Distinct Particle Morphologies Revealed through Comparative Parallel Analyses of Retrovirus-Like Particles

Abstract

The Gag protein is the main retroviral structural protein, and its expression alone is usually sufficient for production of viruslike particles (VLPs). In this study, we sought to investigate-in parallel comparative analyses-Gag cellular distribution, VLP size, and basic morphological features using Gag expression constructs (Gag or Gag-YFP, where YFP is yellow fluorescent protein) created from all representative retroviral genera: Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, and Spumavirus. We analyzed Gag cellular distribution by confocal microscopy, VLP budding by thinsection transmission electron microscopy (TEM), and general morphological features of the VLPs by cryogenic transmission electron microscopy (cryo-TEM). Punctate Gag was observed near the plasma membrane for all Gag constructs tested except for the representative Beta-and Epsilonretrovirus Gag proteins. This is the first report of Epsilonretrovirus Gag localizing to the nucleus of HeLa cells. While VLPs were not produced by the representative Beta-and Epsilonretrovirus Gag proteins, the other Gag proteins produced VLPs as confirmed by TEM, and morphological differences were observed by cryo-TEM. In particular, we observed Deltaretrovirus-like particles with flat regions of electron density that did not follow viral membrane curvature, Lentivirus-like particles with a narrow range and consistent electron density, suggesting a tightly packed Gag lattice, and Spumaviruslike particles with large envelope protein spikes and no visible electron density associated with a Gag lattice. Taken together, these parallel comparative analyses demonstrate for the first time the distinct morphological features that exist among retrovirus-like particles. Investigation of these differences will provide greater insights into the retroviral assembly pathway. IMPORTANCEComparative analysis among retroviruses has been critically important in enhancing our understanding of retroviral replication and pathogenesis, including that of important human pathogens such as human T-cell leukemia virus type 1 (HTLV-1) and HIV-1. In this study, parallel comparative analyses have been used to study Gag expression and virus-like particle morphology among representative retroviruses in the known retroviral genera. Distinct differences were observed, which enhances current knowledge of the retroviral assembly pathway. The Gag polyprotein is the primary retroviral structural protein responsible for orchestrating retrovirus assembly. HIV-1 Gag has arguably been the most extensively studied Gag polyprotein to date (1-7); however, distinct differences in the biology of retroviral Gag proteins have emphasized the importance of comparative analyses in order to gain further insights (8).The Gag polyprotein is functionally conserved across the two retroviral subfamilies (i.e., Orthoretrovirinae and Spumaretrovirinae). While the six genera in the Orthoretrovirinae subfamily (Alpha-, Beta-, Delta-, Epsilon-, Gamma-, and Lentivirus) an...

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

“…In particular, past observations of chimeric Gag proteins have been made for lentivirus-and alpharetrovirus-derived Gag proteins (28). The established differences in VLP assembly and morphology among different retroviral genera may offer an explanation for the differences observed in our present and previous studies (25,28). Furthermore, it is important to note that the CA subdomain chimeras contain marked alterations to the WT CA sequence due to the introduction of CA domains from another genus, which limits our ability to extrapolate our interpretations to WT Gag.…”

“…HeLa cells were cultured in six-well plates on no. 1.5 standard glass coverslips coated with poly-L-lysine, and experiments were performed as previously described (25). Cells were transiently transfected with eYFP-tagged Gag and untagged Gag expression plasmids at a 1:4 weight ratio using GenJet, version II (SignaGen, Gaithersburg, MD).…”

“…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.…”

“…Morphological differences between WT and chimeric Gag-based VLPs. HIV-1 and HTLV-1 WT Gag-based VLPs have established and distinct electron-dense regions characteristic of a Gag lattice (25). HTLV-1-like particles contain flat regions of Gag lattice electron density that do not follow the curvature of the lipid bilayer (Fig.…”

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

“…2B). Sequences were compared against the HTLV-1 reference sequence (RefSeq accession number NC_001436.1) and a codon-optimized gag sequence (28). The provirus harboring a truncated gag sequence on Chr 6 was predicted to have the largest amount of diversity of the three proteins, with significant variation in amino acid sequence, particularly in the MA domain of the protein.…”