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Expression of simian type D retroviral (Mason-Pfizer monkey virus) capsids in insect cells using recombinant
M A Sommerfelt1, C R Roberts, E Hunter
1University of Alabama at Birmingham, Department of Microbiology, Birmingham 35294.
Abstract:
Mason-Pfizer monkey virus (M-PMV) is a primate retrovirus that shows type D morphogenesis in mammalian cells. Immature intracytoplasmic A type particles (ICAPs) preassemble in the infected cell cytoplasm migrate to the plasma membrane and are released by budding. This is in contrast to retroviruses that show type C morphogenesis, where assembly and budding occur concurrently at the plasma membrane. We expressed the M-PMV structural genes (gag-pro-pol) in insect cells using a recombinant baculovirus. The polyprotein precursors assembled predominantly intracellularly, although a small proportion also assembled at the membrane. The protease enzyme was active since mature particles were identified in the culture supernatant. We also expressed the M-PMV mutants, D26N and gag-STOP, which carry a nonfunctional protease or fail to express the protease gene, respectively. These baculovirus recombinants generated a homogeneous population of immature M-PMV capsids having exclusively type D morphogenesis. Sufficient quantities of polyprotein precursors were synthesized to be visualized directly on a Coomassie-stained protein gel, and the capsids were subject to purification. These results provide the first expression of type D retrovirus particles using the baculovirus expression system.
Insights
Researchers successfully expressed Mason-Pfizer monkey virus (M-PMV) type D retrovirus particles in insect cells using baculovirus. This system efficiently produced immature M-PMV capsids, advancing retroviral research.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Mason-Pfizer monkey virus (M-PMV) is a primate retrovirus exhibiting type D morphogenesis.
- M-PMV assembly involves intracellular preassembly of immature intracytoplasmic A type particles (ICAPs) followed by budding.
- This contrasts with type C retroviruses where assembly and budding are concurrent at the plasma membrane.
Purpose of the Study:
- To express M-PMV structural genes (gag-pro-pol) in insect cells using a recombinant baculovirus system.
- To investigate the intracellular assembly and morphogenesis of M-PMV particles.
- To generate and purify M-PMV capsids for further study.
Main Methods:
- Recombinant baculovirus vectors were constructed to express M-PMV gag-pro-pol genes.
- Insect cells were infected with the recombinant baculovirus.
- Expressed proteins and assembled particles were analyzed using protein gels and microscopy.
- Mutants D26N (nonfunctional protease) and gag-STOP (no protease expression) were used to study protease activity.
Main Results:
- The baculovirus expression system successfully produced M-PMV polyprotein precursors in insect cells.
- Polyprotein precursors predominantly assembled intracellularly, consistent with type D morphogenesis.
- Active protease enzyme was detected, leading to mature particles in the supernatant.
- Mutant constructs yielded homogeneous populations of immature M-PMV capsids with type D morphogenesis.
- Sufficient quantities of precursors were synthesized for visualization and purification.
Conclusions:
- The baculovirus expression system is effective for producing M-PMV structural proteins and type D retroviral particles.
- This system allows for the generation of homogeneous immature M-PMV capsids, facilitating detailed structural and functional studies.
- This represents the first successful expression of type D retrovirus particles using the baculovirus system.