Human immunodeficiency virus type 1 hnRNP A/B-dependent exonic splicing silencer ESSV antagonizes binding of U2AF65 to viral polypyrimidine tracts

Human immunodeficiency virus type 1 (HIV-1) exonic splicing silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of splicing by ESSV. Possible splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication are discussed.     

B-cell and plasma-cell splicing differences: a potential role in regulated immunoglobulin RNA processing

The immunoglobulin micro pre-mRNA is alternatively processed at its 3' end by competing splice and cleavage-polyadenylation reactions to generate mRNAs encoding the membrane-associated or secreted forms of the IgM protein, respectively. The relative use of the competing processing pathways varies during B-lymphocyte development, and it has been established previously that cleavage-polyadenylation activity is higher in plasma cells, which secrete IgM, than in B cells, which produce membrane-associated IgM. To determine whether RNA-splicing activity varies during B-lymphocyte development to contribute to micro RNA-processing regulation, we first demonstrate that micro pre-mRNA processing is sensitive to artificial changes in the splice environment by coexpressing SR proteins with the micro gene. To explore differences between the splice environments of B cells and plasma cells, we analyzed the splicing patterns from two different chimeric non-Ig genes that can be alternatively spliced but have no competing cleavage-polyadenylation reaction. The ratio of intact exon splicing to cryptic splice site use from one chimeric gene differs between several B-cell and several plasma-cell lines. Also, the amount of spliced RNA is higher in B-cell than plasma-cell lines from a set of genes whose splicing is dependent on a functional exonic splice enhancer. Thus, there is clear difference between the B-cell and plasma-cell splicing environments. We propose that both general cleavage-polyadenylation and general splice activities are modulated during B-lymphocyte development to ensure proper regulation of the alternative micro RNA processing pathways.     

Simian immunodeficiency virus displays complex patterns of RNA splicing.

The human and simian immunodeficiency viruses encode at least six gene products that apparently serve regulatory functions. To evaluate the regulation of simian immunodeficiency virus gene expression at the level of RNA splicing, we used the polymerase chain reaction to amplify and clone cDNAs corresponding to a large array of mRNAs from infected cells. We identified mRNAs that used splice acceptor sites upstream of the initiator codons for tat, rev, vpr, nef, vif, and vpx, suggesting that these proteins may be expressed from different mRNAs. We also provide hybridization data suggesting that the same splice acceptor site may be used for both rev and env mRNAs. Furthermore, we isolated both tat and rev cDNAs that utilized three alternative splice acceptor sites at the start of coding exon 2, indicating that different versions of these proteins may be encoded. Finally, approximately 10 to 20% of simian immunodeficiency virus mRNAs spliced an intron from their untranslated 5' ends, and sequences contained within this intron constituted a portion of the tat-responsive TAR element. Thus, alternative pre-mRNA splicing adds a level of complexity to simian immunodeficiency virus expression, which may affect several levels of gene regulation.     

Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV.     

Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein.

Fine-structure mapping of the capsid-specific mRNAs from adeno-associated virus (AAV) revealed an alternate splicing pattern in these RNAs. S1 nuclease and primer extension analyses showed that splicing of these mRNAs occurs at acceptor sites at nucleotide 2228 (major splice) or 2201 (minor splice). Both splice acceptors were ligated to the same 55-nucleotide leader in mature mRNAs. Both species were present in equal amounts in mRNA derived from AAV plasmid-transfected cells. However, when adenovirus infection accompanied the DNA transfection, the major splice predominated over the minor splice. Using cDNA clones of both the major and minor spliced mRNAs, we demonstrated that the largest AAV capsid protein, VP1, was derived from the minor spliced mRNA. The other capsid proteins, VP2 and VP3, came predominantly from the major spliced mRNA. These results, which describe the previously undetected minor splice, provide a mechanism for the production of all three AAV virion proteins.     

A naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production.

We have isolated a naturally arising human immunodeficiency type 1 (HIV-1) mutant containing a point mutation within the env gene. The point mutation resulted in complete loss of balanced splicing, with dominant production of aberrant mRNAs. The aberrant RNAs arose via activation of normally cryptic splice sites flanking the mutation within the env terminal exon to create exon 6D, which was subsequently incorporated in aberrant env, tat, rev, and nef mRNAs. Aberrant multiply spliced messages contributed to reduced virus replication as a result of a reduction in wild-type Rev protein. The point mutation within exon 6D activated exon 6D inclusion when the exon and its flanking splice sites were transferred to a heterologous minigene. Introduction of the point mutation into an otherwise wild-type HIV-1 proviral clone resulted in virus that was severely inhibited for replication in T cells and displayed elevated usage of exon 6D. Exon 6D contains a bipartite element similar to that seen in tat exon 3 of HIV-1, consisting of a potential exon splicing silencer (ESS) juxtaposed to a purine-rich sequence similar to known exon splicing enhancers. In the absence of a flanking 5' splice site, the point mutation within the exon 6D ESS-like element strongly activated env splicing, suggesting that the putative ESS plays a natural role in limiting the level of env splicing. We propose, therefore, that exon silencers may be a common element in the HIV-1 genome used to create balanced splicing of multiple products from a single precursor RNA.     

A bidirectional SF2/ASF- and SRp40-dependent splicing enhancer regulates human immunodeficiency virus type 1 rev, env, vpu, and nef gene expression

The integrated human immunodeficiency virus type 1 (HIV-1) genome is transcribed in a single pre-mRNA that is alternatively spliced into more than 40 mRNAs. We characterized a novel bidirectional exonic splicing enhancer (ESE) that regulates the expression of the HIV-1 env, vpu, rev, and nef mRNAs. The ESE is localized downstream of the vpu-, env-, and nef-specific 3' splice site no. 5. SF2/ASF and SRp40 activate the ESE and are required for efficient 3' splice site usage and binding of the U1 snRNP to the downstream 5' splice site no. 4. U1 snRNP binding to the 5' splice site no. 4 is required for splicing of the rev and nef mRNAs and to increase expression of the partially spliced env mRNA. Finally, our results indicate that this ESE is necessary for the recruitment of the U1 snRNP to the 5' splice site no. 4, even when the 5' splice site and the U1 snRNA have been mutated to obtain a perfect complementary match. The ESE characterized here is highly conserved in most viral subtypes.     

Activation of a cryptic splice donor in human immunodeficiency virus type-1

The human immunodeficiency virus type-1 regulatory protein Rev is absolutely required for the production of viral structural proteins. Splice sites have been seen to function ascis-acting repressor sequendes (CRS) and inhibit expression of the Rev-dependent RNAs. In order to analyze the role of a splice donor in Rev dependence, the wild-type 5 splice donor of HIV-1 was mutated in the context of othergag sequences. Following transient transfection, RNA expression by RT-PCR was analyzed. The unspliced RNA produced by the mutant construct still required Rev for the cytoplasmic accumulation of the RNA. Despite deletion of the wild-type 5 splice donor and thetat splice acceptor was used. A cryptic splice donor was identified by PCR and subsequent cloning of the spliced RNA. The cryptic site is 5/9 to the consensus sequence and located immediately downstream of the initiation codon (ATG) for Gag. Analysis of the RNA product containing the cryptic splice donor revealed that the Rev was required for the cytoplasmic accumulation of unspliced RNA, while spliced RNA was Rev independent. Transfection of a wild-type construct also demonstrated usage of the cryptic splice donor. These results indicate that a cryptic splice donor can be activated when the wild-type splice donor is inactivated and that the cryptic splice donor may retain Rev regulation. The findings also suggest the potential for cryptic splice sites to serve as CRS in the determining the Rev dependence of viral RNAs.     

Characterization and expression of novel singly spliced RNA species of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) expresses the Vif, Vpr, Vpu, and Env proteins through complex differential splicing of a single full-length RNA precursor. We used HIV-1-specific oligonucleotide primer pairs in a quantitative polymerase chain reaction procedure on RNA from fresh peripheral blood lymphocytes infected with HIV-1JR-CSF to detect and characterize the singly spliced RNA species which might encode these proteins. The nucleotide sequences at the junctions of splice donor and acceptor sites of these RNAs were determined. One of these RNAs, which has not been previously described, appears to be a novel HIV-1 RNA encoding Env and/or Vpu proteins.     

Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene.

A large proportion of mutations at the human hprt locus result in aberrant splicing of the hprt mRNA. We have been able to relate the mutation to the splicing abnormality in 30 of these mutants. Mutations at the splice acceptor sites of introns 4, 6 and 7 result in splicing out of the whole of the downstream exons, whereas in introns 1, 7 or 8 a cryptic site in the downstream exon can be used. Mutations in the donor site of introns 1 and 5 result in the utilisation of cryptic sites further downstream, whereas in the other introns, the upstream exons are spliced out. Our most unexpected findings were mutations in the middle of exons 3 and 8 which resulted in splicing out of these exons in part of the mRNA populations. Our results have enabled us to assess current models of mRNA splicing. They emphasize the importance of the polypyrimidine tract in splice acceptor sites, they support the role of the exon as the unit of assembly for splicing, and they are consistent with a model proposing a stem-loop structure for exon 8 in the hprt mRNA.     

Comparison of Rous sarcoma virus RNA processing in chicken and mouse fibroblasts: evidence for double-spliced RNA in nonpermissive mouse cells.

Rous sarcoma virus, an avian retrovirus, transforms but does not replicate in mammalian cells. To determine to what extent differences in RNA splicing might contribute to this lack of productive infection, cloned proviral DNA derived from the Prague A strain of Rous sarcoma virus was transfected into mouse NIH 3T3 cells, and the viral RNA was compared by RNase protection with viral RNA from transfected chicken embryo fibroblasts by using a tandem antisense riboprobe spanning the three major splice sites. The levels of viral RNA in NIH 3T3 cells compared with those in chicken embryo fibroblasts were lower, but the RNA was spliced at increased efficiency. The difference in the ratio of unspliced to spliced RNA levels was not due to the increased lability of unspliced RNA in NIH 3T3 cells. Although chicken embryo fibroblasts contained equal levels of src and env mRNAs, spliced viral mRNAs in NIH 3T3 cells were almost exclusively src. In NIH 3T3 cells the env mRNA was further processed by using a cryptic 5' splice site located within the env coding sequences and the normal src 3' splice site to form a double-spliced mRNA. This mRNA was identical to the src mRNA, except that a 159-nucleotide sequence from the 5' end of the env gene was inserted at the src splice junction. Smaller amounts of single-spliced RNA were also present in which only the region between the cryptic 5' and src 3' splice sites was spliced out. The aberrant processing of the viral env mRNA in NIH 3T3 cells may in part explain the nonpermissiveness of these cells to productive Rous sarcoma virus infection.     

Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.

The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown.