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Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy
Published on: November 1, 2011
Isolation and characterization of the Mason-Pfizer monkey virus p12 protein
Zdenek Knejzlík1, Martin Strohalm, Lenka Sedlácková
1Department of Biochemistry and Microbiology and Center for Integrated Genomics, Institute of Chemical Technology, 166 28 Prague, Czech Republic.
Abstract:
The Mason-Pfizer monkey virus (M-PMV) Gag protein, precursor to the structural proteins of the infectious virion, assembles into immature capsid-like particles when expressed at high levels in bacterial cells. Similar capsid-like particles can be obtained by in vitro assembly using a high concentration of isolated Gag. M-PMV Gag contains a p12 protein that has no corresponding analogues in most other retroviruses and has been suggested to contain an internal scaffold domain (ISD). We have expressed and purified p12 and the N- and C-terminal halves (Np12 and Cp12) that are predicted to be structurally independent domains. The behavior of these proteins was analyzed using chemical cross-linking, CD spectroscopy, and electron microscopy. The N-terminal half of p12 is largely alpha-helical although the C-terminal portion lacks any apparent ordered structure. Both p12 and Np12 form high-order oligomers in vitro and when expressed in E. coli produce organized structures that are visible by electron microscopy. Interestingly, Cp12, as well as the whole protein, can form dimers in the presence of SDS. The data show that both domains of p12 contribute to its ability to multimerize with much of this potential residing in its N-terminal part, most probably within the leucine zipper-like (LZL) sequence.
Insights
The Mason-Pfizer monkey virus (M-PMV) Gag protein
Area of Science:
- Retroviral structural biology
- Molecular virology
- Protein biochemistry
Background:
- The Mason-Pfizer monkey virus (M-PMV) Gag protein is essential for assembling infectious virions.
- M-PMV Gag forms immature capsid-like particles in bacteria and in vitro.
- A unique p12 protein within M-PMV Gag is proposed to contain an internal scaffold domain (ISD).
Purpose of the Study:
- To investigate the structural properties and multimerization potential of the M-PMV p12 protein and its domains.
- To determine the contribution of p12's N-terminal (Np12) and C-terminal (Cp12) halves to its oligomerization.
- To identify the specific regions within p12 responsible for its structural organization.
Main Methods:
- Expression and purification of M-PMV p12, Np12, and Cp12 proteins.
- Analysis of protein behavior using chemical cross-linking, Circular Dichroism (CD) spectroscopy, and electron microscopy.
- Assessment of oligomerization states and structural integrity of p12 and its fragments.
Main Results:
- The N-terminal half (Np12) of p12 is predominantly alpha-helical, while the C-terminal half (Cp12) is largely unstructured.
- Both p12 and Np12 exhibit high-order oligomerization in vitro and form organized structures in E. coli.
- Cp12 and the full-length p12 protein can form dimers in the presence of SDS, indicating domain-specific contributions to multimerization.
- The leucine zipper-like (LZL) sequence in the N-terminal region is likely crucial for p12's multimerization potential.
Conclusions:
- The M-PMV p12 protein possesses distinct structural domains that mediate its multimerization.
- The N-terminal, alpha-helical domain, particularly the LZL sequence, plays a significant role in p12's oligomerization.
- These findings provide insights into the assembly mechanisms of M-PMV capsids and the function of unique retroviral Gag domains.
