Expression of simian type D retroviral (Mason-Pfizer monkey virus) capsids in insect cells using recombinant baculovirus.
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.
University of Alabama at Birmingham, Department of Microbiology, Birmingham 35294.
M A
MA
Sommerfelt
C R
CR
Roberts
E
E
Hunter
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002213:Capsid / Q000235:genetics* / Q000648:ultrastructure; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015870:Gene Expression; D015750:Genes, gag; D015751:Genes, pol; D066298:In Vitro Techniques; D016093:Mason-Pfizer monkey virus / Q000235:genetics* / Q000648:ultrastructure; D009024:Morphogenesis; D009036:Moths; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014779:Virus Replication
D012333:RNA, Messenger; D011994:Recombinant Proteins