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. 2009 Dec;83(23):12499-511.
doi: 10.1128/JVI.01754-08. Epub 2009 Sep 23.

Identification and mutational analysis of a Rej response element in Jaagsiekte sheep retrovirus RNA

Takayuki Nitta  1 , Andrew Hofacre, Stacey Hull, Hung Fan
Affiliations

Identification and mutational analysis of a Rej response element in Jaagsiekte sheep retrovirus RNA

Takayuki Nitta et al. J Virol. 2009 Dec.

Abstract

Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus causing a contagious lung cancer of sheep. JSRV encodes unspliced and spliced viral RNAs, among which unspliced RNA encodes Gag and Pol proteins and a singly spliced mRNA encodes Env protein. In another study we found that JSRV encodes a regulatory protein, Rej, that is responsible for synthesis of Gag polyprotein from unspliced viral RNA. Rej is encoded in the 5' end of env, and it enhances nuclear export or accumulation of cytoplasmic unspliced viral RNA in 293T cells but not in most other cell lines (A. Hofacre, T. Nitta, and H. Fan, J. Virol. 83:12483-12498, 2009). In this study, we found that mutations in the 3' end of env in the context of a cytomegalovirus-driven full-length JSRV expression construct abolished Gag protein synthesis and released viruses in 293T cells. These mutants also showed deficits in accumulation of unspliced viral RNA in the cytoplasm. These mutants defined a Rej-responsive element (RejRE). Inhibition of CRM1 but not Tap function prevented nuclear export/accumulation of cytoplasmic unspliced RNA in 293T cells, similarly to other complex retroviruses that express analogous regulator proteins (e.g., human immunodeficiency virus Rev). Structural modeling of the RejRE with Zuker M-fold indicated a region with a predicted stable secondary structure. Mutational analysis in this region indicated the importance of both secondary structures and primary nucleotide sequences in a central stem-bulge-stem structure. In contrast to 293T cells, mutations in the RejRE did not affect the levels of cytoplasmic unspliced RNA in 293 cells, although the unspliced RNA showed partial degradation, perhaps due to lack of translation. RejRE-containing RNA relocalized Rej protein from the nucleus to the cytoplasm in 293 and rat 208F cells, suggesting binding of Rej to the RejRE.

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Figures

FIG. 1.
FIG. 1.
Expression plasmids used. Diagrams of the expression plasmids used in these experiments are shown. (A) pCMVJS21 expresses wild-type JSRV from a cytomegalovirus promoter (CMV-P). The subdomains in Env were separated into three regions as signal peptide (SP), surface (SU), and transmembrane (TM) regions. JSRV Env (ΔGP) expresses wild-type JSRV Env from a cytomegalovirus promoter. ΔGP-HA and ΔGP-Flag have HA and Flag tag sequence at the 3′ end of the TM region. pTN3 has an internal deletion removing the coding sequences for a small portion of the 3′ end of pol and a large portion of env. The JSRV LTR is at the 3′ end of these constructs. (B) The nucleotide and amino acid substitutions in the Env cytoplasmic tail (CT) region are shown. The names of Env CT plasmids were converted from amino acid-based names previously used (18) to nucleotide-based names, due to the importance of nucleotide sequence in these experiments.
FIG. 2.
FIG. 2.
Effects of mutations in the 3′ end of the JSRV env gene on virion production. (A) Ten-centimeter dishes of 293T cells were transiently transfected with JSRV expression plasmids based on pCMV2JS21 (full-length JSRV) containing alanine mutations in the cytoplasmic tail of TM (residues 605 to 615). The released virus was assessed by Western blotting for JSRV CA protein (using a CA-specific monoclonal antibody) in tissue culture supernatants harvested from equal numbers of cells 16 h posttransfection. The region of the gel containing CA protein (23 kDa) is shown. (B) Whole-cell lysates from 293T cells transfected with selected mutants (residues 608 to 612) were prepared 16 h posttransfection and analyzed by SDS-PAGE and Western blotting for JSRV Gag protein with anti-CA monoclonal antibody. Positive and negative controls (pcDNA3, empty vector; pCMVJS21, wild-type JSRV expression vector) are shown in the lanes at the left. The locations of Gag polyprotein (Gag) and processed CA protein are shown. A ca.-60-kDa nonspecific band (also evident in the pcDNA3-transfected extracts) was present in all lanes. For a loading control, the samples were subjected to Western blot assays with anti-β-tubulin. The experiments were repeated several times with the same results.
FIG. 3.
FIG. 3.
Mutations in the 3′ end of the env gene affect export of unspliced viral RNA in 293T cells. (A) The wild-type (pCMVJS21) and mutant JSRV expression vectors were transfected into 293T cells, and cytoplasmic (C) and nuclear (N) RNAs were isolated 16 h after transfection and then subjected to agarose gel electrophoresis and Northern blot analysis with an env-LTR probe. The env-LTR probe showed unspliced full-length (7.5-kb) and spliced (2.4-kb) env mRNA. Rehybridization of the blot with a GAPDH probe confirmed that equal amounts of RNA were loaded. Cell fractionation was checked by Western blot assays using anti-β-tubulin and anti-lamin A/C. (B) Regions of the Northern blots corresponding to full-length unspliced and GAPDH RNA in the autoradiograms were quantified by immunoblot densitometry, and the percentages of unspliced RNA in the cytoplasm relative to pCMVJS21 are shown.
FIG. 4.
FIG. 4.
Complementation of pTN3 for Gag protein synthesis by JSRV Rej expression plasmids. 293T cells were cotransfected with pTN3 with env deleted and Env/Rej expression constructs (ΔGP) harboring nucleotide substitutions at the 3′ end of env. The cells and the media were harvested 16 h after transfection and were subjected to Western blot analysis with anti-CA antibodies. Arrows indicate Gag protein in cell lysates (70-kDa polyprotein) and CA protein (23 kDa) from virus released into the medium. The two leftmost lanes show transfection with pcDNA3 and pTN3 alone. Transfection with the wild-type JSRV expression plasmid pCMVJS21 is shown on the right. The faint band migrating slightly slower than the 70-kDa Gag polyprotein observed in the lane with pTN3 alone represents a nonspecific band that is also observed in cells transfected with pcDNA3. A ca.-60-kDa nonspecific band was also present in all lanes. For a loading control, the samples were subjected to Western blot assays with anti-β-tubulin.
FIG. 5.
FIG. 5.
Effects of inhibitors for CRM1 and Tap on nuclear export of unspliced RNA and Gag synthesis. (A) pCMVJS21 was cotransfected with ΔCAN (defective form of the nucleoporin Nup214/CAN inhibiting CRM1-dependent RNA export) or TapA17-HA (dominant-negative inhibitory form of Tap) into 293T cells. The cells were harvested 24 h after transfection, and cytoplasmic (C) and nuclear (N) RNAs were subjected to agarose gel electrophoresis and Northern blot analysis with a JSRV env-LTR probe. Full-length unspliced RNA (7.5 kb) and Env-spliced RNA are indicated. Rehybridization of the Northern blot with Nup214/CAN and GAPDH probes is shown. (B) Cytoplasmic proteins isolated from 293T cells cotransfected with pCMVJS21 and ΔCAN or TapA17 were subjected to Western blot assays using antibodies against HA epitope and β-tubulin. The whole-cell lysates (whole) extracted from 293T cells transfected with pcDNA3 are shown as a cell fractionation control. (C) The Gag polyprotein in cell lysates and capsid (CA) in medium detected by Western blot assays in the 293T cells cotransfected with ΔCAN or TapA17-HA are shown. For a loading control, the samples were subjected to Western blot assays with anti-β-tubulin. (D) 293T cells were cotransfected with pTN3 and ΔGP-Flag. The cells were incubated for 24 h and then treated with 10 nM LMB for 12 h, followed by harvest of medium (M) and fractionation into nuclei (N) and cytoplasms (C) and Western blotting with anti-CA (upper panel) and anti-Flag (middle panel). The Gag polyprotein in the cytoplasm and cleaved capsid in the medium and Env (SU-TM polyprotein and TM) are indicated. The blots were reprobed with antibodies for nuclear lamin A/C in nucleus and cytoplasmic β-tubulin (bottom panels) to assess the efficacy of cell fractionation. The same results were observed in replicate experiments.
FIG. 6.
FIG. 6.
Prediction of the RejRE secondary structure. The secondary structure of JSRV RNA corresponding to the cytoplasmic tail region of env and the 3′ LTR (nt 7150 to 7288) predicted by the Zuker M-fold program is shown on the left. The predicted structures of four env mutants showing defective Rej/RejRE activity (Fig. 3) are also shown. Subregions of the predicted structure are indicated on the wild-type sequence. In the structures, open arrows indicate nucleotide substitutions in pCMV2JS21 constructs that did not affect Gag synthesis or optimal unspliced RNA export and/or accumulation of cytoplasmic unspliced viral RNA (Rej response), while solid arrows indicate substitutions that abolished or greatly reduced activity.
FIG. 7.
FIG. 7.
Extended mutagenesis of the RejRE. (A) The nucleotide substitutions in additional mutants of the RejRE are shown, with the wild-type sequence and structure as reference. The plasmids JS#2, #7, #13, and #15 in stems A, C, and E were produced from JS a7157g/a7158c, JS a7169g/g7170c, JS g7180u/c7179u, and JS g7182c/c7261g (Fig. 6), respectively, by adding mutations on the opposite strands that were predicted to restore base pairing. JS#9, #11, #16, #17, and #18 contained additional nucleotide substitutions in loops B and D. All of these new mutations were predicted by M-fold computations to have the same secondary structure as that of wild-type JSRV. The open and solid arrows are described in the legend to Fig. 6. (B) The new mutants were transfected into 293T cells, and cytoplasmic RNAs were isolated 16 h after transfection and subjected to Northern blot analysis with a JSRV env-LTR probe. Positive (wild-type JSRV, pCMVJS21) and negative (pcDNA3) controls were analyzed in parallel. Regions of the Northern blots corresponding to full-length unspliced and GAPDH RNA in the autoradiograms were quantified by immunoblot densitometry, and the percentages of unspliced RNA in the cytoplasm relative to pCMVJS21 are shown at the bottom of the panel. (C) Release of viral protein into the medium from 293T cells transfected with the same plasmids as those in panel B was assessed by SDS-PAGE of cell extracts followed by Western blotting with anti-CA antibody. The region of the blot corresponding to cleaved CA protein (23 kDa) is shown.
FIG. 8.
FIG. 8.
Northern blot analysis with RejRE mutants in 293 cells. Wild-type and RejRE mutant JSRV expression vectors were transfected into 293 cells, and cytoplasmic (C) and nuclear (N) RNAs were isolated and then subjected to agarose gel electrophoresis and Northern blot analysis with a gag probe mixed with a GAPDH probe. The unspliced full-length (7.5-kb), full-length prematurely polyadenlylated (6.5-kb) (31), and GAPDH RNAs are indicated. JS c7179t/g7180t and JS g7182c also showed slightly smaller unspliced full-length RNA (asterisks) between the 7.5- and 6.4-kb JSRV RNA bands.
FIG. 9.
FIG. 9.
The RejRE relocalizes Rej protein to the cytoplasm. 293 and 208F cells were transfected with pcDNA3 (empty-vector control), ΔGP-HA (epitope-tagged Env expression plasmid), or ΔGPSP-HA (epitope-tagged Rej expression plasmid) with or without pCMVJS21 or JS c7179t/g7180t. The cells were grown on chamber slides for 40 h prior to fixation and incubated with anti-HA, followed by exposure to FITC-conjugated anti-rabbit immunoglobulin G. To visualize nuclei, the cells were covered with Vectashield mounting medium plus DAPI. Immunofluorescence for HA and DAPI staining on the same fields is shown. Bar, 5 μm.
FIG. 10.
FIG. 10.
Association of RejRE activity and Rej relocalization. 293 and 208F cells were transfected with ΔGPSP-HA along with the RejRE mutants showing different levels of Gag (Fig. 3). The cells were analyzed as described for Fig. 9. Bar, 5 μm.
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