Retroviral RNA Processing.

This review is an accompaniment to a Special Issue on "Retroviral RNA Processing". It discusses post-transcriptional regulation of retroviruses, ranging from the ancient foamy viruses to more modern viruses, such as HIV-1, HTLV-1, Rous sarcoma virus, murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus. This review is not comprehensive. However, it tries to address some of the major questions in the field with examples of how different retroviruses express their genes. It is amazing that a single primary RNA transcript can have so many possible fates: genomic RNA, unspliced mRNA, and up to 50 different alternatively spliced mRNAs. This review will discuss the sorting of RNAs for packaging or translation, RNA nuclear export mechanisms, splicing, translation, RNA modifications, and avoidance of nonsense-mediated RNA decay.

Integration of ALV into CTDSPL and CTDSPL2 genes in B-cell lymphomas promotes cell immortalization, migration and survival.

Avian leukosis virus induces tumors in chickens by integrating into the genome and altering expression of nearby genes. Thus, ALV can be used as an insertional mutagenesis tool to identify novel genes involved in tumorigenesis. Deep sequencing analysis of viral integration sites has identified CTDSPL and CTDSPL2 as common integration sites in ALV-induced B-cell lymphomas, suggesting a potential role in driving oncogenesis. We show that in tumors with integrations in these genes, the viral promoter is driving the expression of a truncated fusion transcript. Overexpression in cultured chick embryo fibroblasts reveals that CTDSPL and CTDSPL2 have oncogenic properties, including promoting cell migration. We also show that CTDSPL2 has a previously uncharacterized role in protecting cells from apoptosis induced by oxidative stress. Further, the truncated viral fusion transcripts of both CTDSPL and CTDSPL2 promote immortalization in primary cell culture.

Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3'UTRs.

All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3' untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3'UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3'UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3'UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5'-3' exonucleolytic (XRN1) function in knockdown experiments in human cells.

The FACT Complex Promotes Avian Leukosis Virus DNA Integration.

All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. In this study, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity in vitro Likewise, in infected cells, the FACT complex promotes ALV integration activity, with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-long-terminal-repeat (2-LTR) circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells.IMPORTANCE The majority of human gene therapy approaches utilize HIV-1- or murine leukemia virus (MLV)-based vectors, which preferentially integrate near genes and regulatory regions; thus, insertional mutagenesis is a substantial risk. In contrast, ALV integrates more randomly throughout the genome, which decreases the risks of deleterious integration. Understanding how ALV integration is regulated could facilitate the development of ALV-based vectors for use in human gene therapy. Here we show that the FACT complex directly binds and regulates ALV integration efficiency in vitro and in infected cells.

A Novel Long Non-Coding RNA in the hTERT Promoter Region Regulates hTERT Expression.

A novel antisense transcript was identified in the human telomerase reverse transcriptase (hTERT) promoter region, suggesting that the hTERT promoter is bidirectional. This transcript, named hTERT antisense promoter-associated (hTAPAS) RNA, is a 1.6 kb long non-coding RNA. hTAPAS transcription is initiated 167 nucleotides upstream of the hTERT transcription start site and is present in both the nucleus and the cytoplasm. Surprisingly, we observed that a large fraction of the hTERT polyadenylated RNA is localized in the nucleus, suggesting this might be an additional means of regulating the cellular abundance of hTERT protein. Both hTAPAS and hTERT are expressed in immortalized B-cells and human embryonic stem cells but are not detected in normal somatic cells. hTAPAS expression inversely correlates with hTERT expression in different types of cancer samples. Moreover, hTAPAS expression is not promoted by an hTERT promoter mutation (-124 C>T). Antisense-oligonucleotide mediated knockdown of hTAPAS results in an increase in hTERT expression. Conversely, ectopic overexpression of hTAPAS down regulates hTERT expression, suggesting a negative role in hTERT gene regulation. These observations provide insights into hTAPAS as a novel player that negatively regulates hTERT expression and may be involved in telomere length homeostasis.

Lack of TERT Promoter Mutations in Human B-Cell Non-Hodgkin Lymphoma.

Non-Hodgkin lymphomas (NHL) are a heterogeneous group of immune cell neoplasms that comprise molecularly distinct lymphoma subtypes. Recent work has identified high frequency promoter point mutations in the telomerase reverse transcriptase (TERT) gene of different cancer types, including melanoma, glioma, liver and bladder cancer. TERT promoter mutations appear to correlate with increased TERT expression and telomerase activity in these cancers. In contrast, breast, pancreatic, and prostate cancer rarely demonstrate mutations in this region of the gene. TERT promoter mutation prevalence in NHL has not been thoroughly tested thus far. We screened 105 B-cell lymphoid malignancies encompassing nine NHL subtypes and acute lymphoblastic leukemia, for TERT promoter mutations. Our results suggest that TERT promoter mutations are rare or absent in most NHL. Thus, the classical TERT promoter mutations may not play a major oncogenic role in TERT expression and telomerase activation in NHL.

Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway.

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD.

Common Viral Integration Sites Identified in Avian Leukosis Virus-Induced B-Cell Lymphomas.

UNLABELLED: Avian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating within or near cancer genes and perturbing their expression. Four genes--MYC, MYB, Mir-155, and TERT--have previously been identified as common integration sites in these virus-induced lymphomas and are thought to play a causal role in tumorigenesis. In this study, we employ high-throughput sequencing to identify additional genes driving tumorigenesis in ALV-induced B-cell lymphomas. In addition to the four genes implicated previously, we identify other genes as common integration sites, including TNFRSF1A, MEF2C, CTDSPL, TAB2, RUNX1, MLL5, CXorf57, and BACH2. We also analyze the genome-wide ALV integration landscape in vivo and find increased frequency of ALV integration near transcriptional start sites and within transcripts. Previous work has shown ALV prefers a weak consensus sequence for integration in cultured human cells. We confirm this consensus sequence for ALV integration in vivo in the chicken genome. IMPORTANCE: Avian leukosis virus induces B-cell lymphomas in chickens. Earlier studies showed that ALV can induce tumors through insertional mutagenesis, and several genes have been implicated in the development of these tumors. In this study, we use high-throughput sequencing to reveal the genome-wide ALV integration landscape in ALV-induced B-cell lymphomas. We find elevated levels of ALV integration near transcription start sites and use common integration site analysis to greatly expand the number of genes implicated in the development of these tumors. Interestingly, we identify several genes targeted by viral insertions that have not been previously shown to be involved in cancer.

Avian retroviral replication.

Avian retroviruses were originally identified as cancer-inducting filterable agents in chicken neoplasms at the beginning of the 20th century. Since their discovery, the study of these simple retroviruses has contributed greatly to our understanding of viral replication and cancer. Avian retroviruses continue to evolve and have great economic importance in the poultry industry worldwide. The aim of this review is to provide a broad overview of the genome, pathology, and replication of avian retroviruses. Notable gaps in our current knowledge are highlighted, and areas where avian retroviruses differ from other retroviruses are emphasized.

Exclusion of exon 2 is a common mRNA splice variant of primate telomerase reverse transcriptases.

Telomeric sequences are added by an enzyme called telomerase that is made of two components: a catalytic protein called telomerase reverse transcriptase (TERT) and an integral RNA template (TR). Telomerase expression is tightly regulated at each step of gene expression, including alternative splicing of TERT mRNA. While over a dozen different alternative splicing events have been reported for human TERT mRNA, these were all in the 3' half of the coding region. We were interested in examining splicing of the 5' half of hTERT mRNA, especially since exon 2 is unusually large (1.3 kb). Internal mammalian exons are usually short, typically only 50 to 300 nucleotides, and most long internal exons are alternatively processed. We used quantitative RT-PCR and high-throughput sequencing data to examine the variety and quantity of mRNA species generated from the hTERT locus. We determined that there are approximately 20-40 molecules of hTERT mRNA per cell in the A431 human cell line. In addition, we describe an abundant, alternatively-spliced mRNA variant that excludes TERT exon 2 and was seen in other primates. This variant causes a frameshift and results in translation termination in exon 3, generating a 12 kDa polypeptide.

Splicing of internal large exons is defined by novel cis-acting sequence elements.

Human internal exons have an average size of 147 nt, and most are <300 nt. This small size is thought to facilitate exon definition. A small number of large internal exons have been identified and shown to be alternatively spliced. We identified 1115 internal exons >1000 nt in the human genome; these were found in 5% of all protein-coding genes, and most were expressed and translated. Surprisingly, 40% of these were expressed at levels similar to the flanking exons, suggesting they were constitutively spliced. While all of the large exons had strong splice sites, the constitutively spliced large exons had a higher ratio of splicing enhancers/silencers and were more conserved across mammals than the alternatively spliced large exons. We asked if large exons contain specific sequences that promote splicing and identified 38 sequences enriched in the large exons relative to small exons. The consensus sequence is C-rich with a central invariant CA dinucleotide. Mutation of these sequences in a candidate large exon indicated that these are important for recognition of large exons by the splicing machinery. We propose that these sequences are large exon splicing enhancers (LESEs).

The structure and function of the rous sarcoma virus RNA stability element.

For simple retroviruses, such as the Rous sarcoma virus (RSV), post-transcriptional control elements regulate viral RNA splicing, export, stability, and packaging into virions. These RNA sequences interact with cellular host proteins to regulate and facilitate productive viral infections. One such element, known as the RSV stability element (RSE), is required for maintaining stability of the full-length unspliced RNA. This viral RNA serves as the mRNA for the Gag and Pol proteins and also as the genome packaged in progeny virions. When the RSE is deleted from the viral RNA, the unspliced RNA becomes unstable and is degraded in a Upf1-dependent manner. Current evidence suggests that the RSE inhibits recognition of the viral gag termination codon by the nonsense-mediated mRNA decay (NMD) pathway. We believe that the RSE acts as an insulator to NMD, thereby preventing at least one of the required functional steps that target an mRNA for degradation. Here, we discuss the history of the RSE and the current model of how the RSE is interacting with cellular NMD factors.

Structural features in the Rous sarcoma virus RNA stability element are necessary for sensing the correct termination codon.

BACKGROUND: Nonsense-mediated mRNA decay (NMD) is an mRNA quality control mechanism that selectively recognizes and targets for degradation mRNAs containing premature termination codons. Retroviral full-length RNA is presented to the host translation machinery with characteristics rarely observed among host cell mRNAs: a long 3' UTR, retained introns, and multiple open reading frames. As a result, the viral RNA is predicted to be recognized by the host NMD machinery and degraded. In the case of the Rous sarcoma virus (RSV), we identified a stability element (RSE), which resides immediately downstream of the gag termination codon and facilitates NMD evasion. RESULTS: We defined key RNA features of the RSE through directed mutagenesis of the virus. These data suggest that the minimal RSE is 155 nucleotides (nts) and functions independently of the nucleotide sequence of the stop codon or the first nucleotide following the stop codon. Further data suggested that the 3'UTRs of the RSV pol and src may also function as stability elements. CONCLUSIONS: We propose that these stability elements in RSV may be acting as NMD insulators to mask the preceding stop codon from the NMD machinery.

Reticuloendotheliosis virus strain T induces miR-155, which targets JARID2 and promotes cell survival.

The oncogenic microRNA miR-155 is upregulated by several oncogenic viruses. The precursor of miR-155, termed bic, was first observed to cooperate with myc in chicken B-cell lymphomas induced by avian leukosis proviral integrations. We identified another oncogenic retrovirus, reticuloendotheliosis virus strain T (REV-T), that upregulates miR-155 in chicken embryo fibroblasts. We also observed very high levels of miR-155 in REV-T-induced B-cell lymphomas. To study the role of miR-155 in these tumors, we identified JARID2/Jumonji, a cell cycle regulator and part of a histone methyltransferase complex, as a target of miR-155. The overexpression of miR-155 decreased levels of endogenous JARID2 mRNA. We confirmed that miR-155 directly targets both human and chicken JARID2 by assaying the repression of reporters containing the JARID2 3'-untranslated regions. Further, the overexpression of a sponge complementary to miR-155 in a tumor cell line increased endogenous JARID2 mRNA levels. The overexpression of JARID2 in chicken fibroblasts led to decreased cell numbers and an increase in apoptotic cells. The overexpression of miR-155 rescued cells undergoing cytopathic effect caused by infection with subgroup B avian retroviruses. Therefore, we propose that miR-155 has a prosurvival function that is mediated through the downregulation of targets including JARID2.

Structural characterization of the Rous sarcoma virus RNA stability element.

In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3' untranslated region (UTR) is often degraded by the nonsense-mediated mRNA decay (NMD) pathway. Despite the presence of a 5- to 7-kb 3' UTR, unspliced retroviral RNA escapes this degradation. We previously identified the Rous sarcoma virus (RSV) stability element (RSE), an RNA element downstream of the gag natural translation termination codon that prevents degradation of the unspliced viral RNA. Insertion of this element downstream of a PTC in the RSV gag gene also inhibits NMD. Using partial RNase digestion and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, we determined the secondary structure of this element. Incorporating RNase and SHAPE data into structural prediction programs definitively shows that the RSE contains an AU-rich stretch of about 30 single-stranded nucleotides near the 5' end and two substantial stem-loop structures. The overall secondary structure of the RSE appears to be conserved among 20 different avian retroviruses. The structural aspects of this element will serve as a tool in the future design of cis mutants in addressing the mechanism of stabilization.

Telomerase reverse transcriptase expression elevated by avian leukosis virus integration in B cell lymphomas.

Simple retroviruses induce tumors by integrating into the host genome, activating cellular oncogenes and microRNAs, or inactivating tumor suppressor genes. The identification of these genes elucidates molecular mechanisms of tumorigenesis. In this study, we identified avian leukosis virus (ALV) proviral integration sites in rapid-onset B cell lymphomas arising <12 weeks after infection of chicken embryos. By using inverse PCR, 28 unique viral integration sites were identified in rapid-onset tumors. Integrations in the telomerase reverse transcriptase (TERT) promoter/enhancer region were observed in four different tumors, suggesting that this is a common integration site. These provirus integrations ranged from 217 to 2,584 bp upstream of the TERT transcription initiation site and were all in the opposite transcriptional orientation to TERT. Southern blots of tumor samples demonstrated that these integrations are clonal and therefore occurred early in the process of tumorigenesis. Real-time RT-PCR showed overexpression of TERT mRNA in tumors harboring viral integrations in the TERT promoter. Telomerase activity was also up-regulated in these tumors; however, telomere-length alterations were not detected. Furthermore, viral LTR sequences directly enhanced the expression of luciferase reporters containing the TERT promoter sequences. This study documents retroviral up-regulation of cellular TERT by insertional activation to initiate or enhance tumor progression.

Tap and Dbp5, but not Gag, are involved in DR-mediated nuclear export of unspliced Rous sarcoma virus RNA.

All retroviruses must circumvent cellular restrictions on the export of unspliced RNAs from the nucleus. While the unspliced RNA export pathways for HIV and Mason-Pfizer monkey virus are well characterized, that of Rous sarcoma virus (RSV) is not. We have previously reported that the RSV direct repeat (DR) elements are involved in the cytoplasmic accumulation of unspliced viral RNA. Here, using fluorescent in situ hybridization (FISH), we demonstrate that unspliced viral RNAs bearing a single point mutation (G8863C) in the DR exhibit a restricted cellular localization in and around the nucleus. In contrast, wild type unspliced viral RNA had a diffuse localization throughout the nucleus and cytoplasm. Since the RSV Gag protein has a transient localization in the nucleus, we examined the effect of Gag over-expression on a DR-mediated reporter construct. While Gag did not enhance DR-mediated nuclear export, the dominant-negative expression of two cellular export factors, Tap and Dbp5, inhibited expression of the same reporter construct. Furthermore, FISH studies using the dominant-negative Dbp5 demonstrated that unspliced wild type RSV RNA was retained within the nucleus. Taken together, these results further implicate the DR in nuclear RNA export through interactions with Tap and Dbp5.

The negative regulator of splicing element of Rous sarcoma virus promotes polyadenylation.

The Rous sarcoma virus gag gene contains a cis-acting negative regulator of splicing (NRS) element that is implicated in viral polyadenylation regulation. To study the mechanism of polyadenylation promotion at the viral poly(A) site located over 8 kb downstream, we performed in vitro polyadenylation analysis. RNA containing only the poly(A) site and flanking sequences in the 3' long terminal repeat (LTR) was not polyadenylated detectably in vitro; however, if the transcript contained the NRS upstream of the LTR, polyadenylation was observed. Insertion of the viral env 3' splice site sequence between the NRS and the LTR did not alter the level of polyadenylation appreciably. We conclude that the NRS promotes polyadenylation in vitro and can do so without formation of a splicing complex with a 3' splice site. We then explored the roles of several cellular factors in NRS-mediated polyadenylation. Mutation of the binding sites of U1 and U11 snRNPs to the NRS did not affect polyadenylation, whereas hnRNP H strongly inhibited polyadenylation. We propose a model in which hnRNP H and SR proteins compete for binding to the NRS. Bound SR proteins may bridge between the NRS and the 3' LTR and aid in the recruitment of the 3'-end processing machinery.

A 3' UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus.

Eukaryotic cells target mRNAs to the nonsense-mediated mRNA decay (NMD) pathway when translation terminates within the coding region. In mammalian cells, this is presumably due to a downstream signal deposited during pre-mRNA splicing. In contrast, unspliced retroviral RNA undergoes NMD in chicken cells when premature termination codons (PTCs) are present in the gag gene. Surprisingly, deletion of a 401-nt 3' UTR sequence immediately downstream of the normal gag termination codon caused this termination event to be recognized as premature. We termed this 3' UTR region the Rous sarcoma virus (RSV) stability element (RSE). The RSE also stabilized the viral RNA when placed immediately downstream of a PTC in the gag gene. Deletion analysis of the RSE indicated a smaller functional element. We conclude that this 3' UTR sequence stabilizes termination codons in the RSV RNA, and termination codons not associated with such an RSE sequence undergo NMD.

Retroviral splicing suppressor sequesters a 3' splice site in a 50S aberrant splicing complex.

Retroviral replication requires both spliced and unspliced mRNAs. Splicing suppression of avian retroviral RNA depends in part upon a cis-acting element within the gag gene called the negative regulator of splicing (NRS). The NRS, linked to a downstream intron and exon (NRS-Ad3'), was not capable of splicing in vitro. However, a double-point mutation in the NRS pseudo-5' splice site sequence converted it into a functional 5' splice site. The wild-type (WT) NRS-Ad3' transcript assembled an approximately 50S spliceosome-like complex in vitro; its sedimentation rate was similar to that of a functional spliceosome formed on the mutant NRS-Ad3' RNA. The five major spliceosomal snRNPs were observed in both complexes by affinity selection. In addition, U11 snRNP was present only in the WT NRS-Ad3' complex. Addition of heparin to these complexes destabilized the WT NRS-Ad3' complex; it was incapable of forming a B complex on a native gel. Furthermore, the U5 snRNP protein, hPrp8, did not cross-link to the NRS pseudo-5' splice site, suggesting that the tri-snRNP complex was not properly associated with it. We propose that this aberrant, stalled spliceosome, containing U1, U2, and U11 snRNPs and a loosely associated tri-snRNP, sequesters the 3' splice site and prevents its interaction with the authentic 5' splice site upstream of the NRS.