TY - JOUR
T1 - Nucleocytoplasmic export of type D simian retrovirus genomic RNA
T2 - Identification of important genetic subregions and interacting cellular proteins
AU - Li, Biao
AU - Wyman, Theodore E.
AU - Moudgil, Tarsem
AU - Marracci, Gail H.
AU - Ju, Chong Fang
AU - MacHida, Curtis A.
N1 - Funding Information:
The authors gratefully acknowledge the generosity of Drs. Jerald Hurwitz (Sloan–Kettering Institute, New York) and Jae Jung (New England Regional Primate Research Center) in providing the RNA heli-case A polyclonal antibody and expression recombinant and the TAP polyclonal antibody, respectively. The authors acknowledge the artistic and photographic support of Joel Ito and Vince Warren and the secretarial assistance of Carol Houser. We also acknowledge the help of Yibing Jia and the ORPRC Molecular Biology Core Facility. We thank Michael Axthelm, Sergio Ojeda, Scott Wong, Philbert Kirigiti, and Yong-feng Yang for discussions and support concerning this work, and we thank Michael Axthelm and Philbert Kirigiti for critically reading the manuscript. This research has been partially described in abstract form at the Fourteenth, Fifteenth, and Sixteenth Annual Symposiums on Nonhuman Primate Models for AIDS, held in Portland, Oregon (October 23–26, 1996), Seattle, Washington (September 3–6, 1997), and Atlanta, Georgia (October 7–10, 1998), respectively. C.A.M. is supported by NIH Grants RR00163, HL 42358, and DK 53462 and was an American Heart Association Established Investigator. This research was also supported by grants to C.A.M. from the Medical Research Foundation of Oregon, from the Collins Medical Trust, and from the ORPRC Core Grant RR00163. L.B. was a 1997 Leukemia Research Foundation Postdoctoral Fellow. T.E.W. is now a medical student at Loma Linda University School of Medicine. G.H.M. was a prior recipient of a N. L. Tartar Research Fellowship.
PY - 1999/11/10
Y1 - 1999/11/10
N2 - The 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.
AB - The 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.
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U2 - 10.1006/viro.1999.9938
DO - 10.1006/viro.1999.9938
M3 - Article
C2 - 10544128
AN - SCOPUS:0033544414
SN - 0042-6822
VL - 264
SP - 37
EP - 54
JO - Virology
JF - Virology
IS - 1
ER -