Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging
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) prote...
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MDPI AG
2017-07-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/9/7/191 |
| _version_ | 1818421323509006336 |
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| author | José O. Maldonado Isaac Angert Sheng Cao Serkan Berk Wei Zhang Joachim D. Mueller Louis M. Mansky |
| author_facet | José O. Maldonado Isaac Angert Sheng Cao Serkan Berk Wei Zhang Joachim D. Mueller Louis M. Mansky |
| author_sort | José O. Maldonado |
| collection | DOAJ |
| description | 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. |
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| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-12-14T13:08:32Z |
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| spelling | doaj.art-ef7895b2293d44cea0dd0d672b49f3df2022-12-21T23:00:15ZengMDPI AGViruses1999-49152017-07-019719110.3390/v9070191v9070191Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-PackagingJosé O. Maldonado0Isaac Angert1Sheng Cao2Serkan Berk3Wei Zhang4Joachim D. Mueller5Louis M. Mansky6Institute for Molecular Virology, D.D.S.-Ph.D. Dual Degree Program, University of Minnesota, Minneapolis, MN 55455, USAInstitute for Molecular Virology, Institute for Molecular Virology Training Program, School of Physics & Astronomy, University of Minnesota, Minneapolis, MN 55455, USAInstitute for Molecular Virology, Department of Diagnostic and Biological Sciences, School of Dentistry, University of Minnesota, Minneapolis, MN 55455, USAInstitute for Molecular Virology, Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USAInstitute for Molecular Virology, Department of Diagnostic and Biological Sciences, School of Dentistry, Characterization Facility, College of Science and Engineering, Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USAInstitute for Molecular Virology, School of Physics & Astronomy, Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USAInstitute for Molecular Virology, Division of Basic Sciences, School of Dentistry, Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USAHuman 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.https://www.mdpi.com/1999-4915/9/7/191scanning transmission electron microscopy (STEM)cryogenic transmission electron microscopy (cryo-TEM)fluorescence fluctuation spectroscopy (FFS)Gag stoichiometryhuman T-cell leukemia virus type 1 (HTLV-1) |
| spellingShingle | José O. Maldonado Isaac Angert Sheng Cao Serkan Berk Wei Zhang Joachim D. Mueller Louis M. Mansky Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging Viruses scanning transmission electron microscopy (STEM) cryogenic transmission electron microscopy (cryo-TEM) fluorescence fluctuation spectroscopy (FFS) Gag stoichiometry human T-cell leukemia virus type 1 (HTLV-1) |
| title | Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging |
| title_full | Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging |
| title_fullStr | Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging |
| title_full_unstemmed | Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging |
| title_short | Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging |
| title_sort | perturbation of human t cell leukemia virus type 1 particle morphology by differential gag co packaging |
| topic | scanning transmission electron microscopy (STEM) cryogenic transmission electron microscopy (cryo-TEM) fluorescence fluctuation spectroscopy (FFS) Gag stoichiometry human T-cell leukemia virus type 1 (HTLV-1) |
| url | https://www.mdpi.com/1999-4915/9/7/191 |
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