Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging

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.

Determinants in the HTLV-1 capsid major homology region that are critical for virus particle assembly

“…Given previous observations that residues in retroviral CA MHR region play a critical role in viral particle assembly, including HIV-1 [22][23][24][25][26][27][28][29][30][31], RSV [31][32][33][34], MPMV [35] and MLV [36], we sought to address this knowledge gap with HTLV-1 CA by conducting alanine-scanning mutagenesis of the entire HTLV-1 CA MHR domain (Figure 1). In order to analyze particle production, we utilized a previously characterized tractable HTLV-1-like particle system to assess the impact of individual mutations on authentic, immature virus particle assembly and release [20,38,39]. With the exception of E142 [37], 19 of the 20 amino acid residues in the MHR domain have not been previously characterized for HTLV-1.…”

Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context

“…In this work we investigated HBV capsid assembly in Huh7 hepatoma mammalian cells by combining FLIM-FRET, FCS and TEM. Combination of modern imaging techniques had previously been used to image the assembly of HTLV, RSV or HIV and to quantify the interaction between viral and cellular partners [47,48,[59][60][61][62][63][64][65][66]. These approaches provided data that revolutionized our knowledge of the dynamic and structural changes involved during viruses infectious cycle (for reviews see [67][68][69][70]) as well as in other fields of biology [71].…”

“…2, lower panels). This combination of plasmids contained 0.7 µg of plasmid coding for unlabeled HBc and 0.3 µg of plasmid coding for eGFP-labelled HBc, to attenuate the impact of eGFP on HBc assembly according to a previously-reported strategy [37,47,48]. Nuclei were stained with DAPI, and proteins were localized through the eGFP fluorescence.…”

Virus-like Particles as Preventive and Therapeutic Cancer Vaccines

“…The HTLV-1 is endemic in several geographic regions, including Japan, the Caribbean, Africa, South America, and the Melanesian islands. The Gag proteins have been used to generate produce HTLV-1-like particles [25,70]. Gag polyprotein is known to be the primary driver for virus particle assembly and release.…”