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Journal of Virology, May 2007, p. 5155-5165, Vol. 81, No. 10
0022-538X/07/$08.00+0     doi:10.1128/JVI.01796-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Incorporation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase into Virus-Like Particles{triangledown}

Wei-Hao Liao,1 Kuo-Jung Huang,1 Yu-Fen Chang,1 Shiu-Mei Wang,1 Ying-Tzu Tseng,1 Chien-Cheng Chiang,1 Jaang-Jiun Wang,2 and Chin-Tien Wang1*

Department of Medical Research and Education, Taipei Veterans General Hospital, and Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan,1 Graduate Institute of Medicine, Fu Jen Catholic University, Taipei, Taiwan2

Received 17 August 2006/ Accepted 20 February 2007

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 Pr55gag. 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.

* Corresponding author. Mailing address: Department of Medical Research and Education, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road, Taipei 11217, Taiwan. Phone: 886-2-28712121, ext. 2655. Fax: 886-2-28742279. E-mail: chintien{at}ym.edu.tw

{triangledown} Published ahead of print on 7 March 2007.

Journal of Virology, May 2007, p. 5155-5165, Vol. 81, No. 10
0022-538X/07/$08.00+0     doi:10.1128/JVI.01796-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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