Related Experiment Videos
Molecular organization of Mason-Pfizer monkey virus capsids assembled from Gag polyprotein in Escherichia coli
Milan V Nermut1, Patrick Bron, Daniel Thomas
1National Institute for Biological Standards and Control, South Mimms, Hertfordshire EN6 3QG, United Kingdom. mvnermut@nibsc.ac.uk
Abstract:
We describe the results of a study by electron microscopy and image processing of Gag protein shells-immature capsids--of Mason-Pfizer monkey virus assembled in Escherichia coli from two truncated forms of the Gag precursor: Deltap4Gag, in which the C-terminal p4Gag was deleted, and Pro(-)CA.NC, in which the N-terminal peptides and proline 1 of the CA domain were deleted. Negative staining of capsids revealed small patches of holes forming a trigonal or hexagonal pattern most clearly visible on occasional tubular forms. The center-to-center spacing of holes in the network was 7.1 nm in Deltap4Gag capsids and 7.4 nm in Pro(-)CA.NC capsids. Image processing of Deltap4Gag tubes revealed a hexagonal network of holes formed by six subunits with a single subunit shared between rings. This organization suggests that the six subunits are contributed by three trimers of the truncated Gag precursor. Similar molecular organization was observed in negatively stained Pro(-)CA.NC capsids. Shadowed replicas of freeze-etched capsids produced by either construct confirmed the presence of a hexagonal network of holes with a similar center-to-center spacing. We conclude that the basic building block of the cage-like network is a trimer of the Deltap4Gag or Pro(-)CA.NC domains. In addition, our results point to a key role of structurally constrained CA domain in the trimeric interaction of the Gag polyprotein.
Insights
Researchers studied Mason-Pfizer monkey virus Gag protein shells using electron microscopy. They found that trimers of truncated Gag proteins form the basic building block of the capsid network.
Area of Science:
- Structural biology
- Virology
- Biochemistry
Background:
- Mason-Pfizer monkey virus (MPMV) is a retrovirus.
- Gag polyprotein self-assembles into immature capsid structures.
- Understanding Gag protein assembly is crucial for viral structure research.
Purpose of the Study:
- To investigate the assembly of Gag protein shells from truncated Gag precursors.
- To determine the basic building block of the Gag protein network.
- To elucidate the role of the CA domain in Gag trimerization.
Main Methods:
- Electron microscopy (negative staining and freeze-etching).
- Image processing of assembled Gag protein shells.
- Assembly of virus-like particles from truncated Gag proteins in E. coli.
Main Results:
- Truncated Gag proteins (Deltap4Gag and Pro(-)CA.NC) assembled into capsids with a hexagonal network of holes.
- The center-to-center spacing of holes was approximately 7.1-7.4 nm.
- Image processing revealed that trimers of truncated Gag proteins form the basic building block of the capsid network.
Conclusions:
- The trimer of Gag domains is the fundamental unit of the cage-like network.
- The CA domain plays a critical role in trimeric interactions of the Gag polyprotein.
- Structural constraints within the CA domain influence Gag protein assembly.