Biochemical and NMR Study on the Competition between Proteins SC35, SRp40, and Heterogeneous Nuclear Ribonucleoprotein A1 at the HIV-1 Tat Exon 2 Splicing Site

Splicing of human immunodeficiency virus type 1 (HIV-1) exon 6D is regulated by the presence of a complex splicing regulatory element (SRE) sequence that interacts with the splicing factors hnRNP H and SC35. In this work, we show that, in the context of the wild-type viral sequence, hnRNP H acts as a repressor of exon 6D inclusion independent of its binding to the SRE. However, hnRNP H binding to the SRE acts as an enhancer of exon 6D inclusion in the presence of a critical T-to-C mutation. These seemingly contrasting functional properties of hnRNP H appear to be caused by a change in the RNA secondary structure induced by the T-to-C mutation that affects the spatial location of bound hnRNP H with respect to the exon 6D splicing determinants. We propose a new regulatory mechanism mediated by RNA folding that may also explain the dual properties of hnRNP H in splicing regulation.Replication of human immunodeficiency virus type 1 (HIV-1) is dependent on the balanced expression of several viral genes. Multiple cellular factors interact with viral sequences to properly process the single viral primary transcript into more than 40 mRNAs (32). These messages fall into three classes; a ϳ2-kb class of multiple spliced mRNAs coding for the viral proteins Tat, Rev, and Nef, a ϳ4 -kb class of partially unspliced mRNAs coding for the viral proteins Env, Vpu, Vif, and Vpr, and the 9.2-kb unspliced transcript coding for the viral proteins Gag and Pol and which is also packaged into new virions as the viral genome (Fig. 1A). The HIV-1 genome is characterized by a high mutation rate that generates a heterogeneous population of viral isolates. This can lead to the appearance of noncanonical splice sites and unusual patterns of splicing. An example of this unusual splicing was described for the 6D exon, located within the env gene. Its inclusion results in the production of a series of aberrant mRNAs yielding aberrant Tat and Rev proteins (2, 33) (Fig. 1A). Although the splicing machinery rarely recognizes this exon, a naturally arising point mutation (T to C) within the env gene that activates its inclusion into most viral messages was identified. This splicing inclusion of exon 6D leads to downregulation of the production of all viral genes and leads to inefficient viral replication (39). Sequence analysis of viral isolates indicates that the sequences regulating exon 6D splicing are highly conserved at the RNA level but do not correlate with conservation of protein sequence (7).We have previously shown that a multiprotein complex, which includes members of the hnRNP H and serine/argininerich (SR) protein families, specifically binds to a tripartite splicing regulatory element (SRE) within exon 6D (7) (Fig. 1C). Members of the SR protein family have been shown to regulate splicing by binding purine-rich sequences and directing the assembly of prespliceosomal and spliceosomal factors to 5Ј and 3Ј splice sites (17). In agreement with these observations, our previous data indicated that several SR proteins bind to a purine-rich ...

Section: Resultsmentioning

confidence: 99%

The Secondary Structure of the Human Immunodeficiency Virus Type 1 Transcript Modulates Viral Splicing and Infectivity

Jablonski

Buratti

Stuani

et al. 2008

“…1C). For this purpose, we created U1 snRNA mutants (U1T1 to U1T6) that were predicted to base pair to 10-nt HIV-1 sequences within the stemloop structure SLS3-A3 in exon 4 (16,19). To investigate whether expression of the modified U1 snRNAs described above increases splicing of HIV-1 RNA, we cotransfected 293T cells with plasmids expressing modified U1 snRNAs together with the infectious HIV-1 plasmid pNL4-3.…”

Section: Resultsmentioning

confidence: 99%

“…However, overlap of a negative regulatory element cannot explain the results with U1T6, which we showed was equally effective in inducing excessive splicing. Another possibility is sequestration of target sequences in regions of base-paired secondary structures (16,19). Insertion of target sequences into duplex structures has previously been shown to greatly reduce the inhibition induced by modified U1 snRNAs targeted to the 3Ј-terminal exons (14).…”

Section: Downloaded Frommentioning

confidence: 99%

Excessive RNA Splicing and Inhibition of HIV-1 Replication Induced by Modified U1 Small Nuclear RNAs

Mandal

Feng

Stoltzfus

2010

HIV-1 RNA undergoes a complex splicing process whereby over 40 different mRNA species are produced by alternative splicing. In addition, approximately half of the RNA transcripts remain unspliced and either are used to encode Gag and Gag-Pol proteins or are packaged into virions as genomic RNA. It has previously been shown that HIV-1 splicing is regulated by cis elements that bind to cellular factors. These factors either enhance or repress definition of exons that are flanked by the HIV-1 3 splice sites. Here we report that expression of modified U1 snRNPs with increased affinity to HIV-1 downstream 5 splice sites and to sequences within the first tat coding exon act to selectively increase splicing at the upstream 3 splice sites in cotransfected 293T cells. This results in a decrease of unspliced viral RNA levels and an approximately 10-fold decrease in virus production. In addition, excessive splicing of viral RNA is concomitant with a striking reduction in the relative amounts of Gag processing intermediates and products. We also show that T cell lines expressing modified U1 snRNAs exhibit reduced HIV-1 replication. Our results suggest that induction of excessive HIV-1 RNA splicing may be a novel strategy to inhibit virus replication in human patients.The HIV-1 primary RNA transcript undergoes a complex splicing pathway leading to the production of over 40 different spliced mRNA species as well as unspliced (US) RNA (30). Unspliced HIV-1 mRNA, which is transported into the cytoplasm by a Rev-dependent process, encodes Gag and Gag-Pol structural proteins. Unspliced viral RNA is also packaged into the virus particles as genome RNA. Spliced HIV-1 mRNAs are either incompletely spliced (IS) and serve as mRNAs for viral Env, Vpu, Vpr, and Vif proteins or completely spliced (CS) to serve as mRNAs for Tat, Rev, or Nef protein. Splicing is a two-step catalytic reaction that occurs within a large complex called the spliceosome. One of the earliest steps in spliceosome assembly is the recognition of 5Ј splice donor sites (5Јss) by cellular U1 snRNP (28, 39). The extent of base pairing between the 5Јss and the 5Ј-terminal sequence of U1 snRNA determines the strength of the 5Јss and thus contributes to the recognition and frequency of U1 snRNP usage (17, 32). In retroviruses, 3Ј splice acceptor sites (3Јss) are weak, and recognition of these sites is assisted by the binding of cellular splicing regulatory proteins which regulate alternative splicing and gene expression. These regulatory proteins (SR proteins and hnRNPs) bind to exonic splicing enhancers (ESE) or exonic or intronic splicing silencers (ESS or ISS) within the viral genome (10, 38). U1 snRNP bound at the 5Јss also activates splicing at the upstream 3Јss by a mechanism referred to "exon definition" or "exon bridging" (18, 31).Our laboratory has shown that splicing of the mRNAs encoding HIV-1 accessory proteins Vif and Vpr is regulated by suboptimal 5Јss D2 and D3 downstream of 3Јss A1 and A2, respectively (Fig. 1A) (13,23,24). Mutations of 5Јss D2 that increa...

“…Although hnRNPs of the A/B family and the SR protein SC35 regulate expression of the Tat1 mRNA as predicted in an in vitro system (15,41), downregulation of the Rev2 and Nef2 mRNAs following overexpression of SF2 and SRp40 does not correlate well with data obtained with a viral mini-gene (4). Discrepancies between the results presented here and the ones obtained in controlled biochemical assays or with partial genomic sequences are likely due to (i) the combinatorial effect of multiple and overlapping viral sequences that may interact with several cellular factors, (ii) pleiotropic effects on mRNA metabolism of hnRNPs and SR proteins, and (iii) secondary effects on cellular genes required for viral gene expression, replication, and infectivity.…”

Section: Downloaded Frommentioning

confidence: 99%

Role of Cellular RNA Processing Factors in Human Immunodeficiency Virus Type 1 mRNA Metabolism, Replication, and Infectivity

Jablonski

Caputi

2009

102

Expression of the human immunodeficiency virus type 1 genome requires several cellular factors regulating transcription, alternative splicing, RNA stability, and intracellular localization of the viral transcripts. In vitro and ex vivo approaches have identified SR proteins and hnRNPs of the A/B and H subfamilies as cellular factors that regulate different aspects of viral mRNA metabolism. To understand the role of these protein families within the context of the full replicating virus, we altered the expression levels of hnRNPs H, F, 2H9, GRSF1, A1, A2, and A3 and SR proteins SC35, SF2, and SRp40 in HEK 293 cells transfected with the proviral clone pNL4-3. Quantitative and semiquantitative PCR analyses showed that overexpression as well as downregulation of these proteins disrupted the balance of alternatively spliced viral mRNAs and may alter viral transcription. Furthermore, expression of hnRNPs H, F, 2H9, A1, and A2 and SR proteins SF2 and SRp40 increased nuclear localization of the unspliced Gag/Pol mRNA, while the same factors increased the cytoplasmic localization of the partially spliced Env mRNA. We also report that overexpression of hnRNPs A1 and A2 and SR proteins SF2, SC35, and SRp40 causes a dramatic decrease in virion production. Finally, utilizing a reporter TZM-bl cell line, we show that virion infectivity may be also impacted by deregulation of expression of most SR proteins and hnRNPs. This work demonstrates that cellular factors regulating mRNA processing have wide-ranging effects on human immunodeficiency virus type 1 replication and should be considered novel therapeutic targets.The complex mechanisms that regulate human immunodeficiency virus type 1 (HIV-1) replication utilize both cellular and viral factors, which interact with several cis-acting elements located within the viral genome. Processing of the HIV-1 transcript provides an important model for human RNA processing pathways and can be key in the characterization of novel therapeutic targets to block viral replication. Transcription of the integrated viral genome generates a single transcript and is mediated by several transcription factors and the viral protein Tat (3, 31). The viral transcript undergoes a complex series of splicing events to generate 22 different mRNAs of approximately 4 kb coding for the Env, Vpu, Vpr, and Vif proteins and 22 different mRNAs of approximately 2 kb coding for the Tat, Rev, Vpr, and Nef proteins ( Fig. 1) (33). The unspliced 9-kb mRNA codes for the Gag and Gag/Pol polyproteins and is packaged within the nascent virions as a viral genome. Alteration of the balanced splicing of the viral mRNAs can have dramatic effects on viral replication and infectivity (20,33,39). HIV-1 splicing regulation relies on the presence of intronic and exonic sequences as well as cellular splicing factors that interact with these elements. To date, four exonic splicing silencers, one intronic splicing silencer, and four splicing exonic enhancers have been identified (37).In addition to having a complex arrangement of spli...