Thermal stability of RNA phage virus-like particles displaying foreign peptides

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

The plasmids and peptide insertions utilized in this study. (A) pDSP1 expresses the MS2 coat protein single-chain dimer from the T7 promoter. The manipulations that resulted in the various peptide insertions utilized Sal I and Kpn I sites uniquely present in the downstream half of the dimer. The plasmid pET2P7K32 is a similar construction that expresses the PP7 single-chain dimer.

Figure 2

The amino acid and nucleotide sequences of MS2 coat protein in the vicinity of the various peptide insertions. Note the positions of Kpn I and Sal I sites (underlined).

Figure 3

PP7 sequences, showing the wild-type, the Kpn I mutant and the Flag peptide insertion. Note that the mutations that introduce a Kpn I site in the AB-loop and also substitute Glu11 with Thru.

Figure 4

Denaturation of MS2 VLPs. (A) Coincidence of VLP disassembly, as determined by loss of the VLP band on an agarose gel, and coat protein denaturation, determined by disappearance of soluble protein as a function of temperature. "MS2 WT" denotes the wild-type coat protein, while "MS2 sc-dimer" refers to the MS2 coat protein single-chain dimer. "Soluble" indicates whether the data points were obtained by measuring the amount of soluble protein remaining after treatment and "VLP" identifies data points showing the quantity of intact VLP. (B) Denaturation of various recombinant VLPs as a function of temperature. "F13/14" and "F13/16" refer to VLPs displaying the Flag epitope at two different locations in the AB-loop (see the text). "ECL2" and "V3" refer to VLPs displaying the extracellular loop 2 of CCR5 and the V3 loop of the HIV envelope protein, respectively. "Random 10mer" identifies results obtained using a complex library of random-sequence 10mer insertions. (C) Denaturation of the indicated MS2 VLPs as a function of time at 55°C.

Figure 5

Comparison of thermal stabilities of PP7 single-chain dimer with and without Flag peptide insertion. (A) Disassembly and aggregation of PP7 single-chain dimer VLPs as a function of temperature in the presence and absence of a reducing agent (DTT). (B) Disassembly and aggregation of the Flag-PP7 single-chain dimer VLP with and without DTT.