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.
Highlights
IntroductionHuman T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus identified, and it is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) and the neurological disorder
Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus identified, and it is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) and the neurological disorderHTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) [1,2]
We found that ratios of 3:1 (Gag:Gag-yellow fluorescence protein (YFP)) or greater resulted in a particle morphology indistinguishable from that of virus-like particle (VLP) produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 83 to 120 MDa as determined by cryo-transmission electron microscopy (TEM) and Scanning transmission electron microscopy (STEM), respectively
Summary
Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus identified, and it is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) and the neurological disorder. HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) [1,2]. It is estimated that 15 million people are infected with HTLV-1 worldwide [3,4]. Regions in the world with high prevalence levels include the Caribbean basin, South America, Central Africa, southwestern Japan, southern. The Middle East [4]. About 5% of HTLV-1 infected individuals develop an aggressive form of ATLL [5,6]. HTLV-1 is a deltaretrovirus and like other retroviruses, requires the expression of the Gag polyprotein—the main retroviral structural protein—to drive particle assembly and release [7].
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