# Centrifugation-Based Purification Protocol Optimization Enhances Structural Preservation of Nucleopolyhedrovirus Budded Virion Envelopes

## TL;DR

An optimized purification method significantly improves the structural preservation of viral envelopes in baculoviruses, aiding structural studies and potential medical applications.

## Contribution

An optimized continuous sucrose density gradient protocol enhances envelope integrity of baculovirus virions.

## Key findings

- Optimized purification increased intact viral envelopes from 36% to 81%.
- Cryo-EM confirmed preserved prefusion conformation of GP64 envelope protein.
- Improved methods support structural studies and applications in gene therapy and vaccines.

## Abstract

Baculoviruses are widely used for scientific and medical applications, but purifying baculovirus virions without damaging their viral envelopes is a challenge. In this study, we compared the effects of three purification methods on the percentage of budded virions (BVs) with intact envelopes. Compared to the traditional protocols, the optimized continuous sucrose density gradient purification increased the percentage of intact viral envelopes from 36% to 81%. Cryo-electron microscopy (cryo-EM) confirmed preserved prefusion conformations of the envelope protein GP64. This advancement supports structural studies of envelope proteins and may improve applications in gene therapy and vaccine development using enveloped viruses.

The structural integrity of viral envelopes is a critical determinant of infectivity for enveloped viruses, directly influencing vector stability, functional accuracy of surface-displayed epitopes, and preservation of native conformational states required for membrane protein studies. However, conventional purification methods often disrupt envelope integrity and cause envelope proteins to lose their activity. Here, we systematically compared discontinuous, continuous, and optimized continuous sucrose density gradient centrifugation protocols for purifying Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Through cryo-EM, we demonstrated that our optimized continuous sucrose gradient protocol significantly increased the proportion of AcMNPV budded virions with intact envelopes from 36% to 81%, while preserving the metastable prefusion conformation of the fusion protein GP64. This advancement should prove useful for structural studies of viral envelope proteins and may enhance applications in gene therapy and vaccine development utilizing enveloped viruses.