Effects of mutant Vpr/Vpx on HIV-2 assembly demonstrated by immunoelectron microscopy.
The virion-associated accessory proteins Vpr and Vpx of human immunodeficiency virus type 2 (HIV-2) are required for efficient viral replication. Vpr could be important for Vpx assembly. To investigate the interaction of Vpr and Vpx with respect to the effects of reverse transcriptase (RT) activity, viral particle information and Vpx expression site directed mutagenesis was carried out to construct Vpr, Vpx and double Vpr/Vpx HIV-2 mutants. These mutants were used for infection of peripheral blood mononuclear cells (PBM), human acute lymphoblastomic leukaemia cells (CEM-CM3) and HeLa CD4+ cells. Visualization of Vpx expression was carried out using FITC and gold labelling by means of laser scanning confocal microscopy and semi quantitative immunoelectron microscopy. Intracellular and extracellular localizations of Vpx were determined by means of fine structural analysis. Up to 80-90% reduction in the RT activity, total number of viral particles, and average Vpx expression was observed after infection of target cells with the Vpr mutant strains. In addition, intracellular Vpx expression was reduced to 51.2% with the Vpr mutant. Only 0.02% Vpx expression was detected after mutation at amino acid 62. These results provide evidence that Vpr or Vpr/Vpx mutants reduce RT activity and interfere with the expression of Vpx in HIV-2 particles during viral assembly. Vpr is efficient for Vpx corporation during viral assembly.
Department and Institute of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan, ROC.
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JJ
Wan
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Ratner
D015704:CD4 Antigens; D002478:Cells, Cultured / Q000821:virology; D018389:Codon, Nonsense; D016336:Gene Products, vpr / Q000502:physiology*; D016334:Genes, vpr; D054303:HIV Reverse Transcriptase; D015498:HIV-2 / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D006367:HeLa Cells / Q000821:virology; D006801:Humans; D007150:Immunohistochemistry; D007963:Leukocytes, Mononuclear / Q000821:virology; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D009024:Morphogenesis; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology; D012194:RNA-Directed DNA Polymerase / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000821:virology; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000502:physiology*; D014771:Virion / Q000648:ultrastructure; D014779:Virus Replication; D054325:vpr Gene Products, Human Immunodeficiency Virus
D015704:CD4 Antigens; D018389:Codon, Nonsense; D016336:Gene Products, vpr; C074286:VPX protein, Human immunodeficiency virus 2; D054334:Viral Regulatory and Accessory Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase