The present invention provides a rapid virus entry/binding detection assay. An enzyme such as luciferase was incorporated at the C-terminal end of viral envelope proteins of the HIV Nef protein that would specifically associate with cell membranes to deliver the enzyme into viral particles upon viral assembly. Virus entry/binding can then be assayed by determining the enzymatic activities in infected cells. The assay allows high-throughput non-radioactive detection of virus entry within 30 minutes after virus-cell contact. This assay provides high signal to noise ratio and is useful for screening compounds that affect virus-cell binding and entry. The design also permits packaging of potential therapeutic proteins into functional virus particles and delivering them to specific cellular targets.