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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 Gag ) from full-length, unspliced mRNA was investigated here. The yield of Pr65 Gag  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 Gag  production and 398 silent nucleotide changes engineered into  gag  rendered Pr65 Gag  synthesis  pol -independent. These results are consistent with  pol- encoded RNA acting in  cis  to promote Pr65 Gag  translation. Two independently-acting  pol  fragments were identified by screening 17  pol  deletion mutations. To determine the mechanism by which  pol  promoted Pr65 Gag  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 Gag  translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for  pol  in promoting Pr65 Gag  synthesis. A  pol  RNA stem-loop resembling the CTE promoted Pr65 Gag  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 Gag  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 Gag  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 Gag  from unspliced mRNA.

Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag-encoded RNA