Baculovirus Envelope Protein Research Articles

Identification of Cellular Genes Involved in Baculovirus GP64 Trafficking to the Plasma Membrane.

The baculovirus envelope protein GP64 is an essential component of the budded virus and is necessary for efficient virion assembly. Little is known regarding intracellular trafficking of GP64 to the plasma membrane, where it is incorporated into budding virions during egress. To identify host proteins and potential cellular trafficking pathways that are involved in delivery of GP64 to the plasma membrane, we developed and characterized a stable Drosophila cell line that inducibly expresses the AcMNPV GP64 protein and used that cell line in combination with a targeted RNA interference (RNAi) screen of vesicular protein trafficking pathway genes. Of the 37 initial hits from the screen, we validated and examined six host genes that were important for trafficking of GP64 to the cell surface. Validated hits included Rab GTPases Rab1 and Rab4, Clathrin heavy chain, clathrin adaptor protein genes AP-1-2β and AP-2μ, and Snap29. Two gene knockdowns (Rab5 and Exo84) caused substantial increases (up to 2.5-fold) of GP64 on the plasma membrane. We found that a small amount of GP64 is released from cells in exosomes and that some portion of cell surface GP64 is endocytosed, suggesting that recycling helps to maintain GP64 at the cell surface. IMPORTANCE While much is known regarding trafficking of viral envelope proteins in mammalian cells, little is known about this process in insect cells. To begin to understand which factors and pathways are needed for trafficking of insect virus envelope proteins, we engineered a Drosophila melanogaster cell line and implemented an RNAi screen to identify cellular proteins that aid transport of the model baculovirus envelope protein (GP64) to the cell surface. For this we developed an experimental system that leverages the large array of tools available for Drosophila and performed a targeted RNAi screen to identify cellular proteins involved in GP64 trafficking to the cell surface. Since viral envelope proteins are often critical for production of infectious progeny virions, these studies lay the foundation for understanding how either pathogenic insect viruses (baculoviruses) or insect-vectored viruses (e.g., flaviviruses, alphaviruses) egress from cells in tissues such as the midgut to enable systemic virus infection.

Interaction with the Receptor SLAM and Baculovirus Surface Display of Peste des petits ruminants Virus Hemagglutinin.

Peste des petits ruminants (PPR) is an acute, highly infectious, and highly pathogenic disease, which mainly damages small ruminants such as goats and sheep. Hemagglutinin protein (H), the main antigenic protein of peste des petits ruminants virus (PPRV), has been a hot spot in the research of genetic engineering vaccine for PPRV. In this study, the silkworm baculovirus surface display technology is combined with the transmembrane structure of the silkworm baculovirus envelope protein GP64 and different characteristics of the promoters to display four kinds of fusion proteins, which contain Pph-H, Pph-HJ, Pie1-H, and Pie1-HJ. The fusion proteins displayed on baculovirus surface have been detected by western blotting, cell surface immunofluorescence, and immunogold electron microscopy. In addition, the dominant form of PPR H displayed on baculovirus surface has been determined which is fusion protein mediated by Pph containing the hemagglutinin protein and full-length GP64, Pph-H. Furthermore, by comparing the fluorescence intensity of binding of hemagglutinin protein and signaling lymphocyte activation molecules (SLAM) in Vero-SLAM cells by immunocytochemistry, Pph-H can be combined with the receptor protein of PPRV, SLAM. It provides technical support for displaying the different structure of hemagglutinin and exploring the key sites of hemagglutinin and SLAM binding. Meanwhile, it is important for exploring the pathogenesis and immune mechanism of PPRV.

Novel GP64 envelope variants for improved delivery to human airway epithelial cells.

Lentiviral vectors pseudotyped with the baculovirus envelope protein GP64 transduce primary cultures of human airway epithelia (HAE) at their apical surface. Our goal in this study was to harness a directed evolution approach to develop a novel envelope glycoprotein with increased transduction properties for HAE. Using error-prone PCR, a library of GP64 mutants was generated and used to prepare a diverse pool of lentiviral virions pseudotyped with GP64 variants. The library was serially passaged on HAE and three GP64 mutations were recovered. Single-, double- and the triple-combination mutant envelope glycoproteins were compared with wild-type GP64 for their ability to transduce HAE. Our results suggest that lentiviral vectors pseudotyped with evolved GP64 transduced HAE with greater efficiency than wild-type GP64. This effect was not observed in primary cultures of porcine airway epithelial cells, suggesting that the directed evolution protocol was species specific. In summary, our studies indicate that serial passage of a GP64 mutant library yielded specific variants with improved HAE cell tropism, yielding tools with the potential to improve the success of gene therapy for airway diseases.

The crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein

Chondroitin lyases have been known as pathogenic bacterial enzymes that degrade chondroitin. Recently, baculovirus envelope protein ODV-E66 was identified as the first reported viral chondroitin lyase. ODV-E66 has low sequence identity with bacterial lyases at <12%, and unique characteristics reflecting the life cycle of baculovirus. To understand ODV-E66’s structural basis, the crystal structure was determined and it was found that the structural fold resembled that of polysaccharide lyase 8 proteins and that the catalytic residues were also conserved. This structure enabled discussion of the unique substrate specificity and the stability of ODV-E66 as well as the host specificity of baculovirus.

Crystallization and X-ray diffraction analysis of chondroitin lyase from baculovirus: envelope protein ODV-E66

Baculovirus envelope protein ODV-E66 (67-704), in which the N-terminal 66 amino acids are truncated, is a chondroitin lyase. It digests chondroitin and chondroitin 6-sulfate efficiently, but does not digest chondroitin 4-sulfate. This unique characteristic is useful for the preparation of specific chondroitin oligosaccharides and for investigation of the mechanism of baculovirus infection. ODV-E66 (67-704) was crystallized; the crystal diffracted to 1.8 Å resolution and belonged to space group P6(2) or P6(4), with unit-cell parameters a = b = 113.5, c = 101.5 Å. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.54 Å(3) Da(-1).

English

In the present study, one recombinant baculovirus BacSC-GP5, expressing His6-tagged GP5 with the&nbsp;transmembrane domain (TM) and cytoplasmic domain (CTD) derived from baculovirus envelope protein gp64, was constructed&nbsp;and&nbsp;its&nbsp;immunogenicity&nbsp;andprotective efficiency&nbsp;was evaluated in&nbsp;piglets. The results obtained show that, His6-tagged recombinant GP5 was expressed and anchored on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. Immunogold electron microscopy demonstrated that, the GP5 glycoprotein was displayed successfully on the viral surface.Piglets immunized with BacSC-GP5 induced successfully GP5-specific enzyme-linked immunosorbent assay (ELISA) antibody, neutralizing antibody and lymphocyte proliferation response at 6 weeks after primary immunization. An&nbsp;in vivo&nbsp;challenge result indicated that piglets immunized with BacSC-GP5 did not show any obvious clinical signs and histological changes, and&nbsp;the&nbsp;quantitative&nbsp;real-time polymerase chain reaction (RT-PCR) also&nbsp;indicated that&nbsp;the&nbsp;porcine reproductive and respiratory syndrome virus (PRRSV)&nbsp;viral load from the serum in&nbsp;BacSC-GP5 group&nbsp;was significantly reduced at 14 and 21 days post-challenge&nbsp;compared to that in the negative control group. These results indicate that baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccine against PRRSV infection. &nbsp; Key words:&nbsp;Baculovirus surface display,&nbsp;porcine reproductive and respiratory syndrome virus (PRRSV), GP5 protein,&nbsp;immunogenicity, protective efficacy.

Baculovirus Envelope Protein ODV-E66 Is a Novel Chondroitinase with Distinct Substrate Specificity

Chondroitin sulfate is a linear polysaccharide of alternating D-glucuronic acid and N-acetyl-D-galactosamine residues with sulfate groups at various positions of the sugars. It interacts with and regulates cytokine and growth factor signal transduction, thus influencing development, organ morphogenesis, inflammation, and infection. We found chondroitinase activity in medium conditioned by baculovirus-infected insect cells and identified a novel chondroitinase. Sequence analysis revealed that the enzyme was a truncated form of occlusion-derived virus envelope protein 66 (ODV-E66) of Autographa californica nucleopolyhedrovirus. The enzyme was a novel chondroitin lyase with distinct substrate specificity. The enzyme was active over a wide range of pH (pH 4-9) and temperature (30-60 °C) and was unaffected by divalent metal ions. The ODV-E66 truncated protein digested chondroitin most efficiently followed by chondroitin 6-sulfate. It degraded hyaluronan to a minimal extent but did not degrade dermatan sulfate, heparin, and N-acetylheparosan. Further analysis using chemo-enzymatically synthesized substrates revealed that the enzyme specifically acted on glucuronate residues in non-sulfated and chondroitin 6-sulfate structures but not in chondroitin 4-sulfate structures. These results suggest that this chondroitinase is useful for detailed structural and compositional analysis of chondroitin sulfate, preparation of specific chondroitin oligosaccharides, and study of baculovirus infection mechanism.

Baculovirus Virions Displaying Infectious Bursal Disease Virus VP2 Protein Protect Chickens Against Infectious Bursal Disease Virus Infection

Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections.

Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.

Baculovirus surface display of E envelope glycoprotein of Japanese encephalitis virus and its immunogenicity of the displayed proteins in mouse and swine models

Japanese encephalitis virus (JEV), an important pathogen in humans and animals, is capable of causing febrile syndrome, encephalitis and death. The E glycoprotein of JEV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. In this work, we have succeeded in construction of one recombinant baculovirus BacSC-E expressing His6-tagged E with the baculovirus envelope protein gp64 TM and CTD. After infection, E was expressed and anchored on the plasma membrane of Sf-9 cells, as demonstrated by Western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the E glycoprotein was successfully displayed on the viral surface. Vaccination of mouse and swine with recombinant baculovirus BacSC-E successfully induced neutralizing antibody response and protective immunity toward a lethal challenge of the JEV. Taken all findings together, our results indicate that the recombinant baculovirus BacSC-E can be a potential vaccine against JEV infections. This finding provides valuable information for establishing subunit vaccines for JEV antigenic complex viruses. This is a fresh research demonstrating the potential of E-pseudotyped baculovirus as a JEV vaccine.

Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64

A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.

Surface display of IgG Fc on baculovirus vectors enhances binding to antigen-presenting cells and cell lines expressing Fc receptors

Recombinant baculoviruses (recBV) were constructed with dual cassettes for constitutive expression of human IgG Fc following infection of insect cells and the structural proteins of hepatitis C virus (core, E1 and E2) following transduction of mammalian cells. The IgG Fc was expressed in insect cells as a fusion protein with the signal sequence and transmembrane region of either the native baculovirus envelope protein gp64 or the human transferrin receptor as a type I or type II integral membrane protein, respectively. The IgG Fc fusion proteins formed functional homodimers on the surface of recBV-infected insect cells and were incorporated into the envelope of recBV particles during egress from the infected cell. Both pseudotyped recBV bound specifically to recombinant soluble FcgammaRIIalpha receptor and to cell lines and antigen-presenting cells expressing Fc receptors (FcRs). These novel baculoviral vectors, which target cells of the immune system that express FcRs, have potential applications for vaccination or gene therapy.

Baculovirus surface display of NS3 nonstructural protein of classical swine fever virus

Classical swine fever virus (CSFV) causes significant losses in the pig industry in many countries. NS3 proteins of CSFV, which include serine protease and RNA helicase/nucleotide triphosphatase (NTPase) activities, are multifunctional proteins involved in polyprotein processing and viral replication. Previous reports showed that NS3 protein can induce apoptosis in host cells that present cytopathic effects (CPE). Baculovirus/insect cell systems are used widely for recombinant protein production. In this study, one recombinant baculovirus BacSC-NS3 expressing histidine-tagged NS3 with the transmembrane domain (TM) and cytoplasmic domain (CTD) derived from baculovirus envelope protein gp64 of baculovirus was constructed. After infection, NS3 was expressed and anchored to the plasma membrane of Sf-9 cells, as demonstrated by Western blot assay and confocal microscopy. Immunogold electron microscopy demonstrated that the NS3 glycoprotein successfully displayed on the baculoviral envelope. Animal vaccine tests showed that recombinant baculovirus BacSC-NS3 elicited significantly higher NS3 antibody titers in the treated mouse models than the control group. This report demonstrated the potential of NS3-pseudotyped baculovirus expression of NS3 protein successfully.

Baculovirus surface display of E rns envelope glycoprotein of classical swine fever virus

Classical swine fever virus (CSFV) causes significant losses in the pig industry in many countries. E rns is an envelope glycoprotein of CSFV which is known to induce virus-neutralizing antibodies and protective immunity in the natural host. In this study, one recombinant baculoviruses BacSC-E rns expressing histidine-tagged E rns with the transmembrane domain (TM) and cytoplasmic domain (CTD) derived from baculovirus envelope protein gp64 was constructed and its immunizing efficacy was evaluated in a mouse model. After infection, E rns was expressed and anchored on the plasma membrane of Sf-9 cells, as demonstrated by Western-blot and confocal microscopy. Immunogold electron microscopy demonstrated that the E rns glycoprotein was successfully displayed on the baculoviral envelope. Vaccine tests in animals showed that BacSC-E rns elicited significantly higher E rns antibody titers in the immunized mouse models than the control group. This demonstrates that the BacSC-E rns vaccine can be used potentially against CSFV infections. This is the first report demonstrating the potential of E rns-pseudotyped baculovirus as a CSFV vaccine.

Baculovirus surface display of E2 envelope glycoprotein of classical swine fever virus and immunogenicity of the displayed proteins in a mouse model

Classical swine fever virus (CSFV) causes significant losses in pig industry in many countries. The E2 glycoprotein of CSFV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. In this study, one recombinant baculoviruses BacSC-E2 expressing histidine-tagged E2 with the CTD and TM derived from baculovirus envelope protein gp64 was constructed and evaluated its vaccine efficacy in mice model. After infection, E2 was expressed and anchored on the plasma membrane of Sf-9 cells, as revealed by confocal microscopy. Immunogold electron microscopy demonstrated that the BacSC-E2 was displayed E2 glycoprotein on the viral surface. Animal vaccine tests showed that BacSC-E2 elicited significantly higher E2 antibody titers in the treated mouse models than the control group. Virus neutralization test showed that serum from the BacSC-E2 treated models had significant levels of virus neutralization activities. This demonstrates that the BacSC-E2 vaccine can be a potential vaccine against CSFV infections. This is the first report demonstrating that the potential of E2-pseudotyped baculovirus as a classical swine fever virus vaccine.

Avian Influenza Virus Hemagglutinin Display on Baculovirus Envelope: Cytoplasmic Domain Affects Virus Properties and Vaccine Potential

Hemagglutinin (HA) is the major immunogen on the envelope of avian influenza virus (AIV). Therefore we constructed two recombinant baculoviruses: Bac-HA, expressing histidine-tagged HA with the cytoplasmic domain (CTD) derived from HA, and Bac-HA64, expressing histidine-tagged HA with the CTD derived from baculovirus envelope protein gp64. After infection, HA with either CTD was expressed and anchored on the plasma membrane of Sf-9 cells, as revealed by confocal microscopy. Immunogold electron microscopy demonstrated that both Bac-HA and Bac-HA64 displayed HA on the viral surface. However, analyses of purified viruses revealed that significantly more HA was incorporated into Bac-HA64 than into Bac-HA. In comparison with Bac-HA, Bac-HA64 significantly improved the gene delivery and transgene expression in mammalian cells, as determined by quantitative real-time polymerase chain reaction and flow cytometry. Bac-HA64 elicited significantly higher hemagglutination inhibition titers in mouse models than Bac-HA and the negative controls. These data collectively confirmed that the gp64 CTD, in comparison with HA CTD, resulted in more efficient HA incorporation into baculovirus, more efficient transgene delivery and expression, and elevated immunogenicity. This is the first report demonstrating the potential of HA-pseudotyped baculovirus as an avian influenza vaccine and that the choice of CTD tremendously affects baculovirus properties and vaccine efficacy.

Cloning and sequence analysis of theAntheraea pernyi nucleopolyhedrovirusgp64 gene

Frequent outbreaks of the purulence disease of Chinese oak silkworm are reported in Middle and Northeast China. The disease is produced by the pathogen Antheraea pernyi nucleopolyhedrovirus (AnpeNPV). To obtain molecular information of the virus, the polyhedra of AnpeNPV were purified and characterized. The genomic DNA of AnpeNPV was extracted and digested with HindIII. The genome size of AnpeNPV is estimated at 128 kb. Based on the analysis of DNA fragments digested with HindIII, 23 fragments were bigger than 564 bp. A genomic library was generated using HindIII and the positive clones were sequenced and analysed. The gp64 gene, encoding the baculovirus envelope protein GP64, was found in an insert. The nucleotide sequence analysis indicated that the AnpeNPV gp64 gene consists of a 1,530 nucleotide open reading frame (ORF), encoding a protein of 509 amino acids. Of the eight gp64 homologues, the AnpeNPV gp64 ORF shared the most sequence similarity with the gp64 gene of Anticarsia gemmatalis NPV, but not Bombyx mori NPV. The upstream region of the AnpeNPV gp64 ORF encoded the conserved transcriptional elements for early and late stage of the viral infection cycle. These results indicated that AnpeNPV belongs to group I NPV and was far removed in molecular phylogeny from the BmNPV.

Baculovirus GP64-pseudotyped HIV-based lentivirus vectors are stabilized against complement inactivation by codisplay of decay accelerating factor (DAF) or of a GP64–DAF fusion protein

The eventual development of efficient gene transfer vectors for in vivo gene delivery will require the development of a number of important new technologies such as stabilization of vectors against protective mechanisms that destroy or otherwise inactivate foreign infectious agents like gene transfer vectors. It is known that the baculovirus envelope protein GP64 of Autographa californica nucleopolyhedrovirus can efficiently pseudotype lentivirus vectors and that modified forms of the baculovirus envelope protein GP64 can also assemble efficiently into baculovirus particles to display functional foreign proteins on the baculovirus surface. In the present study we have combined these techniques to prepare HIV-based lentivirus vectors pseudotyped with GP64 envelope protein and coexpressing a fusion protein of GP64 with the complement-regulatory, decay accelerating factor (DAF, CD55). In addition, we have also prepared GP64-pseudotyped vectors in the presence of a DAF expression plasmid to allow the incorporation of DAF protein into viral particles. Our results demonstrate both the efficient expression and the high-titer production of GP64/GP64-DAF and GP64/DAF-pseudotyped particles and their stability against inactivation by human and nonhuman primate serum.

Novel baculovirus-derived p67 subunit vaccines efficacious against East Coast fever in cattle

Two novel baculovirus-derived recombinant Theileria parva p67 constructs were tested for their vaccine potential against East Coast fever. Boran calves were immunized with a his-GFP-p67 fusion protein (GFP:p67ΔSS) or with GP64:p67C, a protein fusion between a C-terminal domain of p67 and the baculovirus envelope protein GP64. Both GFP:p67ΔSS and GP64:p67C induced antibodies with high ELISA titers that neutralized T. parva sporozoites with high efficiency. Upon challenge, a correlation was observed between the in vitro neutralizing capacity and the reduction in severe ECF for individual animals. A protection level upto 85% was obtained. This level of protection was achieved with only two inoculations of 100 μg per dose, which is a major improvement over previous recombinant p67 products.

Regulation of thyrotropin receptor protein expression in insect cells.

Expression of large quantities of conformationally intact thyrotropin receptor (TSHR) is essential to understand the structure-function relationship of the receptor. We expressed three different constructs of full-length human TSHR in insect cells: (a) a TSHR cDNA lacking signal sequence (TSHR-ns), (b) a TSHR cDNA containing human TSHR signal sequence (TSHR-hs) and (c) a TSHR cDNA with baculovirus envelope protein encoded signal sequence gp-67 (TSHR-gp). No unique protein band, corresponding to any of these recombinant proteins, was visible upon Coomassie Blue staining after SDS-PAGE. However, Western blot using TSHR specific monoclonal antibody showed unique bands around 80, 100 and 100 kDa in TSHR-ns, TSHR-hs and TSHR-gp virus infected insect cells respectively. All three full-length TSHR proteins could neutralize the TSH binding inhibitory immunoglobulin (TBII) activity from sera of experimental animals. However, only glycosylated proteins (TSHR-hs and TSHR-gp) neutralized the TBII activity of sera from autoimmune thyroid patients, confirming the importance of glycosylation for patient autoantibody reactivity. Expression levels of full-length TSHR proteins were much lower than the levels of similarly produced corresponding ectodomains of TSHR proteins. Southern blot and Northern blot analyses showed that DNA and RNA levels in full-length TSHR virus infected insect cells were comparable to the levels found in cells infected with viruses encoding only the ectodomain of TSHR. These data suggest that full-length TSHR expression is very low and is regulated at the translational level.