Nucleocytoplasmic Export of Type D Simian Retrovirus Genomic RNA: Identification of Important Genetic Subregions and Interacting Cellular Proteins

AbstractThe simian retrovirus (SRV) genome contains a constitutive transport element (CTE) within its 3′ intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. The serogroup 2 SRV CTE is predicted to form a stable stem–loop structure containing two major internal loops exhibiting 180° inverse symmetry, with loop face sequences A, A′, B, and B′ and additional minor internal and terminal loops. To begin the identification of potential CTE-interacting proteins and to assess structural requirements for protein interaction, we conducted RNA mobility shift assays using IR fragments that obliterated this region's known stable stem–loop structure. Using immunoblotting assays, we have determined that RNA helicase A, implicated in the nuclear export of unspliced SRV genomic RNA, does not appear to interact directly with either the complete serogroup 2 SRV 3′ IR or the subregion RNAs and that formation of RNA–protein complexes is conferred by interaction with other novel proteins. UV crosslinking of RNA–protein complexes, coupled with RNase T1/A digestion, indicates that a novel protein of 120 kDa molecular weight interacts with the complete CTE or with individual subregion RNAs. Transfection analyses indicate that SRV recombinants containing A, A′, B, or B′ sequences forming the faces for two open loops undergo RNA export; only the complete sense CTE recombinant or a second recombinant containing two subregions in sense orientation that reconstitute the 3′ two-thirds of the 3′ IR, and contain only A′ and B that form the faces for two terminal loops, are capable of SRV RNA export. These experiments indicate that secondary structural determinants of the 3′ IR and multiple protein interactions may be important factors in the nuclear export of unspliced SRV RNA