Alpha-complementation assay for HIV envelope glycoprotein-mediated fusion.
The fusion reaction mediated by viral envelope glycoproteins proceeds through an ordered series of conformational changes in the envelope glycoprotein. Fusion inhibitors have been developed that target glycoprotein subunits, arresting the reaction at different points in the process. We report the development of a novel method for detecting viral glycoprotein-mediated fusion that is based on the principle of alpha-complementation of beta-galactosidase. The method is simple, accurate, has a high signal-to-noise ratio, is suited for high-throughput screening, and does not require new transcription or protein synthesis. Cells expressing a viral envelope glycoprotein and the N-terminal alpha fragment of beta-galactosidase were mixed with cells expressing the C-terminal beta-galactosidase fragment, CD4, CCR5, or CXCR4. Fusion was detected after 30 min and continued to increase to very high levels for more than 5 h. The assay was used to examine the temperature dependence of fusion and the effect of coreceptor and glycoprotein density on inhibitor activity.
Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
Anne U
AU
Holland
Carsten
C
Munk
Ginger R
GR
Lucero
Lucia D
LD
Nguyen
Nathaniel R
NR
Landau
D002459:Cell Fusion; D002460:Cell Line; D005816:Genetic Complementation Test; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008561:Membrane Fusion; D013696:Temperature; D014759:Viral Envelope Proteins / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000378:metabolism*; D014779:Virus Replication; D001616:beta-Galactosidase / Q000737:chemistry
D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins; D001616:beta-Galactosidase