A novel method for in vitro radiolabeling and testing enveloped viruses by phosphatidylethanolamine N-methyltransferase and host cell-specific binding.
The present study developed a novel virus labeling and testing method, referred to as an envelope-labeled virus assay (ELVA), in which virus envelope is labeled in vitro by the action of phosphatidylethanolamine N-methyltransferase (PEMT) and tested through a host cell-specific binding. A recombinant strain (vGFPuv) of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and Spodoptera frugiperda (Sf-9) insect cells were used as a model of viruses and host cells, respectively. The labeling mixture, which contained PEMT, [methyl-3H]S-adenosylmethionine (SAM), and a trace amount of detergent Triton X-100, brought about little change in virus titer of vGFPuv on a 1-h incubation, but was so toxic to Sf-9 cells as to immediately cause cell death. After being incubated with vGFPuv, therefore, the labeling mixture was neutralized by adsorptive removal of PEMT and Triton X-100 before Sf-9 cells were contacted with the mixture to extract the virus. The Sf-9 cells were then washed with a phosphate buffered saline (PBS), and lipid extracts with a 1% SDS solution were subjected to a liquid scintillation analysis for the determination of labeling efficiency. As a result, a significant amount of radioactivity was determined in the extracts, demonstrating the validity of ELVA for labeling and testing enveloped viruses. The conditions for the PEMT reaction and cell-virus binding were examined, and the lower detection limit of AcMNPV by ELVA was found to lie in the order of 10(3) plaque forming unit (pfu) per milliliter. Since the labeling reaction and detection of virus are based on neither immunological nor genetic characteristics of virus, ELVA is also expected to be a convenient and comprehensive test of other enveloped viruses.
Department of Materials-Process Engineering and Applied Chemistry for Environments, Akita University, 1-1 Tegata Gakuen-cho, Akita 010-8502, Japan. tgotoh@ac5.as.akita-u.ac.jp
Takeshi
T
Gotoh
Nozomi
N
Ando
Ken-Ichi
K
Kikuchi
D000818:Animals; D016367:Baculoviridae / Q000302:isolation & purification* / Q000502:physiology*; D001665:Binding Sites; D002460:Cell Line; D007553:Isotope Labeling / Q000379:methods; D050918:Phosphatidylethanolamine N-Methyltransferase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods*; D018411:Spodoptera / Q000821:virology*; D014316:Tritium; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000378:metabolism*; D010948:Viral Plaque Assay / Q000379:methods*
D014759:Viral Envelope Proteins; D014316:Tritium; D050918:Phosphatidylethanolamine N-Methyltransferase