Thermal Stability of RNA Phage Virus-Like Particles Displaying Foreign Peptides

<p>Abstract</p> <p>Background</p> <p>To be useful for genetic display of foreign peptides a viral coat protein must tolerate peptide insertions without major disruption of subunit folding and capsid assembly. The folding of the coat protein of RNA phage MS2 does not normally tolerate insertions in its AB-loop, but an engineered single-chain dimer readily accepts them as long as they are restricted to one of its two halves.</p> <p>Results</p> <p>Here we characterize the effects of peptide insertions on the thermal stabilities of MS2 virus-like particles (VLPs) displaying a variety of different peptides in one AB-loop of the coat protein single-chain dimer. These particles typically denature at temperatures around 5-10°C lower than unmodified VLPs. Even so, they are generally stable up to about 50°C. VLPs of the related RNA phage PP7 are cross-linked with intersubunit disulfide bonds and are therefore significantly more stable. An AB-loop insertion also reduces the stability of PP7 VLPs, but they only begin to denature above about 70°C.</p> <p>Conclusions</p> <p>VLPs assembled from MS2 single-chain dimer coat proteins with peptide insertions in one of their AB-loops are somewhat less stable than the wild-type particle, but still resist heating up to about 50°C. Because they possess disulfide cross-links, PP7-derived VLPs provide an alternate platform with even higher stability.</p

Stability and assembly in vitro of bacteriophage PP7 virus-like particles

<p>Abstract</p> <p>Background</p> <p>The stability of a virus-like particle (VLP) is an important consideration for its use in nanobiotechnology. The icosahedral capsid of the RNA bacteriophage PP7 is cross-linked by disulfide bonds between coat protein dimers at its 5-fold and quasi-6-fold symmetry axes. This work determined the effects of these disulfides on the VLP's thermal stability.</p> <p>Results</p> <p>Measurements of the thermal denaturation behavior of PP7 VLPs in the presence and absence of a reducing agent show that disulfide cross-links substantially stabilize them against thermal denaturation. Although dimers in the capsid are linked to one another by disulfides, the two subunits of dimers themselves are held together only by non-covalent interactions. In an effort to confer even greater stability a new cross-link was introduced by genetically fusing two coat protein monomers, thus producing a "single-chain dimer" that assembles normally into a completely cross-linked VLP. However, subunit fusion failed to increase the thermal stability of the particles, even though it stabilized the isolated dimer. As a step toward gaining control of the internal composition of the capsid, conditions that promote the assembly of PP7 coat protein dimers into virus-like particles <it>in vitro </it>were established.</p> <p>Conclusion</p> <p>The presence of inter-dimer disulfide bonds greatly stabilizes the PP7 virus-like particle against thermal denaturation. Covalently cross-linking the subunits of the dimers themselves by genetically fusing them through a dipeptide linker sequence, offers no further stabilization of the VLP, although it does stabilize the dimer. PP7 capsids readily assemble <it>in vitro </it>in a reaction that requires RNA.</p

A malaria vaccine candidate based on an epitope of the Plasmodium falciparum RH5 protein

BACKGROUND: The Plasmodium falciparum protein RH5 is an adhesin molecule essential for parasite invasion of erythrocytes. Recent studies show that anti-PfRH5 sera have potent invasion-inhibiting activities, supporting the idea that the PfRH5 antigen could form the basis of a vaccine. Therefore, epitopes recognized by neutralizing anti-PfRH5 antibodies could themselves be effective vaccine immunogens if presented in a sufficiently immunogenic fashion. However, the exact regions within PfRH5 that are targets of this invasion-inhibitory activity have yet to be identified. METHODS: A battery of anti-RH5 monoclonal antibodies (mAbs) were produced and screened for their potency by inhibition of invasion assays in vitro. Using an anti-RH5 mAb that completely inhibited invasion as the selecting mAb, affinity-selection using random sequence peptide libraries displayed on virus-like particles of bacteriophage MS2 (MS2 VLPs) was performed. VLPs were sequenced to identify the specific peptide epitopes they encoded and used to raise specific antisera that was in turn tested for inhibition of invasion. RESULTS: Three anti-RH5 monoclonals (0.1 mg/mL) were able to inhibit invasion in vitro by >95%. Affinity-selection with one of these mAbs yielded a VLP which yielded a peptide whose sequence is identical to a portion of PfRH5 itself. The VLP displaying the peptide binds strongly to the antibody, and in immunized animals elicits an anti-PfRH5 antibody response. The resulting antisera against the specific VLP inhibit parasite invasion of erythrocytes more than 90% in vitro. CONCLUSIONS: Here, data is presented from an anti-PfRH5 mAb that completely inhibits erythrocyte invasion by parasites in vitro, one of the few anti-malarial monoclonal antibodies reported to date that completely inhibits invasion with such potency, adding to other studies that highlight the potential of PfRH5 as a vaccine antigen. The specific neutralization sensitive epitope within RH5 has been identified, and antibodies against this epitope also elicit high anti-invasion activity, suggesting this epitope could form the basis of an effective vaccine against malaria

The stability of PP7 virus-like particles under reducing (+DTT) and under non-reducing (-DTT) conditions as measured by the fraction of capsids or soluble coat protein remaining after heating for two minutes at the indicated temperatures

<p><b>Copyright information:</b></p><p>Taken from "Stability and assembly of bacteriophage PP7 virus-like particles"</p><p>http://www.jnanobiotechnology.com/content/5/1/10</p><p>Journal of Nanobiotechnology 2007;5():10-10.</p><p>Published online 26 Nov 2007</p><p>PMCID:PMC2211308.</p><p></p

Purification of DTT/urea-disaggregated PP7 and 2PP7 coat protein by gel filtration chromatography in 10 mM acetic acid, 50 mM NaCl (about pH 4)

<p><b>Copyright information:</b></p><p>Taken from "Stability and assembly of bacteriophage PP7 virus-like particles"</p><p>http://www.jnanobiotechnology.com/content/5/1/10</p><p>Journal of Nanobiotechnology 2007;5():10-10.</p><p>Published online 26 Nov 2007</p><p>PMCID:PMC2211308.</p><p></p> To estimate the molecular weight of the putative dimer species, bovine serum albumin (68 kD), ovalbumin (43 kD), chymotrypsinogen (25.7 kD) and hen lysozyme (14.4 kD) were used as standards. They peaked at fractions 14, 18, 22 and 26 respectively

The stability of PP7 virus-like particles as indicated by the fraction of capsid or soluble protein remaining after heating for the indicated times at 93°C under non-reducing conditions, or at 67°C under reducing conditions

<p><b>Copyright information:</b></p><p>Taken from "Stability and assembly of bacteriophage PP7 virus-like particles"</p><p>http://www.jnanobiotechnology.com/content/5/1/10</p><p>Journal of Nanobiotechnology 2007;5():10-10.</p><p>Published online 26 Nov 2007</p><p>PMCID:PMC2211308.</p><p></p

The sequence at the junction of the 2PP7 duplication is shown at bottom, and the sequences of the 5'- and 3' ends of the PP7 coat sequence are shown above it

<p><b>Copyright information:</b></p><p>Taken from "Stability and assembly of bacteriophage PP7 virus-like particles"</p><p>http://www.jnanobiotechnology.com/content/5/1/10</p><p>Journal of Nanobiotechnology 2007;5():10-10.</p><p>Published online 26 Nov 2007</p><p>PMCID:PMC2211308.</p><p></p> In the 2PP7 construct the C-terminal arginine of the upstream copy is fused through a tyr-gly linker to the serine, residue number 2, of the downstream copy

Stabilities of PP7 and 2PP7 dimers indicated by the disappearance of protein from the soluble fraction as a function of time at 67°C

<p><b>Copyright information:</b></p><p>Taken from "Stability and assembly of bacteriophage PP7 virus-like particles"</p><p>http://www.jnanobiotechnology.com/content/5/1/10</p><p>Journal of Nanobiotechnology 2007;5():10-10.</p><p>Published online 26 Nov 2007</p><p>PMCID:PMC2211308.</p><p></p