptor(s) located on the host cell surface. Evidence to date suggests that receptor usage may be cell and serotype specific, and this study sought to identify dengue virus serotype 1 binding proteins on the surface of liver cells, a known target organ. By using a virus overlay protein binding assay (VOPBA), in both nondenaturing and denaturing gel systems, a putative dengue virus serotype 1 binding protein of approximately 37 kDa expressed on the surface of liver (HepG2) cells was identified. Mass spectrometry analysis identified a candidate protein, the 37/67-kDa high-affinity laminin receptor. Entry of the dengue virus serotype 1 was significantly inhibited in a dose-dependent manner by both antibodies directed against the 37/67-kDa high-affinity laminin receptor and soluble laminin. No inhibition of virus entry was seen with dengue virus serotypes 2, 3, or 4, demonstrating that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells.     

siRNA-mediated silencing of the 37/67-kDa high affinity laminin receptor in Hep3B cells induces apoptosis

The laminin-binding protein, variously called the 37/67-kDa high affinity laminin receptor or p40, mediates the attachment of normal cells to the laminin network, and also has a role as a ribosomal protein. Over-expression of this protein has been strongly correlated with the metastatic phenotype. However, few studies have investigated the cellular consequence of the ablation of this gene's expression. To address this issue, the expression of the 37/67-kDa high affinity laminin receptor was knocked out with several siRNA constructs via RNA interference in transformed liver (Hep3B) cells. In each case where the message was specifically ablated, apoptosis was induced, as determined by annexin V/propidium iodide staining, and by double staining with annexin V and an antibody directed against the 37/67-kDa high affinity laminin receptor. These results suggest that this protein plays a critical role in maintaining cell viability.     

Identification of a putative coreceptor on Vero cells that participates in dengue 4 virus infection

Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (alpha-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.     

High-affinity laminin receptor is a receptor for Sindbis virus in mammalian cells.

Sindbis virus is an alphavirus with a very wide host range, being able to infect many birds and mammals as well as mosquitoes. We have isolated a monoclonal antibody that largely blocks virus binding to mammalian cells. This antibody was found to be directed against the C-terminal domain of the high-affinity laminin receptor, a 67-kDa protein present on the cell surface that binds with high affinity to basement membrane laminin and that is known to be important in development and in tumor invasion. This receptor is believed to be formed from a 295-amino-acid polypeptide that is modified in some unknown way after translation. The primary sequence of this 295-amino-acid protein is highly conserved among mammals. We found the hamster amino acid sequence to be identical to a mouse sequence and to differ at only two amino acids from a human sequence and at two amino acids from a bovine sequence. To verify the importance of the laminin receptor for infection by Sindbis virus, hamster cells were stably transfected with the gene encoding the 295-amino-acid protein under the control of a high-efficiency promoter. Such transfected hamster cells overexpressed the laminin receptor at the cell surface, bound severalfold more Sindbis virions than did the parental cells, and became infected by Sindbis virus with a higher efficiency. In contrast, cells transfected with the antisense gene expressed less laminin receptor on the surface and were less susceptible to the virus. Binding of the virus varied linearly with the amount of laminin receptor on the cell surface, whereas infectivity measured with a plaque assay varied with the 1.4 power of the receptor concentration, suggesting that interaction with more than one receptor aids virus penetration. By these criteria, the laminin receptor functions as the major receptor for Sindbis virus entry into mammalian cells. We also found that the anti-laminin receptor antibody partially blocked Sindbis virus binding to mosquito cells, suggesting that the laminin receptor is conserved in mosquitoes and functions as a Sindbis virus receptor in this host. The wide distribution of this highly conserved receptor may be in part responsible for the broad host range exhibited by the virus, which infects a wide range of mammals and birds as well as its mosquito vector and can infect many different tissues within these hosts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Midkine binds to 37-kDa laminin binding protein precursor, leading to nuclear transport of the complex

Midkine (MK) is a heparin binding multifunctional protein that promotes cell survival and cell migration. MK was found to bind to 37-kDa laminin binding protein precursor (LBP), a precursor of 67-kDa laminin receptor, with K(d) of 1.1 nM between MK and LBP-glutathione-S-transferase fusion protein. The binding was inhibited by laminin, anti-LBP, amyloid beta-peptide, and heparin; the latter two are known to bind to MK. In CMT-93 mouse rectal carcinoma cells, LBP was mostly located in the cytoplasm as revealed by immunostaining with anti-LBP antibody. That a portion of LBP or 67-kDa laminin receptor was located at the surface of these cells was verified by inhibition of cell attachment to laminin-coated dishes by anti-LBP antibody. When MK was added to culture medium of these cells, a part of LBP migrated to the nucleus. The movement occurred concomitantly with nuclear transport of biotin-labeled MK. These findings suggested that the binding of MK to LBP caused nuclear translocation of the molecular complex.     

Structural and functional characterization of a cell surface binding protein of vaccinia virus

The nature of the interaction between the enveloped DNA-containing poxviruses and the surfaces of host cells as a first step in virus infection is not known. In this investigation we have identified and defined structural and functional properties of a 32-kDa protein of vaccinia virus. This protein is part of the virus envelope and binds to the cell surface of various cultured cells. The gene encoding the 32-kDa viral protein was mapped and sequenced. It was found to code a 35,426-Da protein with a large N-terminal domain with sequence homology to carbonic anhydrases and a C-terminal domain with sequences similar to those of the attachment glycoprotein VP7 of rotavirus and to transmembrane proteins. A potential cell surface binding domain was within the last 50 amino acid residues of the C terminus. The 32-kDa protein is basic, predicted pI 8.67, is synthesized at late times post-infection, may form dimers held by disulfide bonds at the single cysteine 262, and is apparently non-glycosylated. The 32-kDa protein is a vaccinia virus antigen, with predicted antigenic sites located near amino acids 108-110 (carbonic anhydrase domain) and 298-299 (transmembrane domain). Several lines of evidence suggest that the 32-kDa protein is needed for efficient virus replication in cultured cells but that in addition to this protein other viral proteins are involved in the process of virus entry into cells.     

Cellular attachment to thrombospondin. Cooperative interactions between receptor systems.

Tumor cell attachment to thrombospondin (TSP) in the extracellular matrix may be of critical importance in the processes of invasion and hematogenous dissemination. To determine the specific receptor systems that mediate the interaction of tumor cells with insoluble TSP, the attachment of HT1080 fibrosarcoma and C32 and G361 melanoma cells to TSP-coated discs was studied in the presence of heparin, Arg-Gly-Asp-Ser, or antibodies to glycoprotein (GP) IV (CD36, GPIIIb), a TSP receptor. HT1080 and C32 cell attachment to TSP was inhibited by the combination of heparin and a monoclonal (or polyclonal) antibody to GPIV but not by either alone. Heparin alone inhibited cell spreading. Neither control monoclonal antibodies nor the cell attachment peptide Arg-Gly-Asp-Ser inhibited tumor cell attachment to TSP, alone or in the presence of heparin. HT1080 cells attached equally as well to a 140-kDa proteolytic TSP fragment lacking the heparin-binding domain as to intact TSP. A monoclonal antibody to GPIV alone inhibited tumor cell attachment to the heparin-domainless 140-kDa TSP fragment. No attachment to the heparin-binding fragment was observed, but the addition of the heparin fragment to 140-kDa heparin-domainless TSP restored the heparin sensitivity of binding. G361 cells that lack GPIV attached well to TSP but were not inhibited by heparin or anti-GPIV alone or in combination. The combination of heparin and Arg-Gly-Asp-Ser inhibited G361 attachment to TSP. These studies suggest that tumor cells may utilize separate receptor systems in a cooperative manner to adhere to TSP. HT1080 fibrosarcoma and C32 melanoma cells utilize GPIV in concert with a heparin-modulated binding systems to attach and spread on TSP. G361 cells, which lack GPIV expression, attach and spread on TSP using an integrin system as well as a heparin-modulated system.     

In vivo expression of the 25-kDa laminin-binding protein of Helicobacter pylori

The gastroduodenal pathogen Helicobacter pylori has been shown to inhibit the interaction between the extracellular matrix protein laminin and its receptor on gastric epithelial cells, potentially contributing to a loss of mucosal integrity. As a 25-kDa outer membrane protein of H. pylori in association with the bacterial lipopolysaccharides (LPS) mediates attachment to laminin, the aim of this study was to determine whether the 25-kDa protein is produced by H. pylori in infected hosts. We examined the immune response to the 25-kDa laminin binding protein in 12 paediatric patients; samples from a H. pylori-negative healthy adult were used as controls. In immunoblotting, antibodies to a 25-kDa protein were found in the serum and saliva of H. pylori-positive individuals only, and using the positive sera and saliva, laminin binding to the 25-kDa protein was inhibited. Thus, the 25-kDa laminin-binding protein is produced by H. pylori in infected hosts.     

Growth suppression of Friend virus-transformed erythroleukemia cells by p53 protein is accompanied by hemoglobin production and is sensitive to erythropoietin.

The murine allele temperature-sensitive (ts) p53Val-135 encodes a ts p53 protein that behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. This ts allele was introduced into the p53 nonproducer Friend erythroleukemia cell line DP16-1. The DP16-1 cell line was derived from the spleen cells of a mouse infected with the polycythemia strain of Friend virus, and like other erythroleukemia cell lines transformed by this virus, it grows independently of erythropoietin, likely because of expression of the viral gp55 protein which binds to and activates the erythropoietin receptor. When incubated at 32 degrees C, DP16-1 cells expressing ts p53Val-135 protein, arrested in the G0/G1 phase of the cell cycle, rapidly lost viability and expressed hemoglobin, a marker of erythroid differentiation. Erythropoietin had a striking effect on p53Val-135-expressing cells at 32 degrees C by prolonging their survival and diminishing the extent of hemoglobin production. This response to erythropoietin was not accompanied by down-regulation of viral gp55 protein.     

Expression of a human cytomegalovirus receptor correlates with infectibility of cells.

Previous studies have demonstrated that human cytomegalovirus (HCMV) specifically binds to a fibroblast membrane glycoprotein(s) with a molecular mass from 30 to 34 kDa. In this study, the distribution of the putative receptor proteins was analyzed in a variety of cell types, including cell types representative of those that are infected in vivo. Using a sensitive microbinding assay (to score virus attachment) and an indirect detection method (to score HCMV-binding proteins), we found that the 34- and 32-kDa HCMV binding proteins are ubiquitous molecules, broadly distributed among diverse cell types. In addition, the level of virus attachment was found to correlate with the abundance of the 34- and 32-kDa cellular proteins, while the ability of the virus to penetrate cells and initiate infection did not. The results support the hypothesis that the 34- and 32-kDa cellular proteins represent the HCMV (attachment) receptor. The data also support the notion that additional cellular components are required for virus entry and fusion.     

The Expression of Membrane-Associated 67-kDa Laminin Receptor (67LR) Is Modulated in Vitro by Cell-Contact Inhibition

The interaction of cells with their substratum is an important determinant of cell behaviour, influencing attachment, proliferation, and motility. Such interactions are mediated by cell surface receptors which bind to attachment factors, like the glycoprotein laminin in basement membranes. We have previously shown that expression of the 67-kDa laminin receptor (67LR) is elevated in proliferating retinal microvasculature compared with mature, quiescent vessels. Here, we examined 67LR mRNA and protein expression in primary cultures of retinal microvascular endothelial cells (RMEC) and in the breast cancer cell-line T47D during stages of contact inhibition. In both cell types, the expression levels of 67LR mRNA and membrane-associated 67LR protein were significantly increased during the proliferative phases of monolayer formation. As the cells achieved contact inhibition, 67LR expression was reduced to comparatively low levels. Thus, the differential expression of 67LR between dividing and contact-inhibited cells may indicate a role for this receptor during proliferative processes.     

Inhibition of murine melanoma cell-matrix adhesion and experimental metastasis by albolabrin, an RGD-containing peptide isolated from the venom of Trimeresurus albolabris

Albolabrin, a 7.5-kDa cysteine-rich protein isolated from the venom of Trimeresurus albolabris, contains the arginine-glycine-aspartic acid (RGD) cell recognition sequence found in many cell adhesion-promoting extracellular matrix proteins, such as fibronectin and laminin. Albolabrin belongs to a family of RGD-containing peptides, termed disintegrins, recently isolated from the venom of various vipers and discovered to be potent inhibitors of both platelet aggregation and cell-substratum adhesion. Here we report that albolabrin inhibited the attachment of B16-F10 mouse melanoma cells to either fibronectin or laminin absorbed on plastic. When immobilized on plastic, albolabrin promoted B16-F10 melanoma cell attachment; this was inhibited by either RGD-serine (RGDS) or antibodies to integrins, suggesting that albolabrin binds via its RGD amino sequence to integrin receptors expressed on the melanoma cell surface. In an in vivo experimental metastasis system, albolabrin at a concentration of 300-600 nM inhibited C57BL/6 mouse lung colonization by tail vein-injected mouse melanoma cells and was at least 2000 times more active than RGDS in this assay. We propose that albolabrin inhibits tumor cell metastasis by inhibiting integrin-mediated attachment of melanoma cells to RGD-containing components of the extracellular matrix in the mouse lung.     

The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein.

Recently, we identified the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP). Here, we show the presence of the 37-kDa LRP and its mature 67-kDa form termed high-affinity laminin receptor (LR) in plasma membrane fractions of N2a cells, whereas only the 37-kDa LRP was detected in baby hamster kidney (BHK) cells. PrP co-localizes with LRP/LR on the surface of N2a cells and Semliki Forest virus (SFV) RNA transfected BHK cells. Cell-binding assays reveal the LRP/LR-dependent binding of cellular PrP by neuronal and non-neuronal cells. Hyperexpression of LRP on the surface of BHK cells results in the binding of exogenous PrP. Cell binding is similar in PrP(+/+) and PrP(0/0) primary neurons, demonstrating that PrP does not act as a co-receptor of LRP/LR. LRP/LR-dependent internalization of PrP is blocked at 4 degrees C. Secretion of an LRP mutant lacking the transmembrane domain (aa 86-101) from BHK cells abolishes PrP binding and internalization. Our results show that LRP/LR acts as the receptor for cellular PrP on the surface of mammalian cells.     

Modulation of growth factor induced fiber outgrowth in rat pheochromocytoma (PC12) cells by a fibronectin receptor antibody

Rat pheochromocytoma PC12 cells respond to the binding of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) by extending neurites in a manner resembling sympathetic neurons. This response requires cell attachment to an appropriate substratum (Fujii et al., J. Neurosci., 2:1157, 1982); attachment factors which function in this capacity include the adhesive proteins fibronectin and laminin. Incubating PC12 cells with a polyclonal antiserum directed against a putative 140-kDa fibroblast cell surface fibronectin receptor (anti-gp140) perturbed spreading but not attachment of the cells to fibronectin and laminin substrates. However, in the presence of anti-gp 140 or its Fab fragments, NGF-stimulated neurite outgrowth was dramatically reduced. The antibody also caused a retraction of previously extended neurites. SDS-PAGE analysis of immunoprecipitates of PC12 cells surface labeled with 125I identified a prominent 120-140-kDa band, suggesting that the site of anti-gp140 action in PC12 cells is also through a fibronectin receptor.     

Insertional inactivation of the vaccinia virus 32-kilodalton gene is associated with attenuation in mice and reduction of viral gene expression in polarized epithelial cells.

The mechanism of poxvirus attachment to cells is poorly understood. We have identified a 32-kDa envelope protein of vaccinia virus which binds to the surface of cultured cells. This binding is specific and selective (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:22174-22180, 1990; C. Lai, S. Gong, and M. Esteban, J. Virol. 65:499-504, 1991). In this investigation, we studied the effect of inactivating the 32-kDa gene (32K gene) on the biology of vaccinia virus. We show that inactivation of the 32K gene decreases by 80% the mortality of mice infected with 32K- vaccinia virus. This reduction in mortality correlates with diminished viral gene expression in target tissues. In highly polarized epithelial cells, viral gene expression of 32K- virus was reduced (50 to 60%) at both the apical and basolateral surfaces in comparison with a 32K+ virus. Restriction of virus gene expression in polarized cell surfaces occurs for both intracellular and extracellular forms of infectious 32K- vaccinia virus. The two infectious forms of vaccinia virus 32K+ infect polarized cells preferentially by the basolateral surface. Our findings provide evidence of the importance of the 32-kDa protein in viral pathogenesis.     

A null mutation in the UL36 gene of herpes simplex virus type 1 results in accumulation of unenveloped DNA-filled capsids in the cytoplasm of infected cells

The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated KDeltaUL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with KDeltaUL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of KDeltaUL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In KDeltaUL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in KDeltaUL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.     

Rift Valley Fever Virus Incorporates the 78 kDa Glycoprotein into Virions Matured in Mosquito C6/36 Cells

Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment coding for four proteins: nonstructural NSm protein, two glycoproteins Gn and Gc and large 78 kDa glycoprotein (LGp) of unknown function. Goat anti-RVFV polyclonal antibody and mouse monoclonal antibody, generated against a polypeptide unique to the LGp within the RVFV proteome, detected this protein in gradient purified RVFV ZH501 virions harvested from mosquito C6/36 cells but not in virions harvested from the mammalian Vero E6 cells. The incorporation of LGp into the mosquito cell line - matured virions was confirmed by immune-electron microscopy. The LGp was incorporated into the virions immediately during the first passage in C6/36 cells of Vero E6 derived virus. Our data indicate that LGp is a structural protein in C6/36 mosquito cell generated virions. The protein may aid the transmission from the mosquitoes to the ruminant host, with a possible role in replication of RVFV in the mosquito host. To our knowledge, this is a first report of different protein composition between virions formed in insect C6/36 versus mammalian Vero E6 cells.