scholarly journals Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors.

1995 ◽  
Vol 15 (11) ◽  
pp. 6480-6480 ◽  
Author(s):  
B A Amendt ◽  
Z H Si ◽  
C M Stoltzfus
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Exon 2 ◽  
Exon Splicing ◽  
Cellular Factors ◽  
Immunodeficiency Virus

scholarly journals Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors.

1995 ◽  
Vol 15 (8) ◽  
pp. 4606-4615 ◽  
Author(s):  
B A Amendt ◽  
Z H Si ◽  
C M Stoltzfus
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Splice Site ◽  
Exon 2 ◽  
Exon Splicing ◽  
Cellular Factors ◽  
Immunodeficiency Virus ◽  
Hiv 1

Human immunodeficiency virus type 1 (HIV-1) pre-mRNA splicing is regulated in order to maintain pools of unspliced and partially spliced viral RNAs as well as the appropriate levels of multiply spliced mRNAs during virus infection. We have previously described an element in tat exon 2 that negatively regulates splicing at the upstream tat 3' splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, Mol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicing enhancer (ESE) elements, we have termed this element an exon splicing silencer (ESS). We show evidence for another negative cis-acting region within tat-rev exon 3 of HIV-1 RNA that has sequence motifs in common with a 20-nt ESS element in tat exon 2. This sequence is juxtaposed to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream tat-rev 3' splice site. Inhibition of the splicing of substrates containing the ESS element in tat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing mediated by the ESS can be specifically abrogated by the addition of competitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and tat-rev exon 3.

scholarly journals The Exon Splicing Silencer in Human Immunodeficiency Virus Type 1 Tat Exon 3 Is Bipartite and Acts Early in Spliceosome Assembly

1998 ◽  
Vol 18 (9) ◽  
pp. 5404-5413 ◽  
Author(s):  
Zhi-hai Si ◽  
Dan Rauch ◽  
C. Martin Stoltzfus
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Splice Site ◽  
Early Step ◽  
Exon 2 ◽  
Exon Splicing ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3′ splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present withintat exon 2 (ESS2) and a suboptimal 3′ splice site together act to inhibit splicing at the 3′ splice site flanking tatexon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3′ splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3′ splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition oftat mRNA splicing mediated by ESS2 and ESS3.

scholarly journals Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression

2008 ◽  
Vol 82 (8) ◽  
pp. 3921-3931 ◽  
Author(s):  
C. M. Exline ◽  
Z. Feng ◽  
C. M. Stoltzfus
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Regulatory Elements ◽  
Mrna Splicing ◽  
Viral Mrnas ◽  
Exon 2 ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5′ splice site (5′ss) downstream of exon 2 (5′ss D2). Here we show that the mutations within 5′ss D2 that are predicted to lower or increase the affinity of the 5′ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5′ss D2 was not necessary for the effect of 5′ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5′ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5′-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5′ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.

scholarly journals The Immunosuppressant Rapamycin Represses Human Immunodeficiency Virus Type 1 Replication

2002 ◽  
Vol 46 (11) ◽  
pp. 3447-3455 ◽  
Author(s):  
Jocelyn Roy ◽  
Jean-Sébastien Paquette ◽  
Jean-François Fortin ◽  
Michel J. Tremblay
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Lymphoid Cells ◽  
Luciferase Reporter ◽  
Exon 2 ◽  
Rapamycin Treatment ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The immunosuppressive macrolide rapamycin is used in humans to prevent graft rejection. This drug acts by selectively repressing the translation of proteins that are encoded by an mRNA bearing a 5′-polypyrimidine tract (e.g., ribosomal proteins, elongation factors). The human immunodeficiency virus type 1 (HIV-1) carries a polypyrimidine motif that is located within the tat exon 2. Treatment of human T lymphoid cells with rapamycin resulted in a marked diminution of HIV-1 transcription when infection was performed with luciferase reporter T-tropic and macrophage-tropic viruses. Replication of fully infectious HIV-1 particles was abolished by rapamycin treatment. The rapamycin-mediated inhibitory effect on HIV-1 production was reversed by FK506. The anti-HIV-1 effect of rapamycin was also seen in primary human cells (i.e., peripheral blood lymphocytes) from different healthy donors. Rapamycin was shown to diminish basal HIV-1 long terminal repeat gene expression, and the observed effect of rapamycin on HIV-1 replication seems to be independent of the virus-specific transactivating Tat protein. A constitutive β-actin promoter-based reporter gene vector was unaffected by rapamycin treatment. Kinetic virus infection studies and exposure to reporter viruses pseudotyped with heterologous envelope proteins (i.e., amphotropic murine leukemia virus and vesicular stomatitis virus G) suggested that rapamycin is primarily affecting the life cycle of HIV-1 at a transcriptional level. Northern blot analysis confirmed that this compound is selectively targeting HIV-1 mRNA synthesis.

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

2008 ◽  
Vol 83 (2) ◽  
pp. 981-992 ◽  
Author(s):  
Joseph A. Jablonski ◽  
Massimo Caputi
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Intracellular Localization ◽  
Sr Proteins ◽  
Mrna Metabolism ◽  
Cellular Factors ◽  
Immunodeficiency Virus

ABSTRACT 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.

scholarly journals Identification of positive and negative splicing regulatory elements within the terminal tat-rev exon of human immunodeficiency virus type 1.

1995 ◽  
Vol 15 (8) ◽  
pp. 4597-4605 ◽  
Author(s):  
A Staffa ◽  
A Cochrane
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Splice Site ◽  
Regulatory Elements ◽  
Control Element ◽  
Exon Splicing ◽  
Immunodeficiency Virus

The requirement of human immunodeficiency virus type 1 to generate numerous proteins from a single primary transcript is met largely by the use of suboptimal splicing to generate over 30 mRNAs. To ensure that appropriate quantities of each protein are produced, there must be a signal(s) that controls the efficiency with which any particular splice site in the RNA is used. To identify this control element(s) and to understand how it operates to generate the splicing pattern observed, we have initially focused on the control of splicing of the tat-rev intron, which spans the majority of the env open reading frame. Previous analysis indicated that a suboptimal branchpoint and polypyridimine tract in this intron contribute to its suboptimal splicing (A. Staffa and A. Cochrane, J. Virol. 68:3071-3079, 1994). In this report, we identify two additional elements within the 3'-terminal exon, an exon-splicing enhancer (ESE) and an exon splicing silencer (ESS), that modulate the overall efficiency with which the 3' tat-rev splice site is utilized. Both elements are capable of functioning independently of one another. Furthermore, while both the ESE and ESS can function in a heterologous context, the function of the ESS is extremely sensitive to the sequence context into which it is placed. In conclusion, it would appear that the presence of a suboptimal branchpoint and a polypyrimidine tract as well as the ESE and ESS operate together to yield the balanced splicing of the tat-rev intron observed in vivo.

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