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Viruses 2016, 8(2), 39; doi:10.3390/v8020039

Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

1
Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697, USA
2
Present address: Gilead Sciences, Inc., Foster City, CA 94404, USA
3
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA
*
Author to whom correspondence should be addressed.
Academic Editor: George Belov
Received: 17 November 2015 / Revised: 18 January 2016 / Accepted: 21 January 2016 / Published: 4 February 2016
(This article belongs to the Special Issue Recent Progress in Enterovirus Research)
View Full-Text   |   Download PDF [5487 KB, uploaded 4 February 2016]   |  

Abstract

Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. View Full-Text
Keywords: enterovirus RNA replication; RNA replication complexes; MS2 hairpin; aptamer; ribonucleoprotein complexes; RNP isolation; RNA affinity enterovirus RNA replication; RNA replication complexes; MS2 hairpin; aptamer; ribonucleoprotein complexes; RNP isolation; RNA affinity
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Flather, D.; Cathcart, A.L.; Cruz, C.; Baggs, E.; Ngo, T.; Gershon, P.D.; Semler, B.L. Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs. Viruses 2016, 8, 39.

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