Rotavirus protein rearrangements in purified membrane-enveloped intermediate particles.
Rotavirus, a double-shelled nonenveloped member of the REoviridae family, becomes transiently membrane enveloped during its maturation process, as single-shelled particles bud from cytoplasmic viroplasm structures into the adjacent endoplasmic reticulum. The present study describes the isolation of these membrane-enveloped viral intermediates from rotavirus SA11-infected Ma104 cells. The enveloped intermediates comprised the proteins VP1, VP2, VP4, VP6, VP7, and NS28 and small amounts of NS35 and NS34. VP7 in the intermediate particles was recognized by either a polyclonal antibody to VP7, which previous studies had shown recognizes the membrane-associated form of VP7, or a monoclonal antibody which recognizes VP7 on mature virus. NS28, VP7, and VP4 could be complexed to a higher-molecular-weight form when the membrane-permeable cross-linker dithiobis(succinimidylproprionate) was used. However, when an impermeable cross-linker was used, the structural proteins, including VP7, were not accessible to cross-linking. Velocity sedimentation of cross-linked immunoisolated enveloped virus particles showed that VP7 and VP4 were located in the same fractions only when the membrane-permeable cross-linker was used, implying their heterooligomeric association during outer capsid formation. When intermediate enveloped virus particles were treated with protease, VP6 and VP7 were protected, but not in the presence of detergent. Taken together, these results support the idea that in the membrane-enveloped intermediate, VP7 is repositioned from its location in the endoplasmic reticulum lumen back across the viral membrane envelope to the inferior of the virus particle during the maturation process.
Poruchynsky
M S
MS
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine--CRI 501, Bronx, New York 10461.
Atkinson
P H
PH
D002213:Capsid / Q000276:immunology / Q000378:metabolism; D003432:Cross-Linking Reagents; D010450:Endopeptidases / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D008854:Microscopy, Electron; D009024:Morphogenesis; D011233:Precipitin Tests; D012401:Rotavirus / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure*; D014758:Viral Core Proteins / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure*; D017361:Viral Nonstructural Proteins; D014779:Virus Replication
D003432:Cross-Linking Reagents; D008562:Membrane Glycoproteins; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; D010450:Endopeptidases