Gammaretroviral pol sequences act in cis to direct polysome loading and NXF 1 / NXT-dependent protein production by gag-encoded RNA

Background: All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated. Results: The mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65) from full-length, unspliced mRNA was investigated here. The yield of Pr65 from a gag–only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65 production and 398 silent nucleotide changes engineered into gag rendered Pr65 synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65 translation. Two independentlyacting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65 synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65 translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65 synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65 synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto polysomes and to increase Pr65 synthesis. Conversely, Gag polyprotein synthesis was decreased by NXF1 knockdown. Finally, overexpression of SRp20, a shuttling protein that binds to NXF1 and promotes NXF1 binding to RNA, also increased gag RNA loading onto polysomes and increased Pr65 synthesis. Conclusion: These experiments demonstrate that gammaretroviral pol sequences act in cis to recruit NXF1 and SRp20 to promote polysome loading of gag RNA and, thereby license the synthesis of Pr65 from unspliced mRNA. Background Retroviruses compress large quantities of genetic information into their relatively small genomes. HIV-1, for example, has a single promoter that drives a primary transcript, from which 9 genes direct the synthesis of at least 15 proteins [1,2]. This is accomplished by exploiting several mechanisms, including the synthesis of essential viral proteins from unspliced or partially spliced mRNAs [1,3,4]. In all retroviruses, the primary, unspliced transcript serves as the viral genomic RNA that is packaged into assembling virions. Unspliced RNA of identical primary sequence also directs translation of the main virion structural elements, the gag–encoded proteins [5]. Assessment of HIV-1 transcripts by conventional methods has revealed nearly 50 variants [6], while newer deep sequencing technology has detected more than 100 [7]. Replication of * Correspondence: jeremy.luban@umassmed.edu Department of Microbiology and Molecular Medicine, University of Geneva, Geneva 1205, Switzerland Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA © 2014 Bartels and Luban; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bartels and Luban Retrovirology 2014, 11:73 http://www.retrovirology. com/content/11/1/73