Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a transmissible lung cancer in sheep, ovine pulmonary adenocarcinoma. JSRV is unique in that the envelope protein functions as an oncogene, since it can morphologically transform fibroblast and epithelial cells in culture and can induce lung tumors in mice. Previous studies indicated that the transmembrane (TM) protein is essential for transformation, and particular attention has focused on a YXXM motif in the cytoplasmic tail. In this study, we carried out systematic mutagenesis of the cytoplasmic tail of JSRV Env. Alanine scanning mutagenesis revealed four classes of mutants: mutants in which transformation was abrogated, those in which transformation was not affected, those with reduced transformation, and those with increased transformation (supertransformers). In general, the alanine mutations did not affect Env protein production or its localization to the plasma membrane. Three functional domains of the cytoplasmic tail were identified: an amphipathic helix at the N-terminal (juxtamembrane) side, a nonessential C-terminal region, and an internal region (including the YXXM motif) where mutations resulted in abrogation, decreases, or increases in transformation. Alanine mutations in the amphipathic helix in both the hydrophobic and hydrophilic faces generally abolished transformation. The mutation R591A showed partial transformation that was consistent with loss of signaling through the Akt-mTOR pathway and signaling predominantly through the Ras-Raf-MEK1/2-extracellular signal-regulated kinase 1/2 pathway. The supertransforming mutants generally showed increased signaling through Akt and reduced activation of p38 MAPK that is inhibitory for transformation. These mutants provide further insight into the role of the TM cytoplasmic tail in JSRV transformation.
Retroviruses utilize an unspliced version of their primary transcription product as an RNA template for synthesis of viral Gag and Pol structural and enzymatic proteins. Cytoplasmic expression of the gag-pol RNA is achieved despite the lack of intron removal and the presence of a long and highly structured 5 untranslated region that inhibits efficient ribosome scanning. In this study, we have identified for the first time that the 5 long terminal repeat ( Retroviruses need to subvert typical cellular posttranscriptional control mechanisms to produce progeny virions. Typical pre-mRNAs are assembled in ribonucleoprotein (RNP) complexes during transcription and RNA maturation that qualify processed transcripts for nuclear export and efficient cytoplasmic expression (11,24,26,30). By contrast, retroviral premRNA recruits viral or cellular posttranscriptional modulators that activate efficient nuclear export and cytoplasmic expression despite lack of intron removal (2,12,25,44). Once in the cytoplasm, the unspliced viral transcript exhibits dual function as mRNA template for translation of Gag precursor protein and Gag-Pol polyprotein and as genomic RNA that is packaged into progeny virions (6). The RNA packaging signal is a series of RNA structural motifs within the 5Ј untranslated region (UTR) that are recognized by the Gag nucleocapsid protein, which directs assembly of the unspliced RNA into progeny virions (1, 35). Structured 5Ј UTRs typically inhibit efficient translation by steric hindrance of ribosome scanning from the 5Ј cap to the proximal start codon (19,29,34). Mutational analysis of structural motifs in the human immunodeficiency virus type 1 (HIV-1) 5Ј UTR has verified that they inhibit efficient translation (18,31,33). Thus, the unspliced viral RNA subverts typical barriers to both nuclear export and efficient translation to achieve productive cytoplasmic expression of viral structural and enzymatic proteins.HIV-1 is a complex retrovirus that encodes the viral posttranscriptional regulatory protein Rev. Rev interacts with newly synthesized viral RNA (22) at the Rev-responsive element (RRE) present in distal intronic sequences to activate nuclear export despite lack of intron removal (8,20,21,28,46). Rev connects RRE-containing RNA to the CRM1/exportin 1 nuclear export receptor, which is typically utilized by 5S rRNA and shuttling proteins that contain a leucine-rich nuclear export sequence (17, 32). Genetically simpler retroviruses lack a viral Rev protein. However, the type D Mason-Pfizer monkey virus (MPMV) and simian retrovirus type 1 contain a functional homolog of RRE, which is designated the constitutive transport element (CTE). The CTE is a redundant stem-loop structure that is positioned in the 3Ј UTR and facilitates nuclear export of intron-containing RNA in transfected cells (5,15,16,38,42). CTE facilitates nuclear export by interaction with Tap and cofactor NXT1, which have been implicated as global modulators of the nuclear export of fully processed cellular mRNAs (4,25,44). Recently, studi...
Jaagsiekte sheep retrovirus (JSRV) is a betaretrovirus that causes ovine pulmonary adenocarcinoma, an infectious lung tumor of sheep (10, 29). Ovine pulmonary adenocarcinoma has morphological resemblance to a human lung cancer, bronchioloalveolar carcinoma, which is only weakly associated with cigarette smoking. In recent years, complete infectious and oncogenic molecular clones of JSRV have been isolated (30). We and others found that the JSRV envelope (Env) protein also functions as an oncogene in that it can induce morphological transformation of fibroblast and epithelial cell lines in culture and tumors in animals (1,24,34). Further studies have demonstrated that amino acids in the cytoplasmic tail of the Env transmembrane (TM) protein are important for transformation, as are multiple domains in the surface (SU) protein (17, 18).The nuclear export of mRNA is a critical step in gene expression. All retroviruses employ unspliced genome-length RNA as mRNA for synthesis of Gag and Pol proteins, while splicing yields mRNA(s) for Env (and other) proteins (15). Thus, genome-length mRNA for Gag and Pol is equivalent to an unspliced precursor for Env mRNA. A key issue for retroviruses is how they transport unspliced genome-length RNA to the cytoplasm. This is accomplished by two general mechanisms. The human immunodeficiency virus type 1 (HIV-1) Rev protein (encoded by a doubly spliced mRNA) specifically binds to a Rev-responsive element (RRE), located in RNA of the env gene. The Rev/RRE complex recruits the cellular CRM1/ Xpo1 protein (as well as other cellular proteins), which results in transport of this RNA-protein complex to the cytoplasm (7). Similarly, human T-cell leukemia virus type 1 (HTLV-1) Rex protein binds a Rex-responsive element on viral RNA, resulting in export via the CRM1 pathway (21). The betaretroviruses mouse mammary tumor virus (MMTV) and human endogenous retrovirus K (HERV-K) also encode analogous regulatory proteins (Rem and Rec, respectively) (19,22,27).In contrast, the betaretroviruses Mason-Pfizer monkey virus (MPMV) and simian retrovirus (SRV) contain constitutive RNA export elements (constitutive transport elements [CTEs]) that facilitate nuclear export of unspliced RNA (4, 41). The MPMV CTE is located between env and the 3Ј long terminal repeat (LTR); it binds to the cellular trans-acting factor NXF1/Tap, which directs nuclear export of the RNAprotein complex to the cytoplasm (14). Rous sarcoma virus and the related avian leukosis viruses contain direct repeat sequences flanking the src gene or in the 3Ј untranslated region of their RNA (28). Structure-function analyses of these RNAexporting elements revealed specific stem-loop structures that are important for activity and for binding of the host cell factors (3).Like other betaretroviruses, JSRV contains the standard genes gag, pro, pol, and env. In addition we recently found that JSRV also encodes a regulatory factor, Rej (17a). Rej is reminiscent of MMTV Rem and HERV-K Rec in that it is encoded in the 5Ј end of env and it is require...
Previous work has shown that spleen necrosis virus (SNV) long terminal repeats (LTRs) are associated with Rex/Rex-responsive element-independent expression of bovine leukemia virus RNA and supports the hypothesis that SNV RNA contains a cis-acting element that interacts with cellular Rex-like proteins. To test this hypothesis, the human immunodeficiency virus type 1 (HIV) Rev/RRE-dependent gag gene was used as a reporter to analyze various SNV sequences. Gag enzyme-linked immunosorbent assay and Western blot analyses reveal that HIV Gag production is enhanced at least 20,000-fold by the 5′ SNV LTR in COS, D17, and 293 cells. Furthermore, SNV RU5 in the sense but not the antisense orientation is sufficient to confer Rev/RRE-independent expression onto a cytomegalovirus-gag plasmid. In contrast, the SNV 3′ LTR and 3′ untranslated sequence between env and the LTR did not support Rev-independent gag expression. Quantitative RNase protection assays indicate that the SNV 5′ RNA terminus enhances cytoplasmic accumulation and polysome association of HIV unspliced and spliced transcripts. However, comparison of the absolute amounts of polysomal RNA indicates that polysome association is not sufficient to account for the significant increase in Gag production by the SNV sequences. Our analysis reveals that the SNV 5′ RNA terminus contains a unique cis-acting posttranscriptional control element that interacts with hypothetical cellular Rev-like proteins to facilitate HIV RNA transport and efficient translation.
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