Ian M. Jones

Protective mucosal immunity elicited by targeted iliac lymph node immunization with a subunit SIV envelope and core vaccine in macaques.

Prevention of sexually transmitted HIV infection was investigated in macaques by immunization with a recombinant SIV (simian immunodeficiency virus) envelope gp120 and core p27 vaccine. In two independent series of experiments, we used the novel targeted iliac lymph node (TILN) route of immunization, aiming close to the iliac lymph nodes draining the genitorectal mucosa. Rectal challenge with the SIVmac 32H J5 molecular clone in two series induced total protection in four out of seven macaques immunized by TILN, compared with infection in 13 of 14 unimmunized macaques or immunized by other routes (P = 0.025). The remaining three macaques showed either a decrease in viral load (>90%) or transient viremia, indicating that all seven TILN–immunized macaques showed total or partial protection (P = 0.001). Protection was associated with significant increase in the iliac lymph nodes of lgA antibody–secreting cells to p27 (P < 0.02), CD8–suppressor factor (P< 0.01), and the chemokines RANTES and MIP–1β (P< 0.01).

Consequences of manganese replacement of copper for prion protein function and proteinase resistance

The prion protein (PrP) binds copper and has antioxidant activity enhancing the survival of neurones in culture. The ability of the PrP to bind other cations was tested and it was found that only manganese could substitute for copper. Although initially manganese‐loaded PrP exhibited similar structure and activity to copper‐loaded PrP, after aging, manganese‐loaded PrP became proteinase resistant and lost function. It was also found that manganese could be incorporated into PrP expressed by astrocytes and that this PrP was partially proteinase resistant. These results show that it is possible to generate proteinase‐resistant PrP from cells and suggest a possible mechanism for the formation of the scrapie isoform of the PrP as generated in sporadic prion disease.

The postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines.

Viral fusion proteins mediate the merger of host and viral membranes during cell entry for all enveloped viruses. Baculovirus glycoprotein gp64 (gp64) is unusual in promoting entry into both insect and mammalian cells and is distinct from established class I and class II fusion proteins. We report the crystal structure of its postfusion form, which explains a number of gp64′s biological properties including its cellular promiscuity, identifies the fusion peptides and shows it to be the third representative of a new class (III) of fusion proteins with unexpected structural homology with vesicular stomatitis virus G and herpes simplex virus type 1 gB proteins. We show that domains of class III proteins have counterparts in both class I and II proteins, suggesting that all these viral fusion machines are structurally more related than previously thought.

Complement activation upon binding of mannan-binding protein to HIV envelope glycoproteins.

ObjectiveRetroviruses can activate the complement system in the absence of antibodies, and the purpose of this study was to examine whether the serum collectin, mannan-binding protein (MBP), could mediate such complement activation. DesignVirus envelope proteins gp120 and gp110 from HIV-1 and HIV-2 were incubated in microtitre wells coated with anti-gp120 or anti-gp110 antibodies. After further incubation with serum, complement activation was measured as deposition of complement factor C4 and C3 onto the wells. Deposited C4 and C3 were detected with enzyme-labelled antibodies. Normal human serum depleted of endogenous lectins by affinity chromatography was used as the complement source. Serum from C1q-deficient patients was used in some experiments. Complement activation was then assessed with and without prior addition of MBP to the wells. Complement activation was also correlated with the quantity of endogenous MBP in a number of normal sera. ResultsComplement activation by HIV envelope glycoproteins was found to be mediated by the binding of MBP to carbohydrates on natural envelope protein produced in virus-infected cells, as well as on glycosylated recombinant envelope proteins produced in insect cells. Non-glycosylated recombinant envelope proteins produced in Escherichia coli did not induce this type of complement activation. ConclusionsActivation of the classical complement pathway by retrovirus envelope proteins can be initiated by the binding of MBP to carbohydrate side chains of envelope glycoproteins.

Protein-disulfide Isomerase-mediated Reduction of Two Disulfide Bonds of HIV Envelope Glycoprotein 120 Occurs Post-CXCR4 Binding and Is Required for Fusion

The human immunodeficiency virus (HIV) envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded molecule that attaches HIV to the lymphocyte surface receptors CD4 and CXCR4. Conformation changes within gp120 result from binding and trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting that redox changes within Env are required. Using a sensitive assay based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5–1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120 exhibited CD4 but not CXCR4 binding capacity. In addition, PDI inhibition did not impair gp120 binding to receptors. We conclude that on average two of the nine disulfides of gp120 are reduced during interaction with the lymphocyte surface after CXCR4 binding prior to fusion and that cell surface PDI catalyzes this process. Disulfide bond restructuring within Env may constitute the molecular basis of the post-receptor binding conformational changes that induce fusion competence.

Developments in the use of baculoviruses for the surface display of complex eukaryotic proteins

The ability to couple genotype to phenotype has proven to be of immense value in systems such as phage display and has allowed genes encoding novel functions to be selected directly from complex libraries. However, the complexity of many eukaryotic proteins places a severe constraint on successful display in Escherichia coli. This restriction could be resolved if a eukaryotic virus could be similarly engineered for display purposes. Preliminary data have suggested that the baculovirus Autographa californica, a multiple nuclear polyhedrosis virus (AcMNPV) is a candidate for eukaryotic virus display because the insertion of peptides into the native virus coat protein, or the expression of foreign proteins as coat protein fusions, results in incorporation of the sequence of interest onto the surface of virus particles. A variety of strategies are currently under investigation to develop further the display capabilities of AcMNPV and to improve the complexity of library that might be accommodated. Several expression vectors for different forms of surface display have been developed and, coupled with improved recombination strategies, represent progress towards a refined tool for use in functional genomics and in vitro protein evolution.

Distinct signals in human immunodeficiency virus type 1 Pr55 necessary for RNA binding and particle formation

The human immunodeficiency virus type 1 (HIV-1) gag gene product Pr55 self-assembles to form virus-like particles when expressed in Spodoptera frugiperda cells using recombinant baculoviruses. The particles resemble immature HIV and are released from the infected cell into the culture medium. Using this system we have progressively truncated the gag open reading frame from the C terminus and examined each deleted gag protein for its particle-producing capability. We show that deletion of Pr6 and deletions that progressively remove the distal region of the Pr7 domain, including one Cys-His box thought to function as an RNA capture signal, do not affect particle formation. However deletion of two Cys-His boxes causes production of slightly larger particles with altered sedimentation properties. Sequence-specific North-Western assays using an RNA probe representative of the HIV-1 packaging signal revealed specific RNA binding by all mutants that maintained both Cys-His boxes. However, deletion of one Cys-His box reduced RNA binding substantially and loss of two Cys-His boxes abolished binding entirely. We conclude that HIV-1 gag particle formation per se does not require viral RNA encapsidation, but that it may act as a cofactor in the condensation of the immature core. Further deletion of gag sequences upstream of the Cys-His boxes led to the abolition of particle-forming ability, and we show that one boundary of the gag sequence necessary for particle formation lies within eight amino acids spanning one of the known protease cleavage sites at the C terminus of Pr24.

Characterization of recombinant gp120 and gp160 from Hiv-1: binding to monoclonal antibodies and soluble Cd4

We compared four preparations of recombinant HIV-1 envelope glycoprotein: mammalian (Chinese hamster ovary cells) gp120 (Celltech); baculovirus gp120 from American Biotechnologies Inc. (ABT) and from MicroGeneSys (MGS); and baculovirus gp160 (Institute of Virology, Oxford, UK). Each envelope glycoprotein binds to a neutralizing monoclonal antibody (MAb) directed against the V3 loop, confirming the integrity of this type-specific neutralization epitope. MGS gp120 binds abnormally well to a MAb which recognizes an epitope preferentially exposed on denatured gp120. Consistent with this finding, MGS gp120 binds to soluble CD4 (sCD4) with an affinity 50-100-fold lower than that of Celltech gp120. The affinity of Celltech gp120 from sCD4 is 2.3 nM, indistinguishable from that of gp120 extracted from HIV-1 virions. Baculovirus gp120 (ABT) and gp160 also have a high affinity for sCD4. A significant proportion of anti-gp120 antibodies in HIV-positive human sera recognize epitopes that are dependent on the mammalian glycosylation pattern, and a human HIV-positive serum inhibits the binding of mammalian gp120 to sCD4 five- to 10-fold more potently than it inhibits baculovirus gp120 binding to sCD4.

Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen.

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.

Roles of Matrix, p2, and N-Terminal Myristoylation in Human Immunodeficiency Virus Type 1 Gag Assembly

ABSTRACT Human immunodeficiency virus type 1 Gag protein is cotranslationally myristoylated at the N terminus and targeted to the plasma membrane, where virus particle assembly occurs. Particle assembly requires the ordered multimerization of Gag proteins, yet there is little direct evidence of intermediates of the reaction or of the domains that lead to each stage of the oligomerization process. In this study, following the expression in insect cells of C-terminally truncated Gag proteins and their purification, both the multimeric nature of each Gag protein and the ability to form Gag virus-like particles (VLP) were analyzed. Our results show that (i) the matrix (MA) domain forms a trimer and contributes to a similar level of oligomerization of the assembly-competent Gag; (ii) the p2 domain, located at the capsid/nucleocapsid junction, is essential for a higher order of multimerization (>1,000 kDa); (iii) the latter multimerization is accompanied by a change in Gag assembly morphology from tubes to spheres and results in VLP production; and (iv) N-terminal myristoylation is not required for either of the multimerization stages but plays a key role in conversion of these multimers to Gag VLP. We suggest that the Gag trimer and the >1,000-kDa multimer are intermediates in the assembly reaction and form before Gag targeting to the plasma membrane. Our data identify a minimum of three stages for VLP development and suggest that each stage involves a separate domain, MA, p2, or N-terminal myristoylation, each of which contributes to HIV particle assembly.