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. 2009 Dec;83(23):12483-98.
doi: 10.1128/JVI.01747-08. Epub 2009 Sep 23.

Jaagsiekte sheep retrovirus encodes a regulatory factor, Rej, required for synthesis of Gag protein

Andrew Hofacre  1 , Takayuki Nitta, Hung Fan
Affiliations

Jaagsiekte sheep retrovirus encodes a regulatory factor, Rej, required for synthesis of Gag protein

Andrew Hofacre et al. J Virol. 2009 Dec.

Abstract

Retroviruses express Gag and Pol proteins by translation of unspliced genome-length viral RNA. For some retroviruses, transport of unspliced viral RNA to the cytoplasm is mediated by small regulatory proteins such as human immunodeficiency virus Rev, while other retroviruses contain constitutive transport elements in their RNAs that allow transport without splicing. In this study, we found that the betaretrovirus Jaagsiekte sheep retrovirus (JSRV) encodes within the env gene a trans-acting factor (Rej) necessary for the synthesis of Gag protein from unspliced viral RNA. Deletion of env sequences from a JSRV proviral expression plasmid (pTN3) abolished its ability to produce Gag polyprotein in transfected 293T cells, and Gag synthesis could be restored by cotransfection of an env expression plasmid (DeltaGP). Deletion analysis localized the complementing activity (Rej) to the putative Env signal peptide, and a signal peptide expression construct showed Rej activity. Two other betaretroviruses, mouse mammary tumor virus (MMTV) and human endogenous retrovirus type K, encode analogous factors (Rem and Rec, respectively) that are encoded from doubly spliced env mRNAs. Reverse transcriptase-PCR cloning and sequencing identified alternate internal splicing events in the 5' end of JSRV env that could signify analogous doubly spliced Rej mRNAs, and cDNA clones expressing two of them also showed Rej activity. The predicted Rej proteins contain motifs similar to those found in MMTV Rem and other analogous retroviral regulatory proteins. Interestingly, in most cell lines, JSRV expression plasmids with Rej deleted showed normal transport of unspliced JSRV RNA to the cytoplasm; however, in 293T cells Rej modestly enhanced export of unspliced viral RNA (2.8-fold). Metabolic labeling experiments with [(35)S]methionine indicated that JSRV Rej is required for the synthesis of viral Gag polyprotein. Thus, in most cell lines, the predominant function of Rej is to facilitate translation of unspliced viral mRNA.

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Figures

FIG. 1.
FIG. 1.
JSRV expression constructs. (A) All constructs are driven by the CMV immediate-early promoter (bold arrows). The organization of the JSRV genome, along with its open reading frames, is detailed for the full-length provirus molecular clone of JSRV, pCMV2JS21. pTN3 has the majority of the env gene deleted, except for a portion that maps to the C terminus of Env. For the ΔGP deletions, deletions are shown as gaps. The nomenclature reflects amino acids deleted within the Env protein. The splice donor (sd) and splice acceptor (sa) nucleotide positions for the JSRV env transcript are indicated. The locations of the putative Env signal peptide (SP), the Env surface (SU) and transmembrane (TM) domains, and the Env membrane-spanning sequence (MS) and CT are shown. (B) The influenza virus HA epitope tag was fused immediately downstream of the Env CT domain for the JSRV Env and Env signal peptide expression constructs (ΔGPHA and ΔGPSPHA, respectively).
FIG. 2.
FIG. 2.
JSRV encodes a trans-acting factor necessary for Gag synthesis. 293T cells were transiently transfected (or cotransfected) with the plasmids indicated, and at 40 h posttransfection, intracellular JSRV Gag polyprotein and/or JSRV particle production (in whole-cell lysates or cell supernatants, respectively) was resolved on 7.5% (lysate) or 10% (supernatant) SDS-polyacrylamide gels followed by Western blotting with a monoclonal antibody to JSRV CA. (A) Transfection with pCMV2JS21, pTN3, and pCDNA3.1. Intracellular JSRV Gag polyprotein (lysate, 74 kDa) and released virion CA (supernatant, 27 kDa) are indicated. A slower-migrating intracellular Gag-specific band (80 kDa) was also detected in cell lysates from both pCMV2JS21- and pTN3-transfected 293T cells and may be a minor nonfunctional form of Gag polyprotein (lanes 2 and 3). (B) Cotransfection of pTN3 with the JSRV env expression construct ΔGP. Intracellular Gag and released virion CA are shown. (C) Cotransfection of pTN3 with different internal deletions of ΔGP. Gag polyprotein in the cellular lysates only is shown. In this initial survey, equivalent amounts of cell lysates were loaded into each lane. (D) Cotransfection of pTN3 with the signal peptide expression construct ΔGPSP rescues Gag synthesis as efficiently as does full-length ΔGP (top panel). To control for equal loading, cell lysates were immunoblotted for β-tubulin (arrow, middle panel). Expression of the processed JSRV Env HA-tagged protein (from ΔGPHA; TM HA, 40 kDa) or from the JSRV Env signal peptide (HA-tagged) region of Env (from ΔGPSPHA; SP HA, 15 kDa) from cotransfection with pTN3 is shown in the cell lysates (bottom panel).
FIG. 3.
FIG. 3.
The JSRV Env signal peptide localizes to the cell nucleus. 208F cells were transfected with the expression constructs for HA-tagged JSRV Env, HA-tagged JSRV Env signal peptide, or empty vector control (ΔGPHA [top panels], ΔGPSPHA [middle panels], or pCDNA3.1 [bottom panels], respectively) and grown on chamber slides for 40 h prior to fixation and incubation with an FITC-conjugated polyclonal antibody to HA and a phycocyanin-conjugated monoclonal antibody to nucleophosmin (B23). To visualize the nuclei of all cells, the cells were also stained with DAPI. Immunofluorescence images were recorded at excitation wavelengths of 350 nm (DAPI), 490 nm for FITC (HA), and 620 nm for phycocyanin (B23); individual and merged images are shown. Bar, 5 μm. Magnification, ×630.
FIG. 4.
FIG. 4.
Identification of alternatively spliced transcripts within the JSRV env gene. (A) RT-PCR analysis for env sequences of cytoplasmic RNA from 293T cells transfected with ΔGP (lane 1). The expected 900-bp band was observed as well as three smaller amplification products (lane 1, bands of <850 bp). 293T cells transfected with pCDNA3.1 were used as a negative control (lane 2); to verify that amplification was from polyadenylated RNAs and reverse transcriptase dependent, an oligo(dT) primer was employed during cDNA synthesis prior to PCR amplification with SU primers (lane 3), and amplification without added reverse transcriptase was performed (lane 4), respectively. ΔGP plasmid DNA control was used as a template in PCR (lane 5); these primers did not amplify fragments of <900 bp. (B) Structure of doubly spliced env mRNAs corresponding to the 650-bp RT-PCR fragments (shown by an arrow in lane 1 of panel A) with the env splice donor (sd) and splice acceptor (sa) nucleotides indicated, as well as the location of the primer pairs (opposing arrows) used in the RT-PCRs. The locations of stop codons are indicated by asterisks. (C) Detection of the alternatively spliced JSRV env mRNAs in JSRV-infected cells and various cell lines by RT-PCR analysis. RNAs from sheep CP cells infected with JSRV particles were used to detect increased levels of spliced env transcripts (650-bp band, shown by an arrow) over endogenous levels (lanes 4 and 3 of left panel, respectively). RNAs from 293T cells transfected with the molecular clone of JSRV, pCMV2JS21, or RNAs from NIH 3T3, 208F, or HeLa cells transfected with ΔGP were used to detect the env (900-bp band) and the spliced env (650-bp band, shown by an arrow) transcripts (lane 2, left panel; lanes 2 and 4, middle panel; and lane 3, right panel). JSRV env-specific RT-PCRs used RNAs from cells transfected with the negative-control plasmid, pCDNA3.1 (lanes 3, left and middle panels, and lane 2, right panel).
FIG. 5.
FIG. 5.
The alternatively spliced env transcripts encode Rej activity. (A) Schematic representation of the doubly spliced env cDNA expression vectors: ΔGPds-envcDNA2 and ΔGPds-envcDNA4 are shown relative to the other expression plasmids shown in Fig. 1. (B) 293T cells were transfected or cotransfected with the plasmids indicated, and at 40 h posttransfection, whole-cell lysates were analyzed on 7.5% SDS-polyacrylamide gels, followed by Western blot analysis with a monoclonal antibody to JSRV CA protein. The arrow indicates the cellular JSRV Gag polyprotein (74 kDa, top panel). Both doubly spliced expression constructs complemented pTN3 for Gag production equivalently to ΔGP and ΔGPSP. In pTN3-transfected cells, a slightly slower-migrating Gag-specific band was also detected, but it was distinct from the Gag polyprotein precursor. β-Tubulin (55 kDa) was used to control for equivalent loading of the cell lysates (bottom panel). (C) Northern blot hybridization analysis using a JSRV env probe indicated the authentic expression of env transcripts from ΔGP (2.4 kb, shown by an arrow) and the truncated env signal peptide from ΔGPSP or the ΔGP-spliced env transcripts expressed from ΔGPds-envcDNA2 and ΔGPds-envcDNA4 (0.7 kb, shown by an arrow).
FIG. 6.
FIG. 6.
Domain structures of the signal peptide and the putative translation products of the doubly spliced JSRV env transcripts. (A) The JSRV Env signal peptide sequence is diagramed, and the locations of predicted sequence motifs are indicated by arrows under the diagram. (B) Amino acid composition of the predicted translations from the doubly spliced env transcripts. The locations of the alternative splice donor and splice acceptor sites are indicated, as well as the location of the stop codon (asterisks) within the second exons (shaded).
FIG. 7.
FIG. 7.
Rej facilitates export of unspliced cytoplasmic RNA in human 293T cells. 293T cells (106) were transfected with 14 μg of pCMV2JS21, pCMV2JS21 SUΔ13-52, or pCDNA3.1; at 40 h posttransfection, the cells were harvested and total RNA or RNA from nuclei and cytoplasm was extracted. (A) Total, nuclear, and cytoplasmic RNAs from equivalent numbers of cells analyzed by agarose gel electrophoresis and Northern blot hybridization with a JSRV gag probe. Bands corresponding to the unspliced 7.5-kb and the 6.5-kb prematurely polyadenylated JSRV genome RNAs are shown (arrows, top panel). The blots were stripped and rehybridized with a GAPDH probe (1.2-kb band, arrow, bottom panel). (B) Western blot analysis of cell lysate fractions 1 to 6 of panel A with antibodies that detect β-tubulin (55 kDa, as a cytoplasmic marker) and lamin A/C (74 kDa, as a nuclear marker) was used to control for nuclear/cytoplasmic separation. Cell lysates were run on 10% SDS-polyacrylamide gels and visualized by immunoblotting; a 55-kDa specific band corresponding to β-tubulin was detected only in cytoplasmic fractions (lanes 1 and 3, top panel), whereas a 74-kDa specific band corresponding to lamin A/C was detected in only nuclear fractions (lanes 2 and 4, bottom panel); both proteins were detected in the total fractions (lanes 5 and 6, top and bottom panels). (C) Cell lysates at 40 h posttransfection from 293T cells transiently transfected with 14 μg of the plasmids indicated were analyzed by Western blotting with anti-JSRV CA monoclonal antibody. Gag polyprotein (74 kDa) was expressed from pCMV2JS21 (Gag, arrow, lane 2) but not from pCMV2JS21 SUΔ13-52 (lane 3) or pCDNA3.1 (lane 1). A nonspecific protein (NSP) which migrated faster than the JSRV poly-Gag protein was detected in all lanes and demonstrates equivalent loading of the cell lysates (60 kDa, arrow).
FIG. 8.
FIG. 8.
Nucleotides in the 3′ region of env (the JREE) are Rej responsive to cytoplasmic export of intron-containing RNA only in human 293T cells. (A) Schematic diagram of pDM128 and pDM/JREE reporter plasmids. The locations of splice donor and acceptor sites (sd and sa, respectively) are shown, as well as coding sequences for CAT, the HIV-1 RRE (RRE), and the JREE. The plasmids are driven by a simian virus 40 early promoter (SV40-P) and utilize the cleavage/poly(A) site of the HIV-1 LTR. (B) pDM128 and pDM/JREE were transfected into human 293T cells, and 48 h posttransfection, CAT activities were measured as described in Materials and Methods. As a positive control, pDM128 was cotransfected with the HIV-1 Rev expression vector, pRev1. To test if the JREE was responsive to Rej, pDM/JREE was cotransfected with the JSRV Rej expression vector, ΔGP, at a ratio of 3 μg to 1 μg DNA, respectively (left panel). This experiment was also performed with variable amounts of ΔGP DNA, ranging from 0.5 μg to 3 μg, while keeping pDM/JREE DNA amounts constant (3 μg) (right panel). To control for transfection efficiency, the pRL-null vector encoding Renilla luciferase was also cotransfected with each of the pDM CAT reporters and the CAT activities were normalized for luciferase activities measured on portions of each transfected cell extract. The results from triplicate experiments are shown (error bars represent standard deviations), and the P value (Student's t test) for the difference between the activities of pDM/JREE and pDM/JREE (cotransfected with Rej; ΔGP) is indicated. (C) Cytoplasmic RNA was isolated from 293T cells 16 h after transfection with the combinations of pDM CAT reporters indicated and subjected to agarose gel electrophoresis and Northern blot hybridization with a 32P-labeled HIV-1 LTR probe followed by phosphorimager analysis. Regions of the blot corresponding to the spliced and unspliced RNA in cytoplasmic RNA are shown (top panel). The blot was stripped and rehybridized with a GAPDH probe (1.2-kb band, arrow, middle panel) and a JSRV env probe (2.4-kb band, arrow, bottom panel) to control for equal loading and expression of rej-containing transcripts, respectively. Phosphorimaging analysis (bar graph) represents the ratio of cytoplasmic unspliced pDM/JREE RNA to pDM128 in the presence or absence of Rej in 293T cells. (D) CAT activity assay in human 293, rat 208F, mouse 3T3, and sheep CP cells using the same constructs and methods as those in the assay in 293T cells (B), described above. Note that a dose-response (for pDM/JREE plus variable amounts of ΔGP DNA) CAT assay was also performed in human 293 cells, identical to what was described above (top right panel).
FIG. 9.
FIG. 9.
Rej does not facilitate the transport of unspliced cytoplasmic RNA in human 293 cells but is absolutely required for JSRV poly-Gag protein synthesis. 293 cells (106) were transfected with 14 μg of pCMV2JS21, pCMV2JS21 SUΔ13-52, or pCDNA3.1; at 40 h posttransfection, the cells were harvested and total RNA or RNA from nuclei and cytoplasm was extracted. (A) Total, nuclear, and cytoplasmic RNAs from equivalent numbers of cells analyzed by agarose gel electrophoresis and Northern blot hybridization with a JSRV gag probe. Bands corresponding to the unspliced 7.5-kb and the 6.5-kb prematurely polyadenylated JSRV genome RNAs are shown (arrows, top panel). The blots were stripped and rehybridized with a GAPDH probe (1.2-kb band, arrow, bottom panel). (B) Western blot analysis of cell lysate fractions 1 to 6 of panel A with antibodies that detect β-tubulin (55 kDa, as a cytoplasmic marker) and lamin A/C (74 kDa, as a nuclear marker) was used to control for nuclear/cytoplasmic separation. Cell lysates were run on 10% SDS-polyacrylamide gels and visualized by immunoblotting; a 55-kDa specific band corresponding to β-tubulin was detected only in cytoplasmic fractions (lanes 1 and 3, top panel), whereas a 74-kDa specific band corresponding to lamin A/C was detected only in nuclear fractions (lanes 2 and 4, bottom panel); both proteins were detected in the total fractions (lanes 5 and 6, top and bottom panels). (C) Cell lysates at 40 h posttransfection from 293 cells transiently transfected with 14 μg of the plasmids indicated were analyzed by Western blotting with anti-JSRV CA monoclonal antibody. Gag polyprotein (74 kDa) was expressed from pCMV2JS21 (Gag, arrow, lane 2) but not from pCMV2JS21 SUΔ13-52 (lane 3) or pCDNA3.1 (lane 1). A nonspecific protein (NSP) that migrated faster than the JSRV poly-Gag protein was detected in all lanes and was used to show equivalent loading of the cell lysates (60 kDa, NSP, arrow).
FIG. 10.
FIG. 10.
(A) Rej is necessary for translation of gag-pol RNA. 293T cells were transfected with pCDNA3.1, pCMV2JS21, or pCMV2JS21 SUΔ13-52; at 16 h posttransfection, the cells were starved for 1 h and then pulse-labeled with [35S]cysteine-methionine for 20 min. Cell lysates were prepared and subjected to immunoprecipitation assay using a rabbit anti-JSRV CA antibody and then resolved by SDS-PAGE followed by phosphorimaging. JSRV-specific 35S-labeled Gag was detected in immunoprecipitates of cells transfected with pCMV2JS21 (middle lane, top panel) but not in cells transfected with pCDNA3.1 or pCMV2JS21 SUΔ13-52 (left and right lanes, respectively, top panel). NSP, nonspecific protein band, immunoprecipitated from all extracts. To control for equivalent amounts of cellular proteins subject to immunoprecipitation, Western blot analysis with anti-β-tubulin antibody was performed (bottom panel). (B) Rej can complement the translation of Gag polyprotein from mutant constructs having Rej deleted. 293T cells were transfected with pCDNA3.1, pCMV2JS21, or pCMV2JS21 SUΔ13-52, or pCMV2JS21 SUΔ13-52 was cotransfected with ΔGP or ΔGPSP (to supply Rej in trans); the cells were treated, labeled, and immunoprecipitated as described above. Gag polyprotein (74 kDa) was expressed from pCMV2JS21 (Gag, arrow, lane 2) but not from pCMV2JS21 SUΔ13-52 (lane 3) or pCDNA3.1 (lane 1); Gag polyprotein was detected from pCMV2JS21 SUΔ13-52 when cotransfected with ΔGP (Gag, arrow, lane 4) or ΔGPSP (Gag, arrow, lane 5).
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References

    1. Arrigo, S. J., and I. S. Chen. 1991. Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs. Genes Dev. 5:808-819. - PubMed
    1. Boese, A., M. Sauter, U. Galli, B. Best, H. Herbst, J. Mayer, E. Kremmer, K. Roemer, and N. Mueller-Lantzsch. 2000. Human endogenous retrovirus protein cORF supports cell transformation and associates with the promyelocytic leukemia zinc finger protein. Oncogene 19:4328-4336. - PubMed
    1. Bogerd, H. P., A. Echarri, T. M. Ross, and B. R. Cullen. 1998. Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1. J. Virol. 72:8627-8635. - PMC - PubMed
    1. Bray, M., S. Prasad, J. W. Dubay, E. Hunter, K. T. Jeang, D. Rekosh, and M. L. Hammarskjold. 1994. A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proc. Natl. Acad. Sci. USA 91:1256-1260. - PMC - PubMed
    1. Butsch, M., S. Hull, Y. Wang, T. M. Roberts, and K. Boris-Lawrie. 1999. The 5′ RNA terminus of spleen necrosis virus contains a novel posttranscriptional control element that facilitates human immunodeficiency virus Rev/RRE-independent Gag production. J. Virol. 73:4847-4855. - PMC - PubMed

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