Abstract
Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly. How these tags affect Gag stoichiometry and particle morphology has not been reported in detail. In this study, we used an HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus as a surrogate for the HTLV-1 Gag-Pol to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle (VLP) morphology and analyzed particles by cryogenic transmission electron microscopy (cryo-TEM). Scanning transmission electron microscopy (STEM) and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, FFS analysis indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 Gag:Gag-YFP ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was is in the range of 1500-2000 molecules per VLP. The observations made in this study indicate that biologically relevant Gag-Gag interactions occur between Gag and Gag-YFP at ratios of 3:1 or higher and create a Gag lattice structure in VLPs that is morphologically indistinguishable from that of VLPs produced with just untagged Gag. This information is useful for the quantitative analysis of Gag-Gag interactions that occur during virus particle assembly and in released immature particles.
| Original language | English (US) |
|---|---|
| Article number | 191 |
| Journal | Viruses |
| Volume | 9 |
| Issue number | 7 |
| DOIs | |
| State | Published - Jul 19 2017 |
Bibliographical note
Funding Information:We thank Joe Wall (Brookhaven National Laboratory, NY, USA) for assistance with the STEM analyses. Cryo-TEM images were recorded using a Tecnai TF30 TEM maintained by the Characterization Facility, College of Science and Engineering, University of Minnesota. We thank Robert Hafner (Characterization Facility) for assisting in the collection images of the fluorescence labeled samples with the iCorr. This work was supported by NIH(National Institutes of Health) grant RO1 GM098550. J.O.M. was supported by NIH grant F30 DE22286 and both J.O.M. and I.A. were supported by NIH grant T32 AI083196 (Institute for Molecular Virology Training Program).
Publisher Copyright:
© 2017 by the authors. Licensee MDPI, Basel, Switzerland.
Keywords
- Cryogenic transmission electron microscopy (cryo-TEM)
- Fluorescence fluctuation spectroscopy (FFS)
- Gag stoichiometry
- Human T-cell leukemia virus type 1 (HTLV-1)
- Scanning transmission electron microscopy (STEM)
