Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry

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Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 cell-to-cell transmission is dependent on the release of infectious virus particles into the virological synapse. The HTLV-1 particle structure is still poorly understood, and previous studies analyzed viruses produced by transformed lymphocytic cell lines chronically infected with HTLV-1, particularly the MT-2 cell line, which harbors truncated proviruses and expresses aberrant forms of the Gag protein. In this study, we demonstrate that the chronically infected SP cell line harbors a relatively low number of proviruses, making it a more promising experimental system for the study of the HTLV-1 particle structure. We first identified the genomic sites of integration and characterized the genetic structure of the gag region in each provirus. We also determined that despite encoding a truncated Gag protein, only the full-length Gag protein was incorporated into virus particles. Cryotransmission electron microscopy analyses of the purified virus particles revealed three classes of particles based upon capsid core morphology: complete cores, incomplete cores, and particles without distinct electron densities that would correlate with the capsid region of a core structure. Observed cores were generally polygonal, and virus particles were on average 115 nm in diameter. These data corroborate particle morphologies previously observed for MT-2 cells and provide evidence that the known poor infectivity of HTLV-1 particles may correlate with HTLV-1 particle populations containing few virus particles possessing a complete capsid core structure. IMPORTANCE Studies of retroviral particle core morphology have demonstrated a correlation between capsid core stability and the relative infectivity of the virus. In this study, we used cryo-transmission electron microscopy to demonstrate that HTLV-1 particles produced from a distinct chronically infected cell line are polymorphic in nature, with many particles lacking organized electron densities that would correlate with a complete core structure. These findings have important implications for infectious HTLV-1 spread, particularly in the context of cell-to-cell transmission, a critical step in HTLV-1 transmission and pathogenesis.KEYWORDS Gag, core morphology, cryoelectron microscopy, human retrovirus, infectivity, retrovirus assembly H uman T-cell leukemia virus type 1 (HTLV-1) was the first-described human retrovirus and is estimated to have emerged in the human population via multiple events of zoonotic transmission from monkeys to humans (1-3). Some of these

“…Defocus values ranged from 4 to 8 m. Virus particles were measured by using ImageJ software. Two perpendicular diameters were measured and averaged for each virus particle (64).…”

Section: Discussionmentioning

confidence: 99%

Polymorphic Nature of Human T-Cell Leukemia Virus Type 1 Particle Cores as Revealed through Characterization of a Chronically Infected Cell Line

Meissner

Mendonça

Zhang

et al. 2017

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“…Codon-optimized HIV-1 and HTLV-1 gag genes in the pN3 and peYFP-N3 vectors have been previously described (25,54). WT and chimeric Gag expression constructs were engineered into vectors with C-terminal eYFP, mCherry, and HA tags using 5= HindIII and 3= BamHI restriction sites or were untagged.…”

Section: Methodsmentioning

confidence: 99%

Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis

Martin

Mendonça

Angert

et al. 2017

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The capsid domain (CA) of the retroviral Gag protein is a primary determinant of Gag oligomerization, which is a critical step for immature Gag lattice formation and virus particle budding. Although the human immunodeficiency virus type 1 (HIV-1) CA carboxy-terminal domain (CTD) is essential for CA-CA interactions, the CA CTD has been suggested to be largely dispensable for human T-cell leukemia virus type 1 (HTLV-1) particle biogenesis. To more clearly define the roles of the HTLV-1 CA amino-terminal domain (NTD) and CA CTD in particle biogenesis, we generated and analyzed a panel of Gag proteins with chimeric HIV-1/HTLV-1 CA domains. Subcellular distribution and protein expression levels indicated that Gag proteins with a chimeric HIV-1 CA NTD/HTLV-1 CA CTD did not result in Gag oligomerization regardless of the parent Gag background. Furthermore, chimeric Gag proteins with the HTLV-1 CA NTD produced particles phenotypically similar to HTLV-1 immature particles, highlighting the importance of the HTLV-1 CA NTD in HTLV-1 immature particle morphology. Taken together, these observations support the conclusion that the HTLV-1 CA NTD can functionally replace the HIV-1 CA CTD, but the HIV-1 CA NTD cannot replace the HTLV-1 CA CTD, indicating that the HTLV-1 CA subdomains provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. Furthermore, we have shown for the first time that HIV-1 and HTLV-1 Gag domains outside the CA (e.g., matrix and nucleocapsid) impact Gag oligomerization as well as immature particle size and morphology. IMPORTANCE A key aspect in virus replication is virus particle assembly, which is a poorly understood process for most viruses. For retroviruses, the Gag structural protein is the primary driver of virus particle biogenesis, and the CA CTD is the primary determinant of Gag-Gag interactions for HIV-1. In this study, the HTLV-1 capsid amino-terminal domain was found to provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. This study provides information that will aid efforts for discovery of therapeutic targets for intervention.

“…The HTLV-1 particles are larger than the virus-like particles previously produced from 293T cells by an HTLV-1 Gag expression construct with yellow fluorescent protein (YFP) fused to the carboxy terminus (18), suggesting that the YFP influenced the particle diameter. The capsid cores in HTLV-1 virions have a polyhedron-like structure.…”

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confidence: 98%

“…Previous structural analyses of HTLV-1 assembly have largely been limited to observing HTLV-1-infected cells by using thin-section transmission electron microscopy (TEM). In those studies, HTLV-1 particles produced from chronically infected MT-2 cells (17) appeared to be assembly intermediates, with the electron densities inside individual particles forming either a half-donut shape (18,19) or a complete donut shape (17) along the inside of the viral membrane. Virus particles were observed that had a distinct capsid core or had the electron densities evenly distributed in the interior of the particle in the absence of a core structure.…”

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confidence: 99%

“…Two new flasks were then each inoculated with ϳ5% of the suspended cells from the first expansion cycle and filled with 25 ml of culture medium supernatant from the previous expansion cycle and 25 ml of fresh medium. The cultures were then grown for an additional week, after which HTLV-1 particles were purified from the culture medium supernatant and concentrated using a procedure modified from that of Grigsby et al (18,19). In particular, after removing large cellular debris by centrifugation and filtration, the sample was pelleted using an 8% OptiPrep (Sigma-Aldrich) cushion and applied to a 10-to-50% OptiPrep gradient for centrifugation.…”

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confidence: 99%

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Analysis of Human T-Cell Leukemia Virus Type 1 Particles by Using Cryo-Electron Tomography

Cao

Maldonado

Grigsby

et al. 2015

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The particle structure of human T-cell leukemia virus type 1 (HTLV-1) is poorly characterized. Here, we have used cryo-electron tomography to analyze 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.