Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.
BACKGROUND
HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm.


RESULTS
Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core.


CONCLUSIONS
The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.
Division of Pediatric Infectious Diseases, Emory University School of Medicine, Atlanta, GA, USA. tanay.desai@emory.edu.
Tanay M
TM
Desai
Division of Pediatric Infectious Diseases, Emory University School of Medicine, Atlanta, GA, USA. mariana.marin@emory.edu.
Mariana
M
Marin
Division of Pediatric Infectious Diseases, Emory University School of Medicine, Atlanta, GA, USA. chetan.sood@emory.edu.
Chetan
C
Sood
Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, 37232, USA. jiong.shi@Vanderbilt.Edu.
Jiong
J
Shi
Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, 37232, USA. fatima.nawaz@vanderbilt.edu.
Fatima
F
Nawaz
Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, 37232, USA. chris.aiken@Vanderbilt.edu.
Christopher
C
Aiken
Division of Pediatric Infectious Diseases, Emory University School of Medicine, Atlanta, GA, USA. gmeliki@emory.edu.|Children's Healthcare of Atlanta, Atlanta, GA, USA. gmeliki@emory.edu.
Gregory B
GB
Melikyan
D021581:Active Transport, Cell Nucleus; D036022:Capsid Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure / Q000821:virology; D003593:Cytoplasm / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D013050:Spectrometry, Fluorescence / Q000379:methods; D014771:Virion / Q000502:physiology; D053586:Virus Internalization; D014779:Virus Replication; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D036022:Capsid Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus