Bicistronic mRNA Research Articles

Adding genes to the RNA genome of vesicular stomatitis virus: positional effects on stability of expression.

Gene expression of the nonsegmented negative strand (NNS) RNA viruses is controlled primarily at the level of transcription by the position of the genes relative to the single transcriptional promoter. We tested this principle by generating engineered variants of vesicular stomatitis virus in which an additional, identical, transcriptional unit was added to the genome at each of the viral gene junctions. Analysis of transcripts confirmed that the level of transcription was determined by the position of the gene relative to the promoter. However, the position at which a gene was inserted affected the replication potential of the viruses. Adding a gene between the first two genes, N and P, reduced replication by over an order of magnitude, whereas addition of a gene at the other gene junctions had no effect on replication levels. All genes downstream of the inserted gene had decreased levels of expression, since transcription of the extra gene introduced an additional transcriptional attenuation event. The added gene was stably maintained in the genome upon repeated passage in all cases. However, expression of the added gene was stable at only three of the four positions. In the case of insertion between the N and P genes, a virus population arose within two passages that had restored replication to wild-type levels. In this population, expression of the additional gene as a monocistronic mRNA was suppressed by mutations at the end of the upstream (N) gene that abolished transcriptional termination. Because transcription is obligatorily sequential, this prevented transcription of the inserted downstream gene as a monocistronic mRNA and resulted instead in polymerase reading through the gene junction to produce a bicistronic mRNA. This eliminated the additional attenuation step and restored expression of all downstream genes and viral replication to wild-type levels. These data show that transcriptional termination is a key element in control of gene expression of the negative strand RNA viruses and a means by which expression of individual genes may be regulated within the framework of a single transcriptional promoter. Further, these results are directly relevant to the use of NNS viruses as vectors and vaccine delivery agents, as they show that the level of expression of an added gene can be controlled by its insertion position but that not all positions of insertion yield stable expression of the added gene.

The repressor for an organic peroxide-inducible operon is uniquely regulated at multiple levels.

ohrR encodes a novel organic peroxide-inducible transcription repressor, and we have demonstrated that ohrR is regulated at the transcriptional and the post-transcriptional levels. Primer extension results show that ohrR transcription initiates at the A residue of the ATG translation initiation codon for the ohrR coding sequence. Thus, the gene has a leaderless mRNA. The ohrR promoter (P1) has high homology to the consensus sequence for Xanthomonas promoters, which is reflected in the high in vivo promoter activity of P1. Deletion of a 139 bp fragment containing the P1 promoter showed that the sequences upstream of -35 regions were required for neither the promoter activity nor OhrR autoregulation. In vitro, purified OhrR specifically binds to the P1 promoter. DNase I footprinting of OhrR binding to the P1 revealed a 44 bp region of protection on both DNA strands. The protected regions include the -35 and -10 regions of P1. We suggest that OhrR represses gene expression by blocking RNA polymerase binding to the promoter. There are two steps in the post-transcriptional regulation of ohrR, namely differential stability and inefficient translation of the mRNA. The bicistronic ohrR-ohr mRNA was highly labile and underwent rapid processing in vivo to give only stable monocistronic ohr mRNA and undetectable ohrR mRNA. Furthermore, the ohrR mRNA was inefficiently translated. We propose that, in uninduced cells, the concentration of OhrR is maintained at low levels by the autoregulation mechanism at the transcriptional levels and by the ohrR mRNA instability coupled with inefficient translation at the post-transcriptional level. Upon exposure to an organic peroxide, the compound probably interacts with OhrR and prevents it from repressing the P1 promoter, thus allowing high-level expression of the ohrR-ohr operon. The rapid processing of bicistronic mRNA gives highly stable ohr mRNA and corresponding high levels of Ohr, which remove an organic per-oxide. Once the peroxide has been removed, the autoregulation mechanism feeds back to inhibit the expression of the operon.

Endostatin gene transfer inhibits joint angiogenesis and pannus formation in inflammatory arthritis.

Rheumatoid arthritis is a prevalent example of an inflammatory angiogenic disease, which is mediated by pro-inflammatory and pro-angiogenic cytokines such as tumor necrosis factor (TNF). To evaluate the effect of the potent anti-angiogenic factor endostatin on TNF-induced inflammatory arthritis, we injected an endostatin-expressing lentiviral vector directly into the joints of human TNF-transgenic mice before the onset of disease. Histological analysis of the injected joints 8 weeks later revealed that endostatin reduced blood vessel density within the synovial tissues and an overall mean arthritis index. In vitro and in vivo examination of the potential mechanism by which endostatin inhibited the arthritis revealed that endostatin blocks TNF-induced activation of JNK and JNK-dependent pro-angiogenic gene expression. These data suggest a novel mechanism by which endostatin inhibits angiogenesis, and demonstrates the potential utility of anti-angiogenic gene therapy for treatment of inflammatory arthritis.

Regulation of Internal Ribosomal Entry Site-mediated Translation by Phosphorylation of the Translation Initiation Factor eIF2α

Initiation of translation from most cellular mRNAs occurs via scanning; the 40 S ribosomal subunit binds to the m(7)G-cap and then moves along the mRNA until an initiation codon is encountered. Some cellular mRNAs contain internal ribosome entry sequences (IRESs) within their 5'-untranslated regions, which allow initiation independently of the 5'-cap. This study investigated the ability of cellular stress to regulate the activity of IRESs in cellular mRNAs. Three stresses were studied that cause the phosphorylation of the translation initiation factor, eIF2alpha, by activating specific kinases: (i) amino acid starvation, which activates GCN2; (ii) endoplasmic reticulum (ER) stress, which activates PKR-like ER kinase, PERK kinase; and (iii) double-stranded RNA, which activates double-stranded RNA-dependent protein kinase (PKR) by mimicking viral infection. Amino acid starvation and ER stress caused transient phosphorylation of eIF2alpha during the first hour of treatment, whereas double-stranded RNA caused a sustained phosphorylation of eIF2alpha after 2 h. The effects of these treatments on IRES-mediated initiation were investigated using bicistronic mRNA expression vectors. No effect was seen for the IRESs from the mRNAs for the chaperone BiP and the protein kinase Pim-1. In contrast, translation mediated by the IRESs from the cationic amino acid transporter, cat-1, and of the cricket paralysis virus intergenic region, were stimulated 3- to 10-fold by all three treatments. eIF2alpha phosphorylation was required for the response because inactivation of phosphorylation prevented the stimulation. It is concluded that cellular stress can stimulate translation from some cellular IRESs via a mechanism that requires the phosphorylation of eIF2alpha. Moreover, there are distinct regulatory patterns for different cellular mRNAs that contain IRESs within their 5'-untranslated regions.

Patterns of Gene Expression and a Transactivation Function Exhibited by the vGCR (ORF74) Chemokine Receptor Protein of Kaposi's Sarcoma-Associated Herpesvirus

The ORF74 or vGCR gene encoded by Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8) has properties of a ligand-independent membrane receptor signaling protein with angiogenic properties that is predicted to play a key role in the biology of the virus. We have examined the expression of vGCR mRNA and protein in primary effusion lymphoma (PEL) cell lines, PEL and multicentric Castleman's disease (MCD) tumors, Kaposi's sarcoma lesions and infected endothelial cell cultures. The vGCR gene proved to be expressed in PEL cell lines as a large spliced bicistronic mRNA of 3.2 kb that also encompasses the upstream vOX2 (K14) gene. This mRNA species was induced strongly by phorbol ester (TPA) and sodium butyrate treatment in the BCBL-1 cell line, but only weakly in the HBL6 cell line, and was classified as a relatively late and low-abundance delayed early class lytic cycle gene product. A complex bipartite upstream lytic cycle promoter for this mRNA was nestled within the intron of the 5'-overlapping but oppositely oriented latent-state transcription unit for LANA1/vCYC-D/vFLIP and responded strongly to both TPA induction and cotransfection with the KSHV RNA transactivator protein (RTA or ORF50) in transient reporter gene assays. A vGCR protein product of 45 kDa that readily dimerized was detected by Western blotting and in vitro translation and was localized in a cytoplasmic and membrane pattern in DNA-transfected Vero and 293T cells or adenovirus vGCR-transduced dermal microvascular endothelial cells (DMVEC) as detected by indirect immunofluorescence assay (IFA) and immunohistochemistry with a specific rabbit anti-vGCR antibody. Similarly, a subfraction of KSHV-positive cultured PEL cells and of KSHV (JSC-1) persistently infected DMVEC cells displayed cytoplasmic vGCR protein expression, but only after TPA or spontaneous lytic cycle induction, respectively. The vGCR protein was also detectable by immunohistochemical staining in a small fraction (0.5 to 3%) of the cells in PEL and MCD tumor and nodular Kaposi's sarcoma lesion specimens that were apparently undergoing lytic cycle expression. These properties are difficult to reconcile with the vGCR protein's playing a direct role in spindle cell proliferation, transformation, or latency, but could be compatible with proposed contributions to angiogenesis via downstream paracrine effects. The ability of vGCR to transactivate expression of both several KSHV promoter-driven luciferase (LUC) reporter genes and an NFkappaB motif containing the chloramphenicol acetyltransferase (CAT) reporter gene may also suggest an unexpected regulatory role in viral gene expression.

Kaposi's Sarcoma-associated Herpesvirus K8 Exon 3 Contains Three 5′-Splice Sites and Harbors a K8.1 Transcription Start Site

Kaposi's sarcoma-associated herpesvirus (KSHV) K8 and K8.1 open reading frames are juxtaposed and span from nucleotide (nt) 74850 to 76695 of the virus genome. A K8 pre-mRNA overlaps the entire K8.1 coding region, and alternative splicing of KSHV K8 and K8.1 pre-mRNAs each produces three isoforms (alpha, beta, and gamma) of the mRNAs. We have mapped the 5' end of the K8.1 RNA in butyrate-induced KSHV-positive JSC-1 cells to nt 75901 in the KSHV genome and have shown that exon 3 of the K8 pre-mRNA in JSC-1 cells covers most part of the intron 3 defined previously and has three 5'-splice sites (ss), respectively, at nt 75838, 76155, and 76338. Selection of the nt 75838 5'-ss dictates the K8 mRNA production and overwhelms the RNA processing. Alternative selection of other two 5'-ss is feasible and leads to production of two additional bicistronic mRNAs, K8/K8.1alpha and -beta. However, the novel bicistronic K8/K8.1 mRNAs translated a little K8 and no detectable K8.1 proteins in 293 cells. Data suggest that production of the K8/K8.1 mRNAs may be an essential way to control K8 mRNAs, especially K8alpha, to a threshold at RNA processing level.

A proposed vestigial translation initiation motif in VP1 of hepatitis A virus

The internal ribosome entry site (IRES) of picornaviruses has a 3′ polypyrimidine tract (PPT) 16–24 bases upstream of an AUG triplet (PPT/AUG motif). This motif is critical in determining the efficiency of cap-independent translation. HAV has a conserved PPT/AUG motif consisting of a nine base sequence (AGGUUUUUC) 23 bases upstream of the preferred AUG start codon. This HAV-specific PPT/AUG motif is repeated and conserved in VP1 of HAV, but not of other picornaviruses. We proposed that the PPT/AUG motif in the open reading frame initiated translation and/or had an impact on the life cycle of the virus. In vitro translation of mutant bicistronic mRNAs and growth in cell culture of mutant viruses provided no evidence that the VP1 PPT/AUG motif had any impact on either translation or growth. HAV differs from other picornaviruses in its inefficient growth in cell culture. Since the HAV-specific PPT/AUG motif is found in only 1 in 300,000 reported viral sequences outside the hepatovirus genus, this motif may be a vestigial translation initiation element and may have played a role in determining the unusual phenotype of HAV.

Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins.

Now that it is likely that all yeast nucleoporins are known, one of the ultimate goals is the in vitro assembly of the entire nuclear pore complex from its approximately 30 individual components. Here, we report the reconstitution of seven proteins (Nup133p, Nup145p-C, Nup120p, Nup85p, Nup84p, Seh1p and Sec13p) into a heptameric 0.5 MDa nuclear pore subcomplex. We found that double plasmid transformation combined with bi-cistronic mRNA translation allow the expression and assembly of distinct subcomplexes of up to five nucleoporins in a single Escherichia coli cell. During the sequential reconstitution of the Nup84p complex, smaller assembly intermediates can be isolated, which exhibit modular structures determined by electron microscopy that finally make up the whole Y-shaped Nup84p complex. Importantly, a seventh subunit, Nup133p, was incorporated into the complex through its interaction with Nup84p, thereby elongating one arm of the Y-shaped assembly to an approximately 40 nm long stalk. Taken together, our data document that the Nup84p-Nup133p complex self-assembles in a modular concept from distinct smaller nucleoporin construction sets.

Functional Organization of MIR2, a Novel Viral Regulator of Selective Endocytosis

Kaposi's sarcoma-associated herpesvirus encodes two related proteins, MIR1 and MIR2, that lead to reduction of the cell surface levels of major histocompatibility complex class I and other polypeptides involved in immune recognition. MIR1 and MIR2 do not affect the assembly or transport of their target proteins through the secretory pathway; rather, they act to enhance the selective endocytosis of target chains from the cell surface. Sequence inspection reveals that the modulator of immune recognition (MIR) proteins contain an NH(2)-terminal zinc finger of the plant homeodomain (PHD) subfamily, two transmembrane (TM) domains, and a C-terminal conserved region (CR). Here we examine the transmembrane topology and functional organization of MIR2. Both the PHD domain and the CR are disposed cytosolically and are essential for MIR-mediated endocytosis. MIR proteins form homo-oligomers; this activity is independent of the PHD and CR elements and maps instead to the TM regions. Analysis of chimeras between MIR1 and MIR2 reveals that the TM regions also mediate target selectivity. Mutations that ablate the PHD or CR regions generate dominant negative phenotypes for major histocompatibility complex class I endocytosis. These findings suggest a domain organization for the MIR proteins, with the TM regions involved in target selection and the cytosolic PHD and CR domains involved in the possible recruitment of cellular machinery that directly or indirectly regulates internalization of target molecules.

The development of a bicistronic plasmid DNA vaccine for B-cell lymphoma

Tumor vaccines are a promising alternative to chemotherapy for the treatment of metastatic cancer. To be effective and safe, a therapeutic cancer vaccine should specifically target antigens expressed only on metastatic tumor cells. A vaccine directed against the unique surface immunoglobulin or idiotype expressed on non-Hodgkin’s B-cell lymphoma fulfills these criteria, as both primary and metastatic tumor cells express tumor specific immunoglobulins. Using the murine 38C13 B-cell lymphoma tumor as a model system, a plasmid DNA vaccine was designed to express a bicistronic mRNA encoding both the light and heavy tumor immunoglobulin (idiotype) proteins expressed on the surface of the 38C13 tumor. To increase the immunogenicity of the plasmid DNA vaccine, each of the murine variable domains (light and heavy) were fused to their respective human immunoglobulin constant domains. In addition, a eukaryotic expression cassette was constructed to effect both high-level expression of the mouse/human chimeric immunoglobulin, and to elicit a protective immune response in vivo. Unique Sfi I restriction sites were used for the rapid cloning of any tumor specific immunoglobulin idiotype domains and a series of plasmid constructs were made to test changes to the J domain and/or the human C domain to insert the Sfi I restriction sites. Such changes were found to have significant effects on both expression and immunogenicity. Vaccination of mice with prototype idiotype vaccines was found to generate a protective immune response to the 38C13 tumor. This study indicates that a novel bicistronic plasmid DNA-based vaccine can be used to develop a tumor specific vaccine against B-cell lymphoma.

Regulation of Internal Ribosome Entry Site-mediated Translation by Eukaryotic Initiation Factor-2α Phosphorylation and Translation of a Small Upstream Open Reading Frame

Adaptation to amino acid deficiency is critical for cell survival. In yeast, this adaptation involves phosphorylation of the translation eukaryotic initiation factor (eIF) 2alpha by the kinase GCN2. This leads to the increased translation of the transcription factor GCN4, which in turn increases transcription of amino acid biosynthetic genes, at a time when expression of most genes decreases. Here it is shown that translation of the arginine/lysine transporter cat-1 mRNA increases during amino acid starvation of mammalian cells. This increase requires both GCN2 phosphorylation of eIF2alpha and the translation of a 48-amino acid upstream open reading frame (uORF) present within the 5'-leader of the transporter mRNA. When this 5'-leader was placed in a bicistronic mRNA expression vector, it functioned as an internal ribosomal entry sequence and its regulated activity was dependent on uORF translation. Amino acid starvation also induced translation of monocistronic mRNAs containing the cat-1 5'-leader, in a manner dependent on eIF2alpha phosphorylation and translation of the 48-amino acid uORF. This is the first example of mammalian regulation of internal ribosomal entry sequence-mediated translation by eIF2alpha phosphorylation during amino acid starvation, suggesting that the mechanism of induced Cat-1 protein synthesis is part of the adaptive response of cells to amino acid limitation.

Inhibition of carcinoma cell growth and metastasis by a vesicular stomatitis virus G-pseudotyped retrovector expressing type I insulin-like growth factor receptor antisense.

A replication-defective, vesicular stomatitis virus G-pseudotyped, Moloney murine leukemia virus retroviral vector (vLTR-IGF-IR(AS)) was generated in which a type I insulin-like growth factor receptor (IGF-IR) antisense fragment is expressed in a bicistronic mRNA with an enhanced green fluorescent protein (EGFP) reporter under the control of a potent long terminal repeat (LTR). The suitability of these retroparticles for gene therapy was tested with highly metastatic, carcinoma H-59 cells, which depend on IGF-IR expression for tumorigenicity and metastasis. Transduction with these, but not with control retroviral particles expressing EGFP only, resulted in a 70% reduction in IGF-IR levels and the loss of IGF-IR-regulated functions. Moreover, the ability of vLTR-IGF-IR(AS) retroparticle-transduced tumor cells to form experimental hepatic metastases was significantly reduced relative to controls. The results identify retrovector-mediated delivery of IGF-IR antisense as a potential strategy for cancer gene therapy.

Novel bicistronic retroviral vector expressing gamma-glutamylcysteine synthetase and the multidrug resistance protein 1 (MRP1) protects cells from MRP1-effluxed drugs and alkylating agents.

We have constructed two retroviral vectors, one expressing multidrug resistance protein 1 (MRP1) alone (SF91MRP) and the other expressing MRP1 and gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione biosynthesis (SF91GCS-MRP). We have utilized the hybrid FMEV (Friend mink cell focus-forming/murine embryonic stem cell virus) backbone, previously shown to be efficient in early hematopoietic cells, even when coexpressing two distinct genes. In SF91GCS-MRP, the cDNAs were combined via an internal ribosomal entry site (IRES) sequence from poliovirus, resulting in a bicistronic mRNA produced via the long terminal repeat (LTR). Producer Fly-eco clones were established by trans-infection with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped retroviral supernatants. Drug-resistant producer clones were subsequently selected with antimony potassium tartrate, a nonmutagenic MRP1 substrate. By RNA slot-blot and transduction of 3T3 fibroblasts, titers of both SF91MRP and SF91GCS-MRP were found to be greater than 10(6) viral particles/ml. The correct viral integration in the genome was established by Southern blotting. By flow cytometry, both MRP1 and bicistronic clones showed an increase in expression of the MRP1 protein. The bicistronic producer clones, as well as 3T3 cells transduced with SF91GCS-MRP, presented an increase in intracellular glutathione levels, compared with the parental counterparts. Producer cells, 3T3 fibroblasts transduced with either SF91MRP or SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were resistant to MRP1-effluxed drugs. However, only bicistronic producers, 3T3 fibroblasts transduced with SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were also resistant to alkylating agents. We conclude that the retrovirus SF91GCS-MRP has features that make it a suitable vector to induce bone marrow resistance to multiple classes of chemotherapeutic agents. The strategy of coexpressing gamma-GCS and MRP1 may help to design an effective in vivo selection for various clinical protocols of gene therapy.

MDR1 bicistronic vectors: analysis of selection stringency, amplified gene expression, and vector stability in cell lines

The human multidrug resistance-1 gene (MDR1) is a dominant selectable and amplifiable marker in mammalian tissue culture cells. MDR1 is also being investigated as a gene therapy tool, both to protect normal cells against chemotherapy-related toxicity and to serve as an in vivo selectable marker for the overexpression of non-selectable therapeutic genes. The success of these strategies will depend on whether MDR1 expression can be sustained at levels high enough to confer a survival advantage on target cells. However, the MDR1 selection system is quite stringent, requiring high gene expression for transduced cells to survive in the presence of drug. The current report is a detailed molecular analysis of MDR1 selection stringency compared with the common neo selectable marker. A bicistronic vector encoding MDR1 and neo genes linked through an internal ribosome entry site was transferred into NIH 3T3 mouse fibroblasts and K562 human leukemia cells; cells were then exposed to colchicine (to select for MDR1 expression) or to G418 (to select for neo expression). Surviving populations and individual clones of cells were analyzed for expression levels of MDR1 and neo gene products; resistance to colchicine, paclitaxel, and G418; level and integrity of bicistronic mRNA; and structural integrity, integration number, and copy number of vector DNA. These studies provide direct evidence that colchicine selection is more stringent than G418 selection; that increased selection pressure with colchicine leads to increased gene expression; that increased gene expression can be accommodated primarily by gene amplification, even within an individual transduced clone and starting from a single-copy proviral integration event; and that the clonal diversity of a transduced population of cells is influenced significantly by the stringency of selection. Taken together, these results have important implications for the potential utility of MDR1 as a selectable marker and as a gene therapy tool in hematopoietic cells.

Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs.

In addition to the cap-dependent mechanism, eukaryotic initiation of translation can occur by a cap-independent mechanism which directs ribosomes to defined start codons enabled by internal ribosome entry site (IRES) elements. IRES elements from poliovirus and encephalomyocarditis virus are often used to construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA. We found that while cap-dependent translation initiation from bicistronic mRNAs remains comparable to monocistronic expression, internal initiation mediated by these viral IRESs is often very inefficient. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cytokines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bicistronic setting is crucial for IRES-dependent translation of the second cistron. A systematic examination of expression of reporter cistrons from bicistronic mRNAs with respect to position was carried out. Using the dual luciferase assay system we show that the composition of reading frames on a bicistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the nature of the IRES element influence translation strength the dominant determinant is the nature and the arrangement of cistrons on the mRNA.

Complex regulation of the organic hydroperoxide resistance gene (ohr) from Xanthomonas involves OhrR, a novel organic peroxide-inducible negative regulator, and posttranscriptional modifications.

Analysis of the sequence immediate upstream of ohr revealed an open reading frame, designated ohrR, with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression. ohrR was transcribed as bicistronic mRNA with ohr, while ohr mRNA was found to be 95% monocistronic and 5% bicistronic with ohrR. Expression of both genes was induced by tert-butyl hydroperoxide (tBOOH) treatment. High-level expression of ohrR negatively regulated ohr expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the ohrR promoter (P1) has organic peroxide-inducible, strong activity, while the ohr promoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR. ohr primer extension results revealed three major primer extension products corresponding to the 5' ends of ohr mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical Xanthomonas promoter. Instead, the regions can form a stem-loop secondary structure with the 5' ends of ohr mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the ohrR-ohr operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of ohr mRNA, while ohrR mRNA is rapidly degraded.

P27 Doesn't Need a Cap

In quiescent cells that have withdrawn from the cell cycle, protein translation is generally depressed by virtue of inhibition of the activity of the cap-dependent translation factor, eukaryotic initiation factor eIF4. One exception is the cyclin-dependent kinase inhibitor p27, which has high rates of expression in quiescent cells. Through analysis of the translation of a reporter from a bicistronic mRNA containing the p27 5′ untranslated region (UTR), Miskimins et al. show that p27 contains an internal ribosome entry sequence. In proliferating cells, the expression of an epitope-tagged p27 from the complete 5′ UTR was low, but could be stimulated by treatment with a drug to induce withdrawal from the cell cycle. In cells expressing a construct lacking the complete 5′ UTR, expression was high in both proliferating and quiescent cells. Thus, translation from the internal ribosome entry sequence can be regulated and provides a mechanism by which proteins can be differentially upregulated despite inhibition of cap-dependent translation. W. K. Miskimins, G. Wang, M. Hawkinson, R. Miskimins, Control of cyclin-dependent kinase inhibitor p27 expression by cap-independent translation. Mol. Cell. Biol. 21 , 4960-4967 (2001). [Abstract] [Full Text]

Binding Sites for Rev and ASF/SF2 Map to a 55-Nucleotide Purine-rich Exonic Element in Equine Infectious Anemia Virus RNA*

The equine infectious anemia virus (EIAV) Rev protein (ERev) negatively regulates its own synthesis by inducing alternative splicing of its mRNA. This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. When Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. The interaction of ERev with its cis-acting RNA response element, the RRE, is also essential for nuclear export of intron-containing viral mRNAs that encode structural and enzymatic gene products. The primary ERev binding site and the manner in which ERev interacts with RNA or cellular proteins to exert its regulatory function have not been defined. We have performed in vitro RNA binding experiments to show that recombinant ERev binds to a 55-nucleotide, purine-rich tract proximal to the 5' splice site of exon 3. Because of its proximity to the 5' splice site and since it contains elements related to consensus exonic splicing enhancer sequences, we asked whether cellular proteins recognize the EIAV RRE. The cellular protein, ASF/SF2, a member of the serine- and arginine-rich family of splicing factors (SR proteins) bound to repeated sequences within the 55-nucleotide RRE region. Electrophoretic mobility shift and UV cross-linking experiments indicated that ERev and SR proteins bind simultaneously to the RRE. Furthermore, in vitro protein-protein interaction studies revealed an association between ERev and SR proteins. These data suggest that EIAV Rev-induced exon skipping observed in vivo may be initiated by simultaneous binding of Rev and SR proteins to the RRE that alter the subsequent assembly or catalytic activity of the spliceosomal complex.

Herpesvirus saimiri.

Herpesvirus saimiri (saimiriine herpesvirus 2) is the classical prototype of the gamma(2)-herpesviruses or rhadinoviruses, which also contains a human member, the Kaposi's sarcoma-associated herpesvirus. The T-lymphotropic Herpesvirus saimiri establishes specific replicative and persistent conditions in different primate host species. Virtually all squirrel monkeys (Saimiri sciureus) are persistently infected with this virus. In its natural host, the virus does not cause disease, whereas it induces fatal acute T-cell lymphoma in other monkey species after experimental infection. The virus can be isolated by cocultivation of permissive epithelial cells with peripheral blood cells from naturally infected squirrel monkeys and from susceptible New World monkeys during the virus-induced disease. Tumour-derived and in vitro-transformed T-cell lines from New World monkeys release virus particles. Herpesvirus ateles is a closely related virus of spider monkeys (Ateles spp.) and has similar pathogenic properties to Herpesvirus saimiri in other New World primate species. Similar to other rhadinoviruses, the genome of Herpesvirus saimiri harbours a series of virus genes with pronounced homology to cellular counterparts including a D-type cyclin, a G-protein-coupled receptor, an interleukin-17, a superantigen homologue, and several inhibitors of the complement cascade and of different apoptosis pathways. Preserved function has been demonstrated for most of the homologues of cellular proteins. These viral functions are mostly dispensable for the transforming and pathogenic capability of the virus. However, they are considered relevant for the apathogenic persistence of Herpesvirus saimiri in its natural host. A terminal region of the non-repetitive coding part of the virus genome is essential for pathogenicity and T-cell transformation. Based on the pathogenic phenotypes and the different alleles of this variable region, the virus strains have been assigned to three subgroups, termed A, B and C. In the highly oncogenic subgroup C strains, the two virus genes stpC and tip are transcribed from one bicistronic mRNA and are essential for transformation and leukaemia induction. stpC fulfils the typical criteria of an oncogene; its product interacts with Ras and tumour necrosis factor-associated factors and induces mitogen-activated protein kinase and nuclear factor kappa B activation. Tip interacts with the RNA transport factor Tap, with signal transduction and activation of transcription factors, and with the T-cellular tyrosine kinase Lck, which is activated by this interaction and phosphorylates Tip as a substrate. It is of particular interest that certain subgroup C virus strains such as C488 are capable of transforming human T lymphocytes to stable growth in culture. The transformed human T cells harbour multiple copies of the viral genome in the form of stable, non-integrated episomes. The cells express only a few virus genes and do not produce virus particles. The transformed cells maintain the antigen specificity and many other essential functions of their parental T-cell clones. Based on the preserved functional phenotype of the transformed T cells, Herpesvirus saimiri provides useful tools for T-cell immunology, for gene transfer and possibly also for experimental adoptive immunotherapy.

Avian polyomavirus agnoprotein 1a is incorporated into the virus particle as a fourth structural protein, VP4.

Agnoproteins, encoded by the 5'-region of the late bicistronic mRNA of some polyomaviruses, are small proteins with largely unknown functions. In avian polyomavirus (APV)-infected cells, mRNAs of seven putative agnoproteins have been observed. Recently, it has been shown that agnoprotein 1a and its truncated variant agnoprotein 1b, encoded by the predominant mRNA species, are essential for APV replication. Here, the presence of agnoprotein 1a is demonstrated in the nucleus of APV-infected cells and in purified APV particles. Interaction between agnoprotein 1a and the major structural protein, VP1, was demonstrated by co-immunoprecipitation experiments using lysates of recombinant baculovirus-infected insect cells. With proteins expressed in E. coli, binding to double-stranded DNA in a sequence-unspecific manner was shown for agnoprotein 1a, whereas agnoprotein 1b failed to bind. A leucine zipper-like motif present in agnoprotein 1a is considered to be involved in DNA binding. Due to the absence of any structural or functional homologies between APV agnoprotein 1a and the agnoproteins of mammalian polyomaviruses, it is suggested that this protein should be renamed VP4, indicating its function as a fourth structural protein of APV.