HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription Sites
The ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency vir...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-11-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/12/11/1281 |
| _version_ | 1827702530930376704 |
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| author | Kevin M. Tuffy Rebecca J. Kaddis Maldonado Jordan Chang Paul Rosenfeld Alan Cochrane Leslie J. Parent |
| author_facet | Kevin M. Tuffy Rebecca J. Kaddis Maldonado Jordan Chang Paul Rosenfeld Alan Cochrane Leslie J. Parent |
| author_sort | Kevin M. Tuffy |
| collection | DOAJ |
| description | The ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency virus, FIV; Mason-Pfizer monkey virus, MPMV; mouse mammary tumor virus, MMTV; murine leukemia virus, MLV; and prototype foamy virus, PFV) have also been observed in the nucleus, little is known about what, if any, role nuclear trafficking plays in those viruses. In the case of HIV-1, the Gag protein interacts in nucleoli with the regulatory protein Rev, which facilitates nuclear export of gRNA. Based on the knowledge that RSV Gag forms viral ribonucleoprotein (RNPs) complexes with unspliced viral RNA (USvRNA) in the nucleus, we hypothesized that the interaction of HIV-1 Gag with Rev could be mediated through vRNA to form HIV-1 RNPs. Using inducible HIV-1 proviral constructs, we visualized HIV-1 Gag and USvRNA in discrete foci in the nuclei of HeLa cells by confocal microscopy. Two-dimensional co-localization and RNA-immunoprecipitation of fractionated cells revealed that interaction of nuclear HIV-1 Gag with USvRNA was specific. Interestingly, treatment of cells with transcription inhibitors reduced the number of HIV-1 Gag and USvRNA nuclear foci, yet resulted in an increase in the degree of Gag co-localization with USvRNA, suggesting that Gag accumulates on newly synthesized viral transcripts. Three-dimensional imaging analysis revealed that HIV-1 Gag localized to the perichromatin space and associated with USvRNA and Rev in a tripartite RNP complex. To examine a more biologically relevant cell, latently infected CD4+ T cells were treated with prostratin to stimulate NF-κB mediated transcription, demonstrating striking localization of full-length Gag at HIV-1 transcriptional burst site, which was labelled with USvRNA-specific riboprobes. In addition, smaller HIV-1 RNPs were observed in the nuclei of these cells. These data suggest that HIV-1 Gag binds to unspliced viral transcripts produced at the proviral integration site, forming vRNPs in the nucleus. |
| first_indexed | 2024-03-10T14:58:37Z |
| format | Article |
| id | doaj.art-33ba0114dad24f4482398d2079ab8307 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T14:58:37Z |
| publishDate | 2020-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-33ba0114dad24f4482398d2079ab83072023-11-20T20:20:35ZengMDPI AGViruses1999-49152020-11-011211128110.3390/v12111281HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription SitesKevin M. Tuffy0Rebecca J. Kaddis Maldonado1Jordan Chang2Paul Rosenfeld3Alan Cochrane4Leslie J. Parent5Division of Infectious Diseases and Epidemiology, Department of Medicine, Penn State College of Medicine, Hershey, PA 17033, USADivision of Infectious Diseases and Epidemiology, Department of Medicine, Penn State College of Medicine, Hershey, PA 17033, USADivision of Infectious Diseases and Epidemiology, Department of Medicine, Penn State College of Medicine, Hershey, PA 17033, USAThe Institute of Medical Sciences, University of Toronto, Toronto, ON MS5 1A8, CanadaThe Institute of Medical Sciences, University of Toronto, Toronto, ON MS5 1A8, CanadaDivision of Infectious Diseases and Epidemiology, Department of Medicine, Penn State College of Medicine, Hershey, PA 17033, USAThe ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency virus, FIV; Mason-Pfizer monkey virus, MPMV; mouse mammary tumor virus, MMTV; murine leukemia virus, MLV; and prototype foamy virus, PFV) have also been observed in the nucleus, little is known about what, if any, role nuclear trafficking plays in those viruses. In the case of HIV-1, the Gag protein interacts in nucleoli with the regulatory protein Rev, which facilitates nuclear export of gRNA. Based on the knowledge that RSV Gag forms viral ribonucleoprotein (RNPs) complexes with unspliced viral RNA (USvRNA) in the nucleus, we hypothesized that the interaction of HIV-1 Gag with Rev could be mediated through vRNA to form HIV-1 RNPs. Using inducible HIV-1 proviral constructs, we visualized HIV-1 Gag and USvRNA in discrete foci in the nuclei of HeLa cells by confocal microscopy. Two-dimensional co-localization and RNA-immunoprecipitation of fractionated cells revealed that interaction of nuclear HIV-1 Gag with USvRNA was specific. Interestingly, treatment of cells with transcription inhibitors reduced the number of HIV-1 Gag and USvRNA nuclear foci, yet resulted in an increase in the degree of Gag co-localization with USvRNA, suggesting that Gag accumulates on newly synthesized viral transcripts. Three-dimensional imaging analysis revealed that HIV-1 Gag localized to the perichromatin space and associated with USvRNA and Rev in a tripartite RNP complex. To examine a more biologically relevant cell, latently infected CD4+ T cells were treated with prostratin to stimulate NF-κB mediated transcription, demonstrating striking localization of full-length Gag at HIV-1 transcriptional burst site, which was labelled with USvRNA-specific riboprobes. In addition, smaller HIV-1 RNPs were observed in the nuclei of these cells. These data suggest that HIV-1 Gag binds to unspliced viral transcripts produced at the proviral integration site, forming vRNPs in the nucleus.https://www.mdpi.com/1999-4915/12/11/1281HIV-1 Gagviral RNAnucleusRev |
| spellingShingle | Kevin M. Tuffy Rebecca J. Kaddis Maldonado Jordan Chang Paul Rosenfeld Alan Cochrane Leslie J. Parent HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription Sites Viruses HIV-1 Gag viral RNA nucleus Rev |
| title | HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription Sites |
| title_full | HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription Sites |
| title_fullStr | HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription Sites |
| title_full_unstemmed | HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription Sites |
| title_short | HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription Sites |
| title_sort | hiv 1 gag forms ribonucleoprotein complexes with unspliced viral rna at transcription sites |
| topic | HIV-1 Gag viral RNA nucleus Rev |
| url | https://www.mdpi.com/1999-4915/12/11/1281 |
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