Impaired RNA incorporation and dimerization in live attenuated leader-variants of SIVmac239

James B Whitney,Mark A Wainberg

doi:10.1186/1742-4690-3-96

Abstract

BackgroundThe 5' untranslated region (UTR) or leader sequence of simian immunodeficiency virus (SIVmac239) is multifunctional and harbors the regulatory elements for viral replication, persistence, gene translation, expression, and the packaging and dimerization of viral genomic RNA (vRNA). We have constructed a series of deletions in the SIVmac239 leader sequence in order to determine the involvement of this region in both the packaging and dimerization of viral genomic RNA. We also assessed the impact of these deletions upon viral infectiousness, replication kinetics and gene expression in cell lines and monkey peripheral blood mononuclear cells (PBMC).ResultsRegions on both sides of the major splice donor (SD) were found to be necessary for the efficiency and specificity of viral genome packaging. However, stem-loop1 is critical for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the initiation site of SIV-Gag have additive effects on RNA packaging and contribute to a lesser degree to RNA dimerization. The targeted disruption of structures on both sides of the SD also severely impacts viral infectiousness, gene expression and replication in both CEMx174 cells and rhesus PBMC.ConclusionIn the leader region of SIVmac239, stem-loop1 functions as the primary determinant for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the translational initiation site of SIV-Gag are classified as secondary determinants and play a role in dimerization. Collectively, these data signify a linkage between the primary encapsidation determinant of SIVmac239 and RNA dimerization.

Highlights

The 5' untranslated region (UTR) or leader sequence of simian immunodeficiency virus (SIVmac239) is multifunctional and harbors the regulatory elements for viral replication, persistence, gene translation, expression, and the packaging and dimerization of viral genomic RNA
RNA structural domains on both sides of the major splice donor regulate the efficiency and specificity of SIVmac239 RNA packaging and dimerization We have constructed a series of proviral mutants within the leader of SIVmac239 (Fig. 1A and 1B) and have assessed viral genomic RNA (vRNA) packaging efficiency through both replicate slot blotting and multiplex reverse transcriptase (RT)-PCR experiments
We have determined the impact of these mutations on vRNA dimerization by recovery of RNA from mature virions and analysis on native Northern gels

Summary

Introduction

The 5' untranslated region (UTR) or leader sequence of simian immunodeficiency virus (SIVmac239) is multifunctional and harbors the regulatory elements for viral replication, persistence, gene translation, expression, and the packaging and dimerization of viral genomic RNA (vRNA). The 5' untranslated region (UTR) or leader sequence of lentivirus possess multiple structural and functional domains that include the regulatory elements for the initiation of reverse transcription, integration of the proviral genome, the trans-activation of RNA transcription, and protein translation. This region is critical for both the encapsidation and dimerization of viral genomic RNA (vRNA). While HIV-1 has been shown capable of packaging the genomic RNA of either human immunodeficiency virus type-2 (HIV-2) or simian immunodeficiency virus (SIV) [10,11], the converse is not true, highlighting the important differences in the mechanism of RNA selection [10,12]

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Conclusion