Incorporation of Pol into Human Immunodeficiency Virus Type 1 Gag Virus-Like Particles Occurs Independently of the Upstream Gag Domain in Gag-Pol

Shan Cen, Meijuan Niu, Jenan Saadatmand, Fei Guo, Yue Huang, Gary J. Nabel, Lawrence Kleiman

Research output: Contribution to journalArticle

Abstract

By using particle-associated reverse transcriptase (RT) activity as an assay for Pol incorporation into human immunodeficiency virus type 1 (HIV-1) Gag virus-like particles (VLPs), it has been found that truncated, protease-negative, Gag-Pol missing cis Gag sequences is still incorporated into Gag VLPs, albeit at significantly reduced levels (10 to 20% of the level of wild-type Gag-Pol). In this work, we have directly measured the incorporation of truncated Gag-Pol species into Gag VLPs and have found that truncated Gag-Pol that is missing all sequences upstream of RT is still incorporated into Gag VLPs at levels approximating 70% of that achieved by wild-type Gag-Pol. Neither protease nor integrase regions in Pol are required for its incorporation, implying an interaction between Gag and RT sequences in the Pol protein. While the incorporation of Gag-Pol into Gag VLPs is reduced 12-fold by the replacement of the nucleocapsid within Gag with a leucine zipper motif, this mutation does not affect Pol incorporation. However, the deletion of p6 in Gag reduces Pol incorporation into Gag VLPs four- to fivefold. Pol shows the same ability as Gag-Pol to selectively package tRNALys into Gag VLPs, and primer tRNA3 Lys is found annealed to the viral genomic RNA. These data suggest that after the initial separation of Gag from Pol during cleavage of Gag-Pol by viral protease, the Pol species still retains the capacity to bind to both Gag and tRNA3 Lys, which may be required for Pol and tRNA3 Lys to be retained in the assembling virion until budding is completed.

Original languageEnglish (US)
Pages (from-to)1042-1049
Number of pages8
JournalJournal of Virology
Volume78
Issue number2
DOIs
StatePublished - Jan 2004
Externally publishedYes

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virus-like particles
Human immunodeficiency virus 1
Virion
HIV-1
RNA-directed DNA polymerase
RNA-Directed DNA Polymerase
proteinases
Peptide Hydrolases
pol Gene Products
RNA, Transfer, Lys
nucleocapsid
leucine zipper
Integrases
Leucine Zippers
Nucleocapsid
virion
Viral RNA
amino acid sequences
RNA
mutation

ASJC Scopus subject areas

  • Immunology

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Incorporation of Pol into Human Immunodeficiency Virus Type 1 Gag Virus-Like Particles Occurs Independently of the Upstream Gag Domain in Gag-Pol. / Cen, Shan; Niu, Meijuan; Saadatmand, Jenan; Guo, Fei; Huang, Yue; Nabel, Gary J.; Kleiman, Lawrence.

In: Journal of Virology, Vol. 78, No. 2, 01.2004, p. 1042-1049.

Research output: Contribution to journalArticle

Cen, Shan ; Niu, Meijuan ; Saadatmand, Jenan ; Guo, Fei ; Huang, Yue ; Nabel, Gary J. ; Kleiman, Lawrence. / Incorporation of Pol into Human Immunodeficiency Virus Type 1 Gag Virus-Like Particles Occurs Independently of the Upstream Gag Domain in Gag-Pol. In: Journal of Virology. 2004 ; Vol. 78, No. 2. pp. 1042-1049.
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abstract = "By using particle-associated reverse transcriptase (RT) activity as an assay for Pol incorporation into human immunodeficiency virus type 1 (HIV-1) Gag virus-like particles (VLPs), it has been found that truncated, protease-negative, Gag-Pol missing cis Gag sequences is still incorporated into Gag VLPs, albeit at significantly reduced levels (10 to 20{\%} of the level of wild-type Gag-Pol). In this work, we have directly measured the incorporation of truncated Gag-Pol species into Gag VLPs and have found that truncated Gag-Pol that is missing all sequences upstream of RT is still incorporated into Gag VLPs at levels approximating 70{\%} of that achieved by wild-type Gag-Pol. Neither protease nor integrase regions in Pol are required for its incorporation, implying an interaction between Gag and RT sequences in the Pol protein. While the incorporation of Gag-Pol into Gag VLPs is reduced 12-fold by the replacement of the nucleocapsid within Gag with a leucine zipper motif, this mutation does not affect Pol incorporation. However, the deletion of p6 in Gag reduces Pol incorporation into Gag VLPs four- to fivefold. Pol shows the same ability as Gag-Pol to selectively package tRNALys into Gag VLPs, and primer tRNA3 Lys is found annealed to the viral genomic RNA. These data suggest that after the initial separation of Gag from Pol during cleavage of Gag-Pol by viral protease, the Pol species still retains the capacity to bind to both Gag and tRNA3 Lys, which may be required for Pol and tRNA3 Lys to be retained in the assembling virion until budding is completed.",
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