Incorporation of human immunodeficiency virus type 1 reverse transcriptase into virus-like particles.
We demonstrate that a genetically engineered human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) composed mainly of p66 or p51 subunits can be incorporated into virus-like particles (VLPs) when coexpressed with HIV-1 Pr55(gag). VLP-associated RT exhibited a detergent-resistant association with immature cores during sucrose gradient equilibrium centrifugation, suggesting that RT is incorporated into VLPs. However, RT that retains downstream integrase (IN) is severely inhibited in terms of incorporation into VLPs. Results from immunofluorescence tests reveal that RT-IN is primarily localized at the perinuclear area and exhibits poor colocalization with Gag. IN removal leads to a redistribution of RT throughout the cytoplasm and improved RT incorporation into VLPs. Similar results were observed for RT-IN in which alanine was substituted for 186-Lys-Arg-Lys-188 residues of the IN putative nuclear localization signal, suggesting that IN karyophilic properties may partly account for the inhibitory effect of IN on RT incorporation. Although the membrane-binding capacity of RT was markedly reduced compared to that of wild-type Gag or Gag-Pol, the correlation of membrane-binding ability with particle incorporation efficiency was incomplete. Furthermore, we observed that membrane-binding-defective myristylation-minus RT can be packaged into VLPs at the same level as its normal myristylated counterpart. This suggests that the incorporation of RT into VLPs is independent of membrane affinity but very dependent on RT-Gag interaction. Results from a genetic analysis suggest that the Gag-interacting regions of RT mainly reside in the thumb subdomain and that the RT-binding domains of Gag are located in the matrix (MA) and p6 regions.
Liao
Wei-Hao
WH
Department of Medical Research and Education, Taipei Veterans General Hospital, Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei 11217, Taiwan.
Huang
Kuo-Jung
KJ
Chang
Yu-Fen
YF
Wang
Shiu-Mei
SM
Tseng
Ying-Tzu
YT
Chiang
Chien-Cheng
CC
Wang
Jaang-Jiun
JJ
Wang
Chin-Tien
CT
D019943:Amino Acid Substitution; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism*; D019065:Virus Assembly
D015683:Gene Products, gag; D019913:Nuclear Localization Signals; D011498:Protein Precursors; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase