Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane.

Eunice C Chen,Rebecca J Kaddis Maldonado,Leslie J Parent

doi:10.3390/v13050903

Eunice C Chen, Rebecca J Kaddis Maldonado + Show 1 more

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https://doi.org/10.3390/v13050903

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Journal: Viruses	Publication Date: May 13, 2021
Citations: 4	License type: CC BY 4.0

Affiliation: Penn State Milton S. Hershey Medical Center

Abstract

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.

Highlights

Retroviruses are unique in that they package their single-stranded RNA genomes as non-covalently linked dimers
In murine leukemia virus (MLV), genome dimerization occurs in the nucleus in a co-transcriptional fashion, resulting in the preferential formation of genetically identical genome homodimers [25,26,27,28,29]
To detect genome heterodimers composed of two genetically distinct unspliced viral RNA (vRNA) within cells, we used three complementary approaches using a combination of two techniques: one to selectively label vRNA using single molecule fluorescence in situ hybridization (smFISH) and the second using fluorescentlylabeled proteins bound to aptamer sequences inserted into vRNA

Summary

Introduction

Retroviruses are unique in that they package their single-stranded RNA genomes as non-covalently linked dimers. During the early stage of retrovirus infection, the retroviral reverse transcriptase mediates two distinct strand transfer reactions that switch between the genomes to generate the double-stranded DNA provirus [11]. These transfer reactions facilitate recombination between the two non-identical copies of the genome, allowing for repair of damage to the fragile RNA genome or acquisition of novel mutations, both actions that encourage increased genetic diversity [7,8,9,10,11,12,13]. DIS structures have been characterized in most retroviruses, as reviewed in [7]

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