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Journal of Virology, July 2009, p. 7210-7220, Vol. 83, No. 14
0022-538X/09/$08.00+0     doi:10.1128/JVI.02603-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Uncoupling Human Immunodeficiency Virus Type 1 gag and pol Reading Frames: Role of the Transframe Protein p6* in Viral Replication{triangledown}

Andreas Leiherer, Christine Ludwig, and Ralf Wagner*

Institute of Medical Microbiology and Hygiene, Molecular Microbiology and Gene Therapy Unit, University of Regensburg, 93053 Regensburg, Germany

Received 17 December 2008/ Accepted 21 April 2009

Apart from its regulatory role in protease (PR) activation, little is known about the function of the human immunodeficiency virus type 1 transframe protein p6* in the virus life cycle. p6* is located between the nucleocapsid and PR domains in the Gag-Pol polyprotein precursor and is cleaved by PR during viral maturation. We have recently reported that the central region of p6* can be extensively mutated without abolishing viral infectivity and replication in vitro. However, mutagenesis of the entire p6*-coding sequence in the proviral context is not feasible without affecting the superimposed frameshift signal or the overlapping p1-p6gag sequences. To overcome these limitations, we created a novel NL4-3-derived provirus by displacing the original frameshift signal to the 3' end of the gag gene, thereby uncoupling the p6* gene sequence from the p1-p6gag reading frame. The resulting virus (AL) proved to be replication competent in different cell cultures and thus represents an elegant tool for detailed analysis of p6* function. Hence, extensive deletions or substitutions were introduced into the p6* gene sequence of the AL provirus, and effects on particle release, protein processing, and viral infectivity were evaluated. Interestingly, neither the deletion of 63% of all p6* residues nor the partial substitution by a heterologous sequence affected virus growth and infectivity, suggesting that p6* is widely dispensable for viral in vitro replication. However, the insertion of a larger reporter sequence interfered with virus production and maturation, implying that the length or conformation of this spacer region might be critical for p6* function.

* Corresponding author. Mailing address: Molecular Microbiology and Gene Therapy Unit, Institute of Medical Microbiology and Hygiene, University of Regensburg, 93053 Regensburg, Germany. Phone: 49 941 944 6452. Fax: 49 941 944 6484. E-mail: ralf.wagner{at}klinik.uni-regensburg.de

{triangledown} Published ahead of print on 29 April 2009.

Journal of Virology, July 2009, p. 7210-7220, Vol. 83, No. 14
0022-538X/09/$08.00+0     doi:10.1128/JVI.02603-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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