Studies on the Assembly and Morphology of Human T-Cell Leukemia Virus Type 1

Abstract

The group-specific antigen (Gag) polyprotein is an essential retrovirus structural protein required for the assembly and release of virus particles. Present knowledge of Gag biology has been limited to a few retroviruses. Furthermore, current understanding of the diversity in the nature of Gag structure and function in virus particle assembly is limited. Human T-cell leukemia virus type 1 (HTLV-1) is a deltaretrovirus that causes an adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated-myelopathy/tropical spastic paraparesis (HAM/TSP), and other neurotropic conditions. HTLV-1 has infected approximately 15 million individuals worldwide. A general knowledge gap exists regarding the details of HTLV-1 replication, including particle assembly. To address this, and to test the overarching hypothesis that HTLV-1 particle assembly is distinct from that of other retroviruses, this dissertation focused on investigating three key aspects of HTLV-1 immature and mature particle morphology. First, an analysis of the morphology and Gag stoichiometry of HTLV-1-like particles and authentic, mature HTLV-1 particles by using cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission electron microscopy (STEM) was conducted. HTLV-1-like particles mimicked the morphology of immature authentic HTLV-1 virions. Importantly, it was observed for the first time that the morphology of these virus-like particles (VLPs) has the unique local feature of a flat Gag lattice that does not follow the curvature of the viral membrane, resulting in an enlarged distance between the Gag lattice and the viral membrane. Measurement of the average size and mass of VLPs and authentic HTLV-1 particles suggested a consistent range of size and Gag copy numbers in these two groups of particles. The unique local flat Gag lattice morphological feature observed suggests that HTLV-1 Gag could be arranged in a lattice structure that is distinct from that of other retroviruses characterized to date. Second, the effects of Gag proteins labeled at the carboxy terminus with a fluorophore protein were analyzed for their influence on particle morphology. In particular, a HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus was used as a surrogate for the HTLV-1 Gag-Pro to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle morphology and particles were analyzed by cryo-TEM. STEM and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. Ratios of 3:1 (Gag:Gag-YFP) or greater were found to result 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. This information is useful for the quantitative analysis of Gag-Gag interactions that occur during virus particle assembly and in released immature particles. Third, cryo-electron tomography (cryo-ET) was used to analyze mature HTLV-1 particle morphology. Particles produced from MT-2 cells were polymorphic, roughly spherical, and varied in size. Capsid cores, when present, were typically poorly defined polyhedral structures with at least one curved region contacting the inner face of the viral membrane. Most of the particles observed lacked a defined capsid core, which likely impacts HTLV-1 particle infectivity. Taken together, the findings of this dissertation provide new insights into the nature of immature and mature HTLV-1 assembly and morphology and provide foundational knowledge towards an advanced understanding of the HTLV particle assembly pathway.