Identification of domains in canine parvovirus VP2 essential for the assembly of virus-like particles

Canine parvovirus capsids are composed of 60 copies of VP2 and 6 to 10 copies of VPl. To locate essential sites of interaction between VP2 monomers, we have analyzed the effects of a number of VP2 deletion mutants representing the amino terminus and the four major loops of the surface, using as an assay the formation of virus-like particles (VLPs) expressed by recombinant baculoviruses. For the amino terminus we constructed three mutants with progressively larger deletions, i.e., 9, 14, and 24 amino acids. Deletions of 9 and 14 amino acids did not affect the morphology and assembly capabilities of the mutants. However, the mutant with the 24-amino-acid deletion did not show hemagglutination properties or correct VLP morphology, stressing again the relevance of the RNER domain in canine parvovirus functionality. Three of the four mutants with deletions in the loops failed to make correct VLPs, indicating that these regions are essential for correct capsid assembly and morphology. Only the mutant with the deletion in loop 2 was able to assemble in regular VLPs, suggesting that this loop has little or no effect in capsid morphogenesis. Further research has demonstrated that this region can tolerate the insertion of foreign epitopes that are correctly exposed in the surface of the capsid. This result opens the door to the use of these VLPs for antigen delivery.     

The matrix protein of HIV-1 is not sufficient for assembly and release of virus-like particles

There is an expectation for Nursing Development Units (NDUs) to explore and develop the clinical leadership role. This paper describes how 28 Department of Health funded NDUs in England sought to meet this objective. We describe the personal and professional characteristics of NDU clinical leaders (CLs) and their perceived responsibilities. We identify 10 elements which appear to be central to the CL role and together form a core role set of the leader which is applicable, to some extent, across clinical specialties and settings. The position of the CL in the organizational hierarchy emerges as crucial to his/her ability to act on these responsibilities and fulfil a leadership role.     

Monomolecular collapse of plasmid DNA into stable virus-like particles

Cationic lipids are being widely used for cell transfection in vitro. The lipid/DNA complexes, however, tend to aggregate into large and polydisperse particle mixtures; this hampers their use in vivo. Cationic detergents, on the contrary, do not mediate cell transfection per se, yet are capable of condensing individual DNA molecules into discrete entities. We have taken (only) the interesting features of both types of amphiphiles for the two-step formation of stable core particles reminiscent of viruses. Individual anionic plasmid molecules were cooperatively collapsed with a carefully tailored cationic cysteine-based detergent. The resulting 23-nm particles were then simply "frozen" by spontaneous aerobic dimerization of the cysteine-detergent into a cystine-lipid on the template DNA. The population of spherical particles is monodisperse and stable over days, in physiological conditions. Together with a negative surface potential, these properties should ensure good tissue dissemination and escape from the blood stream after i.v. injection.     

Analysis of minimal human immunodeficiency virus type 1 gag coding sequences capable of virus-like particle assembly and release

We have constructed a series of human immunodeficiency virus (HIV) gag mutants by progressive truncation of the gag coding sequence from the C terminus and have combined these mutants with an assembly-competent matrix domain deletion mutation (DeltaMA). By using several methods, the particle-producing capabilities of each mutant were examined. Our analysis indicated that truncated Gag precursors lacking most of C-terminal gag gene products assembled and were released from 293T cells. Additionally, a mutant with a combined deletion of the MA (DeltaMA) and p6 domains even produced particles at levels comparable to that of the wild-type (wt) virus. However, most mutants derived from combination of the DeltaMA and the C-terminal truncation mutations did not release particles as well as the wt. Our smallest HIV gag gene product capable of virus-like particle formation was a 28-kDa protein which consists of a few MA amino acids and the CA-p2 domain. Sucrose density gradient fractionation analysis indicated that most mutants exhibited a wt retrovirus particle density. Exceptions to this rule were mutants with an intact MA domain but deleted downstream of the p2 domains. These C-terminal truncation mutants possessed particle densities of 1.13 to 1.15 g/ml, lower than that of the wt. The N-terminal portions of the CA domain, which have been shown to be dispensable for core assembly, became critical when most of the MA domain was deleted, suggesting a requirement for an intact CA domain to assemble and release particles.     

Mapping the functional domains of bacteriophage lambda integrase protein

Bacteriophage lambda encodes a site-specific recombination system that promotes the movement of the phage genome into and out of the host bacterial chromosome. The phage-encoded integrase (Int) is composed of 356 amino acid residues and carries out the required strand exchanges by means of a type I topoisomerase activity. Int also contains two distinct DNA-binding domains that interact with two different, specific sequences (arm-type and core-type sites) on DNA. In order to help understand the mechanism of site-specific recombination, we have used a genetic approach to isolate mutants defective in different steps in the recombination reaction. We developed a genetic screen for Int mutants that are defective in catalyzing excisive recombination in vivo. These mutants were screened for proficiency in binding to the P'123 arm-type sites using the bacteriophage P22 challenge-phage assays. In all, 78 such mutants were isolated and the mutational changes mapped and sequenced. These mutants have been further characterized (1) for their ability to bind the P'1 and P'123 arm-type sites and for their ability to form the attL complex in vivo, (2) for negative dominance in vitro, (3) for the presence of type I topoisomerase activity, and (4) for the ability to resolve artificially constructed recombination intermediates. We found that (1) residues in a stretch of 88 amino acids in the middle of the protein may be involved in Int-Int interactions, (2) a region around Arg212 is involved in the catalytic site, (3) residues near the carboxyl terminus play a role in enhancing Int binding to its arm-type sites, possibly by interacting with the small amino-terminal region that has been shown to be responsible for specific recognition of the arm-type sites, and (4) residues at the very carboxyl end of the protein may be involved in modulating the cleavage or religation activities of the Int protein.     

Recombinant human immunodeficiency Pr55gag virus-like particles presenting chimeric envelope glycoproteins induce cytotoxic T-cells and neutralizing antibodies

Very recently, we demonstrated that the replacement of the human immunodeficiency virus type-1 (HIV-1) gp41 transmembrane protein by an Epstein-Barr virus gp220/350-derived membrane anchor resulted in the incorporation of chimeric envelope (Env) oligomers into Pr55gag virus-like particles (VLPs), exceeding that of wild-type gp160 by a factor of 10. In this study, we examined the immunostimulatory properties of Pr55gag VLPs to both (i) chimeric HIV-1 gp120 external envelope proteins and (ii) full-length gp160 presented on the outer surface of the particles. Immunization studies carried out with VLPs presenting different derivatives of the chimeric and wild-type Env proteins elicited a consistent anti-Pr55gag as well as anti-Env antibody response in complete absence of additional adjuvants. In both cases, the immune sera exhibited an in vitro neutralizing activity against homologous HIV-1 infection in MT4 cells. Noteworthy, these VLPs were also capable of inducing a strong CD8+ cytotoxic T-cell (CTL) response in immunized BALB/c mice that was directed toward a known CTL epitope in the third variable domain V3 of the gp120 external glycoprotein. However, the induction of V3-loop-specific CTLs critically depended on the amounts of Env proteins that were presented by the Pr55gag VLPs. Moreover, the CD8+ CTL response was not significantly altered by adsorbing the VLPs to alum or by repeated booster immunizations. These results illustrate that Pr55gag VLPs provide a safe and effective means of enhancing neutralizing humoral responses to particle-entrapped gp120 proteins and are also capable of delivering these proteins to the MHC class I antigen processing and presentation pathway. Therefore, antigenically expanded Pr55gag VLPs represent an attractive approach in the design of vaccines for which specific stimulation of neutralizing antibodies and cytotoxic effector functions to complex glycoproteins is desired.     

Increased incorporation of chimeric human immunodeficiency virus type 1 gp120 proteins into Pr55gag virus-like particles by an Epstein-Barr virus gp220/350-derived transmembrane domain

Noninfectious Pr55gag virus-like particles containing high quantities of oligomeric human immunodeficiency virus type 1 (HIV-1) envelope (Env) proteins represent potential candidate immunogens for a vaccine against HIV-1 infection. Thus, chimeric env genes were constructed encoding the HIV-1 exterior glycoprotein gp120 which was covalently linked at different C-terminal positions to a transmembrane domain (TM) from the Epstein-Barr virus (EBV) major Env glycoprotein gp220/ 350. All chimeric Env-TM polypeptides as well as the wild-type HIV Env proteins were equally produced and incorporated at the outer surface of insect cells using the baculovirus expression system. In the presence of coexpressed HIV Pr55gag polyproteins significantly decreased amounts of wild-type Env proteins were presented at the cell surface, whereas the membrane incorporation of the Env-TM chimeras was not affected. Biochemical and immunoelectron microscopical analysis of particles that were efficiently released from these cells displayed the incorporation of both wild-type Env and chimeric Env-TM proteins on the surface of VLPs. However, the quantities of particle-associated chimeric Env-TM proteins exceeded those of incorporated wild-type Env proteins by a factor of 5-10. Chemical cross-linking and subsequent polyacrylamide gel electrophoresis of VLP-entrapped Env proteins revealed that the chimeric Env-TM proteins form homodimers and a higher-order oligomer, similar to that observed for wild-type Env proteins. Thus, the results of this study clearly demonstrate that the replacement of the gp41 transmembrane protein of gp160 by a heterologous, EBV gp220/350-derived membrane anchor provides an effective strategy to incorporate high quantities of oligomeric HIV gp120 proteins on the surface of Pr55gag virus-like particles.     

Mapping the RNA binding sites for human immunodeficiency virus type-1 gag and NC proteins within the complete HIV-1 and -2 untranslated leader regions

Encapsidation of HIV-1 genomic RNA is mediated by specific interactions between the RNA packaging signal and the Gag protein. During maturation of the virion, the Gag protein is processed into smaller fragments, including the nucleocapsid (NC) domain which remains associated with the viral genomic RNA. We have investigated the binding of glutathione- S -transferase (GST) Gag and NC fusion proteins from HIV-1, to the entire HIV-1 and -2 leader RNAencompassing the packaging signal. We have mapped the binding sites at conditions where only about two complexes are formed and find that GST-Gag and GST-NC fusion proteins bind specifically to discrete sites within the leader. Analysis of the HIV-1 leader indicated that GST-Gag strongly associates with the PSI stem-loop and to a lesser extent with regions near the primer binding site. GST-NC binds the same regions but with reversed preferences. The HIV-1 proteins also interact specifically with the 5'-leader of HIV-2 and the major site of interaction mapped to a stem-loop, with homology to the HIV-1 PSI stem-loop structure. The different specificities of Gag and NC may reflect functionally distinct roles in the viral replication, and suggest that the RNA binding specificity of NC is modulated by its structural context.     

Cryo-electron microscopy structure of yeast Ty retrotransposon virus-like particles

The virus-like particles (VLPs) produced by the yeast retrotransposon Ty1 are functionally related to retroviral cores. These particles are unusual in that they have variable radif. A paired mass-radius analysis of VLPs by scanning transmission electron microscopy showed that many of these particles form an icosahedral T-number series. Three-dimensional reconstruction to 38-A resolution from cryo-electron micrographs of T = 3 and T = 4 shells revealed that the single structural protein encoded by the TYA gene assembles into spiky shells from trimeric units.     

The immunosuppressive peptide of HIV-1: functional domains and immune response in AIDS patients

OBJECTIVES: To study the biological properties of the immunosuppressive peptide (ISU-peptide) of HIV-1, a 17-mer corresponding to the amino-acid domain 583-599 of the transmembrane glycoprotein gp41 of HIV-1. This peptide exhibits sequence homology to the highly conserved ISU-peptide of type C and D retroviruses. Also, to study the immune response against the corresponding gp41 epitope in AIDS patients. DESIGN: The ISU-peptide and control peptides were synthesized and tested for immunosuppressive activity in different in vitro lymphocyte proliferation assays. Antibody responses were tested using a peptide enzyme-linked immunosorbent assay. A new property of the ISU-peptide, inhibition of HIV-1 replication, was investigated using a cytopathogenicity assay. RESULTS: The ISU-peptide of HIV-1 and the immunosuppressive peptides of type C and type D retroviruses possess similar functional properties. They inhibit mitogen-induced and lymphokine-dependent T-lymphocyte proliferation, they are interspecies-reactive, they must be conjugated to a carrier protein in order to be immunosuppressive, and their N-terminal octamers represent the minimal immunosuppressive domain. HIV-infected individuals develop antibodies against an epitope located at the C-terminal end of the ISU-peptide and the number of responders and antibody titres decrease during progression to AIDS. In addition to its immunosuppressive activity, the ISU-peptide of HIV-1 inhibits the cytopathic effect of HIV-1 on human MT4 cells, suggesting interference with virus replication. CONCLUSIONS: The immunosuppressive property of the ISU-peptide suggests that gp41 might contribute to the development of AIDS. The evolutionary conservation of the immunosuppressive domain and the ability of the corresponding ISU-peptide to inhibit HIV replication suggest that this domain plays an important role in the life cycle of HIV-1.     

The isolation and characteristics of monoclonal antibodies to recombinant proteins of HIV-1 and HIV-2--the env and gag gene products

Ten hybridomas secreting monoclonal antibodies (Mab) against recombinant HIV-1 and HIV-2 antigens were produced (3 Mab anti-gag protein, 2 anti-env1, and 5 anti-env2). In the immunoblotting assay all the anti-gag Mabs reacted with HIV capsid protein p24, whereas one of them reacted also with p55 protein and with 7 other polypeptides. Another anti-gag Mab cross-reacted with the antigen of subpopulation of human peripheral blood lymphocytes. The third one interacted with the antigen of both HIV-1 and HIV-2. All the 10 Mabs interacted with natural HIV antigens and can be used for identification and differentiation of HIV-1 and HIV-2.     

In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: identification of the p10 domain as a morphological determinant in the formation of spherical particles

Retroviruses are unusual in that expression of a single protein, Gag, leads to budding of virus-like particles into the extracellular space. We have developed conditions under which virus-like particles are formed spontaneously in vitro from fragments of Rous sarcoma virus (RSV) Gag protein purified after expression in Escherichia coli. The CA-NC fragment of Gag was shown previously to assemble into hollow cylinders (S. Campbell and V. M. Vogt, J. Virol. 69:6487-6497, 1995). We have now extended these studies to larger Gag proteins. In every case examined, assembly into regular structures required RNA. A nearly full-length Gag missing only the C-terminal PR domain, as well as similar proteins missing in addition the N-terminal half of MA, the C-terminal half of MA, the entire MA sequence, or the entire p2 sequence, all assembled into spherical particles resembling RSV in size. By contrast, proteins missing p10 assembled into cylindrical particles like those formed by CA-NC alone. Thin section electron microscopy showed that each of these Gag proteins formed in the expressing E. coli cells particles similar in shape to those seen in vitro. We conclude from these results that neither the sequences required for membrane binding in vivo, near the N terminus of Gag, nor the sequences required for a late step in budding, in the p2 portion of Gag, are essential for formation of virus-like particles in this system. Furthermore, we postulate the existence of a shape-determining sequence in p10, which provides or facilitates interactions required for the growing particle to be constrained to a spherical shape.     

B-cell epitopes in HIV-1 Tat and Rev proteins colocalize with T-cell epitopes and with functional domains

We describe the characterization of the B-cell epitopes of HIV-1 regulatory proteins Tat and Rev. The prevalence of antibodies to these proteins among human immunodeficiency virus (HIV)-1-infected individuals was examined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The Tat and Rev antibody-positive sera were selected for epitope mapping performed with partially overlapping synthetic peptides bound to polyethylene pins. Eighteen and twelve percent of HIV-infected individuals had antibodies against Tat or Rev, respectively. In Tat, four epitopic regions were identified, situated within amino acids 6-10 (PRLEP), 21-37 (ACTNCYCKKCCFHCQVC), 39-58 (ITKALGISYGRKKRRQRRRA) and 74-82 (TSQSRGDPT). The most frequently recognized epitopic regions were located in the middle of the protein. In Rev, the two most frequently recognized epitopic regions were near the amino terminus of the protein within amino acids 12-20 (LIRTVRLIK) and 38-49 (RRNRRRRWRERQ). A third epitope was mapped around amino acids 55-62 (ISERILGT) and a fourth around amino acids 78-83 (LERLTU). To analyze the specificity of Tat and Rev epitopes, soluble synthetic peptides representing the identified epitopes were used in an ELISA assay, and the recognition of most epitopes was shown to be specific for HIV-1-infected individuals. In addition, many of the Tat and Rev epitopes were shown to overlap with regions having functional activity or with regions previously identified as T-cell epitopes.     

Detection of double-stranded RNA molecules and virus-like particles in different Mucor species

Malignant mesenchymal tumors of the larynx constitute 0.3% to 1% of all laryngeal cancers, and of these osteosarcoma is the rarest. Our review of the literature identified only 12 cases of osteosarcoma of the larynx, all of which occurred in men. Rarely, osteosarcoma of the larynx may follow radiation therapy for squamous cell carcinoma, or the larynx may be the site of metastatic osteosarcoma. We report a case of primary laryngeal osteosarcoma arising in the cricoid cartilage and the first case of osteosarcoma of the larynx occurring in a woman.     

Different functional domains of GLUT2 glucose transporter are required for glucose affinity and substrate specificity

GLUT2 is the major glucose transporter in pancreatic beta-cells and hepatocytes. It plays an important role in insulin secretion from beta-cells and glucose metabolism in hepatocytes. To better understand the molecular determinants for GLUT2's distinctive glucose affinity and its ability to transport fructose, we constructed a series of chimeric GLUT2/GLUT3 proteins and analyzed them in both Xenopus oocytes and mammalian cells. The results showed the following. 1) GLUT3/GLUT2 chimera containing a region from transmembrane segment 9 to part of the COOH-terminus of GLUT2 had Km values for 3-O-methylglucose similar to those of wild-type GLUT2. Further narrowing of the GLUT2 component in the chimeric GLUTs lowered the Km values to those of wild-type GLUT3. 2) GLUT3/GLUT2 chimera containing a region from transmembrane segment 7 to part of the COOH-terminus of GLUT2 retained the ability to transport fructose. Further narrowing of this region in the chimeric GLUTs resulted in a complete loss of the fructose transport ability. 3) Chimeric GLUTs with the NH2-terminal portion of GLUT2 were unable to express glucose transporter proteins in either Xenopus oocytes or mammalian RIN 1046-38 cells. These results indicate that amino acid sequences in transmembrane segments 9-12 are primarily responsible for GLUT2's distinctive glucose affinity, whereas amino acid sequences in transmembrane segments 7-8 enable GLUT2 to transport fructose. In addition, certain region(s) of the amino-terminus of GLUT2 impose strict structural requirements on the carboxy-terminus of the glucose transporter protein. Interactions between these regions and the carboxy-terminus of GLUT2 are essential for GLUT2 expression.     

Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.     

Rapid monitoring of virus-like particles using an optical biosensor: a feasibility study

The mature major microneme protein of Sarcocystis muris cyst merozoites, which is known as a dimeric lectin with high affinity to galactose and some of its derivatives, was expressed in Escherichia colias a histidine-tagged fusion protein. The recombinant polypeptide, which was recognized by a monoclonal antibody directed against the native lectin, was purified from inclusion bodies after solubilization and refolding, using a combination of metal chelate and lactose affinity chromatography. The apparent molecular mass of the refolded polypeptide as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoreses was 16 kDa, whereas gel filtration chromatography clearly demonstrated that the recombinant protein, like its native counterpart, exists as a homodimer of two non-covalently associated subunits. Inhibition of haemagglutination suggests that the combining site of the recombinant lectin recognizes N-acetyl-galactosamine as the dominant sugar, thus confirming the correct folding of the monosaccharide combining site in the renatured lectin. To the best of our knowledge, this work represents the first reported detailed characterization of a recombinant lectin from apicomplexan parasites, and may contribute to a better understanding of the process of host cell recognition and invasion by these obligate intracellular protozoa.     

Role of beta-subunit domains in the assembly, stable expression, intracellular routing, and functional properties of Na,K-ATPase

The beta-subunit of Na,K-ATPase (betaNK) interacts with the catalytic alpha-subunit (alphaNK) in the ectodomain, the transmembrane, and the cytoplasmic domain. The functional significance of these different interactions was studied by expressing alphaNK in Xenopus oocytes along with N-terminally modified betaNK or with chimeric betaNK/betaH,K-ATPase (betaHK). Complete truncation of the betaNK N terminus allows for cell surface-expressed, functional Na,K-pumps that exhibit, however, reduced apparent K+ and Na+ affinities as assessed by electrophysiological measurements. A mutational analysis suggests that these functional effects are not related to a direct interaction of the beta N terminus with the alphaNK but rather that N-terminal truncation induces a conformational change in another functionally relevant beta domain. Comparison of the functional properties of alphaNK.betaNK, alphaNK.betaHK, or alphaNK. betaNK/betaHK complexes shows that the effect of the betaNK on K+ binding is mainly mediated by its ectodomain. Finally, betaHK/NK containing the transmembrane domain of betaHK produces stable but endoplasmic reticulum-retained alphaNK.beta complexes, while alphaNK/betaHK complexes can leave the ER but exhibit reduced ouabain binding capacity and transport function. Thus, interactions of both the transmembrane and the ectodomain of betaNK with alphaNK are necessary to form correctly folded Na,K-ATPase complexes that can be targeted to the plasma membrane and/or become functionally competent. Furthermore, the beta N terminus plays a role in the beta-subunit's folding necessary for correct interactions with the alpha-subunit.